2022年11月, コーセーコスメトロジー研究財団, コーセーコスメトロジー奨励賞　　　　　　　　　　　　　　　

2015年10月, セラミド研究会, JSC Award　　　　　　　　　　　　　　　
木原 章雄

2015年03月, 北海道大学, 北海道大学研究総長賞奨励賞　　　　　　　　　　　　　　　
木原 章雄

2005年, 日本生化学会, 平成１７年度 日本生化学会奨励賞　　　　　　　　　　　　　　　
木原章雄, 日本国

Correlations between Skin Condition Parameters and Ceramide Profiles in the Stratum Corneum of Healthy Individuals.
Fuminari Akiyama, Natsumi Takahashi, Yuto Ueda, Shizuno Tada, Nobuyuki Takeuchi, Yusuke Ohno, Akio Kihara
International journal of molecular sciences, 25, 15, 2024年07月29日, ［国際誌］
英語, 研究論文（学術雑誌）, Ceramides are essential lipids for skin barrier function, and various classes and species exist in the human stratum corneum (SC). To date, the relationship between skin conditions and ceramide composition in healthy individuals has remained largely unclear. In the present study, we measured six skin condition parameters (capacitance, transepidermal water loss, scaliness, roughness, multilayer exfoliation, and corneocyte cell size) for the SC of the cheeks and upper arms of 26 healthy individuals and performed correlation analyses with their SC ceramide profiles, which we measured via liquid chromatography-tandem mass spectrometry. In the cheeks, high levels and/or ratios of two free ceramide classes containing an extra hydroxyl group in the long-chain moiety and a protein-bound ceramide class containing 6-hydroxysphingosine correlated with healthy skin conditions. In contrast, the ratios of two other free ceramide classes, both containing sphingosine, and a protein-bound ceramide class containing 4,14-sphingadiene correlated with unhealthy skin conditions, as did shortening of the carbon chain of the fatty acid portion of two ceramide classes containing non-hydroxy fatty acids. Thus, our findings help to elucidate the relationship between skin conditions and ceramide composition.

Alteration of epidermal lipid composition as a result of deficiency in the magnesium transporter Nipal4
Marino Yamaji, Yusuke Ohno, Madoka Shimada, Akio Kihara
Journal of Lipid Research, 2024年06月
研究論文（学術雑誌）

Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.
Hideo Hagihara, Hirotaka Shoji, Satoko Hattori, Giovanni Sala, Yoshihiro Takamiya, Mika Tanaka, Masafumi Ihara, Mihiro Shibutani, Izuho Hatada, Kei Hori, Mikio Hoshino, Akito Nakao, Yasuo Mori, Shigeo Okabe, Masayuki Matsushita, Anja Urbach, Yuta Katayama, Akinobu Matsumoto, Keiichi I Nakayama, Shota Katori, Takuya Sato, Takuji Iwasato, Haruko Nakamura, Yoshio Goshima, Matthieu Raveau, Tetsuya Tatsukawa, Kazuhiro Yamakawa, Noriko Takahashi, Haruo Kasai, Johji Inazawa, Ikuo Nobuhisa, Tetsushi Kagawa, Tetsuya Taga, Mohamed Darwish, Hirofumi Nishizono, Keizo Takao, Kiran Sapkota, Kazutoshi Nakazawa, Tsuyoshi Takagi, Haruki Fujisawa, Yoshihisa Sugimura, Kyosuke Yamanishi, Lakshmi Rajagopal, Nanette Deneen Hannah, Herbert Y Meltzer, Tohru Yamamoto, Shuji Wakatsuki, Toshiyuki Araki, Katsuhiko Tabuchi, Tadahiro Numakawa, Hiroshi Kunugi, Freesia L Huang, Atsuko Hayata-Takano, Hitoshi Hashimoto, Kota Tamada, Toru Takumi, Takaoki Kasahara, Tadafumi Kato, Isabella A Graef, Gerald R Crabtree, Nozomi Asaoka, Hikari Hatakama, Shuji Kaneko, Takao Kohno, Mitsuharu Hattori, Yoshio Hoshiba, Ryuhei Miyake, Kisho Obi-Nagata, Akiko Hayashi-Takagi, Léa J Becker, Ipek Yalcin, Yoko Hagino, Hiroko Kotajima-Murakami, Yuki Moriya, Kazutaka Ikeda, Hyopil Kim, Bong-Kiun Kaang, Hikari Otabi, Yuta Yoshida, Atsushi Toyoda, Noboru H Komiyama, Seth G N Grant, Michiru Ida-Eto, Masaaki Narita, Ken-Ichi Matsumoto, Emiko Okuda-Ashitaka, Iori Ohmori, Tadayuki Shimada, Kanato Yamagata, Hiroshi Ageta, Kunihiro Tsuchida, Kaoru Inokuchi, Takayuki Sassa, Akio Kihara, Motoaki Fukasawa, Nobuteru Usuda, Tayo Katano, Teruyuki Tanaka, Yoshihiro Yoshihara, Michihiro Igarashi, Takashi Hayashi, Kaori Ishikawa, Satoshi Yamamoto, Naoya Nishimura, Kazuto Nakada, Shinji Hirotsune, Kiyoshi Egawa, Kazuma Higashisaka, Yasuo Tsutsumi, Shoko Nishihara, Noriyuki Sugo, Takeshi Yagi, Naoto Ueno, Tomomi Yamamoto, Yoshihiro Kubo, Rie Ohashi, Nobuyuki Shiina, Kimiko Shimizu, Sayaka Higo-Yamamoto, Katsutaka Oishi, Hisashi Mori, Tamio Furuse, Masaru Tamura, Hisashi Shirakawa, Daiki X Sato, Yukiko U Inoue, Takayoshi Inoue, Yuriko Komine, Tetsuo Yamamori, Kenji Sakimura, Tsuyoshi Miyakawa
eLife, 12, 2024年03月26日, ［国際誌］
英語, 研究論文（学術雑誌）, Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases
Ryoya Kato, Yuka Takenaka, Yusuke Ohno, Akio Kihara
Journal of Biological Chemistry, 300, 2, 105656, 105656, Elsevier BV, 2024年02月
研究論文（学術雑誌）

Comparison of skin barrier abnormalities and epidermal ceramide profiles among three ω-O-acylceramide synthesis-deficient mouse strains.
Yuta Yamamoto, Takayuki Sassa, Akio Kihara
Journal of dermatological science, 113, 1, 10, 17, 2024年01月, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: The epidermis contains many structurally diverse ceramides, which form the skin permeability barrier (skin barrier). Mutations in genes involved in the synthesis of ω-O-acylceramides (acylceramides) and protein-bound ceramides cause ichthyosis. OBJECTIVE: We aimed to elucidate the relationship between the degree of skin barrier impairment and changes in epidermal ceramide profiles caused by mutations in acylceramide synthesis genes. METHODS: Knockout (KO) mice of three genes-fatty acid (FA) ω-hydroxylase Cyp4f39 (human CYP4F22 ortholog), FA elongase Elovl1, and acyl-CoA synthetase Fatp4-were subjected to transepidermal water loss measurement, toluidine blue staining, and epidermal ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: Transepidermal water loss was highest in Cyp4f39 KO mice, followed by Elovl1 KO and Fatp4 KO mice, and Cyp4f39 KO mice also showed the strongest degree of toluidine blue staining. In Cyp4f39 KO, Elovl1 KO, and Fatp4 KO mice, acylceramide levels were 0.6%, 1.6%, and 12%, respectively, of those in wild-type mice. Protein-bound ceramide levels were 0.2%, 30%, and 33%, respectively, of those in wild-type mice. We also observed a near-complete absence of ω-hydroxy ceramides in Cyp4f39 KO mice, reduced total ceramide levels and shortened FA moieties in Elovl1 KO mice, and increased hydroxylated ceramide levels and slightly shortened FA moieties in Fatp4 KO mice. CONCLUSIONS: The degree of reduction in protein-bound ceramide levels is probably related to the severity of skin barrier defects in these three strains. However, reduced acylceramide levels and other changes in ceramide composition unique to each KO strain are also involved.

Determining the structure of protein-bound ceramides, essential lipids for skin barrier function.
Yusuke Ohno, Tetsuya Nakamura, Takafumi Iwasaki, Akira Katsuyama, Satoshi Ichikawa, Akio Kihara
iScience, 26, 11, 108248, 108248, 2023年11月17日, ［国際誌］
英語, 研究論文（学術雑誌）, Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the β-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides.

Metabolism of sphingadiene and characterization of the sphingadiene-producing enzyme FADS3.
Keisuke Jojima, Akio Kihara
Biochimica et biophysica acta. Molecular and cell biology of lipids, 1868, 8, 159335, 159335, 2023年08月, ［国際誌］
英語, 研究論文（学術雑誌）, Of the long-chain bases (LCBs) that comprise the ceramides (CERs) present in mammals, only 4,14-sphingadiene (sphingadiene; SPD) has a cis double bond (at C14). Because of this unique structure, the metabolism of SPD may differ from that of other LCBs, but whether this is the case remains unclear. FADS3 is responsible for introducing the cis double bond in SPD. However, the substrate specificity of FADS3 and cofactors involved in the FADS3-catalyzed reaction are also unknown. In the present study, a cell-based assay using a ceramide synthase inhibitor and an in vitro experiment showed that FADS3 is active toward sphingosine (SPH)-containing CERs (SPH-CERs) but not toward free SPH. FADS3 exhibits specificity with respect to the chain length of the SPH moiety of SPH-CERs (active toward C16-20), but not that of the fatty acid moiety. Furthermore, FADS3 is active toward straight-chain and iso-branched-chain SPH-containing CERs but not toward anteiso-branched forms. In addition to SPH-CERs, FADS3 also shows activity toward dihydrosphingosine-containing CERs, but this activity is approximately half of that toward SPH-CERs. It uses either NADH or NADPH as an electron donor, and the electron transfer is facilitated by cytochrome b5. The metabolic flow of SPD to sphingomyelin is predominant over that to glycosphingolipids. In the metabolic pathway from SPD to fatty acids, the chain length of the SPD is reduced by two carbons and the trans double bond at C4 is saturated. This study thus elucidates the enzymatic properties of FADS3 and the metabolism of SPD.

Ceramide synthase homolog Tlc4 maintains nuclear envelope integrity via its Golgi translocation
Yasuhiro Hirano, Yusuke Ohno, Yoshino Kubota, Tatsuo Fukagawa, Akio Kihara, Tokuko Haraguchi, Yasushi Hiraoka
Journal of Cell Science, 2023年05月15日, ［査読有り］
研究論文（学術雑誌）

Involvement of ω-O-acylceramides and protein-bound ceramides in oral permeability barrier formation.
Takayuki Sassa, Akio Kihara
Cell reports, 112363, 112363, 2023年04月05日, ［国際誌］
英語, 研究論文（学術雑誌）, The permeability barrier present in the oral cavity is critical for protection from infection. Although lipids have properties suitable for permeability barrier formation, little is known about their role in oral barrier formation. Here, we show the presence of ω-O-acylceramides (acylceramides) and protein-bound ceramides, which are essential for the formation of permeability barriers in the epidermis, in the oral mucosae (buccal and tongue mucosae), esophagus, and stomach in mice. Conditional knockout of the fatty acid elongase Elovl1, which is involved in the synthesis of ≥C24 ceramides including acylceramides and protein-bound ceramides, in the oral mucosae and esophagus causes increased pigment penetration into the mucosal epithelium of the tongue and enhanced aversive responses to capsaicin-containing water. We find acylceramides in the buccal and gingival mucosae and protein-bound ceramides in the gingival mucosa in humans. These results indicate that acylceramides and protein-bound ceramides are important for oral permeability barrier formation.

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.
Satarupa Bhaduri, Analine Aguayo, Yusuke Ohno, Marco Proietto, Jasmine Jung, Isabel Wang, Rachel Kandel, Narinderbir Singh, Ikran Ibrahim, Amit Fulzele, Eric J Bennett, Akio Kihara, Sonya E Neal
The EMBO journal, 42, 4, e112275, 2023年02月15日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Incomplete Elongation of Ultra-long-chain Polyunsaturated Acyl-CoAs by the Fatty Acid Elongase ELOVL4 in Spinocerebellar Ataxia Type 34.
Yuka Tamura, Takayuki Sassa, Takumi Nishizawa, Akio Kihara
Molecular and cellular biology, 43, 2, 1, 17, 2023年02月07日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4, which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F. Next, we generated knock-in mouse embryonic stem cells that contained heterozygous Q180P, heterozygous W246G, or homozygous W246G mutations. Neuronal differentiation-dependent production of ULC-PCs was reduced in heterozygous Q180P and homozygous W246G cells relative to control cells, and we observed shortening of the FA moiety in all mutant cells. This FA shortening was consistent with our prediction that amino acid residues substituted by SCA34 mutations are located in the transmembrane helices that interact with the ω-end region of the FA moiety of the substrate acyl-CoA. Hence, reduced levels and shortening of ULC-PCs in neurons may cause SCA34, and incomplete elongation of ULC polyunsaturated acyl-CoAs by mutated ELOVL4 may induce dominant inheritance.

Ceramide analysis in combination with genetic testing may provide a precise diagnosis for self-healing collodion babies.
Takuya Takeichi, Yusuke Ohno, Kana Tanahashi, Yasutoshi Ito, Ken Shiraishi, Ryo Utsunomiya, Satoshi Yoshida, Kenta Ikeda, Hayato Nomura, Shin Morizane, Koji Sayama, Tomoo Ogi, Yoshinao Muro, Akio Kihara, Masashi Akiyama
Journal of lipid research, 63, 12, 100308, 100308, 2022年11月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Self-healing collodion baby (SHCB), also called "self-improving collodion baby", is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79*))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs*3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain fatty acid (FA) ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79*) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with non-invasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.

Optimising the method for visualising mouse meibomian gland using eyelid whole-mount lipid staining.
Yuki Hamada, Lena Sasaki, Hikari Uehara, Tomo Suzuki, Shigeru Kinoshita, Kento Otsuka, Akio Kihara, Yoshiaki Yamaguchi, Takahito Miyake, Masao Doi
The ocular surface, 26, 268, 270, 2022年10月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Ceramide profiling of stratum corneum in Sjögren-Larsson syndrome.
Ayami Arai, Takuya Takeichi, Hiroyuki Wakamoto, Takayuki Sassa, Yasutoshi Ito, Yuya Murase, Tomoo Ogi, Masashi Akiyama, Akio Kihara
Journal of dermatological science, 107, 3, 114, 122, 2022年09月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Sjögren-Larsson syndrome (SLS) is a neurocutaneous disorder whose causative gene is the fatty aldehyde dehydrogenase ALDH3A2 and of which ichthyosis is the major skin symptom. The stratum corneum contains a variety of ceramides, among which ω-O-acylceramides (acylceramides) and protein-bound ceramides are essential for skin permeability barrier formation. OBJECTIVES: To determine the ceramide classes/species responsible for SLS pathogenesis and the enzymes that are impaired in SLS. METHODS: Genomic DNA was collected from peripheral blood samples from an SLS patient and her parents, and whole-genome sequencing and Sanger sequencing were performed. Lipids were extracted from stratum corneum samples from the SLS patient and healthy volunteers and subjected to ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: A duplication (c.55_130dup) and a missense mutation (p.Lys447Glu) were found in the patient's ALDH3A2 gene. The patient had reduced levels of all acylceramide classes, with total acylceramide levels at 25 % of healthy controls. Reductions were also observed for several nonacylated ceramides: ceramides with phytosphingosine or 6-hydroxysphingosine in the long-chain base moiety were reduced to 24 % and 41 % of control levels, respectively, and ceramides with an α-hydroxy fatty acid as the fatty acid moiety were reduced to 29 %. The fatty acid moiety was shortened in many nonacylated ceramide classes. CONCLUSION: These results suggest that reduced acylceramide levels are a primary cause of the ichthyosis symptoms of SLS, but reductions in other ceramide classes may also be involved.

Impaired production of skin barrier lipid acylceramides and abnormal localization of PNPLA1 due to ichthyosis-causing mutations in PNPLA1.
Tomohiro Nohara, Yusuke Ohno, Akio Kihara
Journal of dermatological science, 107, 2, 89, 94, 2022年08月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: PNPLA1 is a causative gene of autosomal recessive congenital ichthyosis. The transacylase PNPLA1 produces ω-O-acylceramides (acylceramides), lipids essential for the skin barrier function, by catalyzing the transfer of a linoleic acid from triglycerides to ω-hydroxyceramides. OBJECTIVE: We aimed to validate the involvement of PNPLA1 mutations found in ichthyosis patients in the pathogenesis and elucidate the correlation between the effects of these mutations on acylceramide-producing activity and ichthyosis pathology. METHODS: Acylceramide-producing activity of PNPLA1 mutants was investigated using a cell-based assay system, in which wild-type PNPLA1 or each PNPLA1 mutant was co-overexpressed with the enzymes involved in acylceramide synthesis. The effect of each mutation on the ABHD5-dependent lipid droplet localization of PNPLA1 was examined through indirect immunofluorescence microscopy. RESULTS: Of 16 PNPLA1 missense mutations, 15 mutations, except the C216R mutation, resulted in a complete loss of acylceramide-producing activity, while the C216R mutation weakly affected this activity. Intracellular localization of mutants with no activity varied among mutants. Two mutants (S19L and D172N) localized in lipid droplets, and eight mutants (S53L, S53W, A59V, T125N, D129E, R166C, P234S, and P235L) partially localized there. Five mutants (A34P, A34T, S53P, K141E, and P163L) localized throughout the cytosol. CONCLUSION: The PNPLA1 missense mutations examined in this study are responsible for ichthyosis pathology. The weak effect of C216R mutation on acylceramide-producing activity correlates with the mild symptoms of the ichthyosis patient. Sixteen PNPLA1 mutants were classified into four groups based on their acylceramide-producing activity and localization.

Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.
Madoka Suzuki, Yusuke Ohno, Akio Kihara
Journal of lipid research, 63, 7, 100235, 100235, 2022年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date. Ceramides containing an LCB whose chain length was C16-26 were present in the human SC. Of these, C18 (28.6%) was the most abundant, followed by C20 (24.8%) and C22 (12.8%). Each ceramide class had a characteristic distribution of LCB chain lengths and was divided into five groups according to this distribution. There was almost no difference in FA composition between the ceramide species containing LCBs of different chain lengths. Furthermore, we demonstrated that one of the serine palmitoyltransferase (SPT) complexes, SPTLC1/SPTLC3/SPTSSB, was able to produce C16-24 LCBs. The expression levels of all subunits constituting the SPT complexes increased during keratinocyte differentiation, resulting in the observed chain-length diversity of LCBs in the human SC. This study provides a molecular basis for elucidating human SC ceramide diversity and the pathogenesis of skin disorders.

Hypomyelinating spastic dyskinesia and ichthyosis caused by a homozygous splice site mutation leading to exon skipping in ELOVL1.
Taiko Takahashi, Sevcan Mercan, Takayuki Sassa, Günseli Bayram Akçapınar, Kanay Yararbaş, Seda Süsgün, Sibel Aylin Uğur İşeri, Akio Kihara, Nihan Hande Akçakaya
Brain & development, 44, 6, 391, 400, 2022年04月01日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, INTRODUCTION: Next generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human. METHODS: We have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ELOVL1 protein structure was computationally modelled. RESULTS: The novel c.376-2A > G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC-MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides. DISCUSSION: It has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.

Formation of fatty alcohols—components of meibum lipids—by the fatty acyl-CoA reductase FAR2 is essential for dry eye prevention　　　　　　　　　　　　　　　
Kento Otsuka, Megumi Sawai-Ogawa, Akio Kihara
FASEB Journal, in press, 2022年, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）

Impaired Skin Barrier Function Due to Reduced ω-O-Acylceramide Levels in a Mouse Model of Sjögren-Larsson Syndrome.
Koki Nojiri, Shuhei Fudetani, Ayami Arai, Takuya Kitamura, Takayuki Sassa, Akio Kihara
Molecular and cellular biology, 41, 10, e0035221, 2021年09月24日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double-knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω-O-acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild-type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2. Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base-derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide production. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

Protein-bound ceramide levels in the epidermis of transglutaminase 1-deficient mice.
Takayuki Sassa, Yasutomo Imai, Akio Kihara, Kiyofumi Yamanishi
The Journal of dermatology, 2021年08月17日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）

Amlexanox enhances the antitumor effect of anti-PD-1 antibody.
Kazuhiko Takeda, Koji Yano, Kaoru Yamada, Akio Kihara
Biochemical and biophysical research communications, 560, 1, 6, 2021年06月30日, ［査読有り］, ［最終著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Cancer immunotherapy, especially treatment with monoclonal antibodies (mAbs) that block programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling, has attracted attention as a new therapeutic option for cancer. However, only a limited number of patients have responded to this treatment approach. In this study, we searched for compounds that enhance the efficacy of anti-PD-1 mAb using mixed lymphocyte reaction (MLR), which is a mixed culture system of the two key cells (dendritic and T cells) involved in tumor immunity. We found that amlexanox enhanced production of interferon (IFN)-γ, an indicator of T cell activation, by anti-PD-1 mAb. Amlexanox also induced PD-L1 expression in dendritic cells in MLR, whereas it did not stimulate interleukin-2 production by Jurkat T cells. These results suggest that amlexanox acts on dendritic cells, not T cells, in MLR. Furthermore, it enhanced the antitumor effect of the anti-PD-1 mAb in vivo in a mouse tumor-bearing model. The combination of amlexanox and anti-PD-1 mAb increased the expression of Ifng encoding IFN-γ, IFN-γ-related genes, Cd274 encoding PD-L1, and cytotoxic T cell-related genes in tumors. In conclusion, amlexanox stimulates the antitumor effect of anti-PD-1 mAb by acting on dendritic cells, which in turn activates cytotoxic T cells in tumors.

Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface
Megumi Sawai, Keisuke Watanabe, Kana Tanaka, Wataru Kinoshita, Kento Otsuka, Masatoshi Miyamoto, Takayuki Sassa, Akio Kihara
iScience, 24, 5, 102478, 102478, Elsevier BV, 2021年05月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations.
Moe Yamamoto, Takayuki Sassa, Yuki Kyono, Hiroyasu Uemura, Masaaki Kugo, Hideki Hayashi, Yasutomo Imai, Kiyofumi Yamanishi, Akio Kihara
The Journal of Dermatology, 48, 4, 447, 456, 2021年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The stratum corneum (SC) of the epidermis acts as a skin permeability barrier, and abnormalities in SC formation lead to several skin disorders. Lipids, especially the epidermis-specific ceramide classes ω-O-acylceramides (acylceramides) and protein-bound ceramides, are essential for skin barrier formation. Ceramide synthase 3 (CERS3) is involved in the synthesis of acylceramides and protein-bound ceramides, and CERS3 mutations cause autosomal recessive congenital ichthyosis. In the present study, we measured ceramide synthase activity and performed comprehensive SC ceramide profiling in an ichthyosis patient with compound heterozygous CERS3 mutations: nonsense mutation p.Arg75* and missense mutation p.Arg229His. The activity of p.Arg75* and p.Arg229His mutant CERS3 proteins was reduced to 4% and 56%, respectively, of the wild-type protein. In the patient's SC, acylceramide levels were greatly reduced, but the levels of protein-bound ceramides remained almost unchanged. Non-acylated ceramide levels were also affected in the patient; in particular, the levels of ceramides composed of sphingosine and non-hydroxy or α-hydroxy fatty acid were substantially higher than in healthy controls. These results suggest that a reduction in acylceramide levels alone leads to ichthyosis. Although protein-bound ceramides are synthesized from acylceramides, levels of acylceramides and protein-bound ceramides are not necessarily correlated.

Improvement of evaporative dry eye with meibomian gland dysfunction in model mice by treatment with ophthalmic solution containing mineral oil
Keisuke Watanabe, Masataka Yoshida, Takashi Okumura, Takayuki Sassa, Akio Kihara, Akira Uchiyama
Translational vision science & technology, 10, 4, 21, 21, 2021年04月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Purpose: Meibomian gland dysfunction (MGD) is a major cause of evaporative dry eye. The purpose of this study is to assess the efficacy of a mineral oil-containing ophthalmic solution (MO) in mitigating the evaporative dry eye phenotypes in a mouse model in which fatty acid elongase Elovl1 is disrupted. Methods: Elovl1-deficient mice were assessed in terms of number of plugged meibomian gland orifices, tear film breakup time (BUT), corneal fluorescein staining (CFS) score, tear quantity, and histology. The effects of the MO on the dry eye phenotypes were compared with those in groups not treated or treated with blank ophthalmic solution (BL). Results: Untreated Elovl1-deficient mice exhibited dry eye phenotypes with MGD symptoms such as plugging of meibomian gland orifices (P = 0.002 compared with control mice), high CFS scores (P = 0.002), and shortened BUT (P < 0.001). Among three groups of Elovl1-deficient mice (MO treated, BL treated, and untreated), the MO-treated group exhibited fewer plugged orifices (MO treated, 7.6; BL treated, 10.5 [P = 0.033]; untreated, 13.0 [P < 0.001]), lower CFS scores (MO treated, 1.1; BL treated, 2.7 [P = 0.013]; untreated, 2.5 [P = 0.050]), and improved BUT (MO treated, 19.4 seconds; BL treated, 8.3 seconds [P = 0.098]; untreated, 1.5 seconds [P = 0.008]). Conclusions: Elovl1-deficient mice exhibited multiple MGD symptoms, which were improved by MO. Translational Relevance: Our findings reveal the usefulness of Elovl1-deficient mice as a model for dry eye with MGD and suggest the potential of mineral oil eye drops as a treatment for this condition.

Direct uptake of sphingosine-1-phosphate independent of phospholipid phosphatases.
Hirotaka Goto, Masatoshi Miyamoto, Akio Kihara
The Journal of biological chemistry, 100605, 100605, 2021年03月27日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple knockout (TKO) HAP1 cells that lacked all PLPPs (PLPP1-3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared to the wild type cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [32P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of SPNS2 or of MFSD2B, both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas knockout of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and are capable of not only exporting, but also importing S1P. Furthermore, we observed that MEDEP-E14 cells, erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.

Impaired production of the skin barrier lipid acylceramide by CYP4F22 ichthyosis mutations.
Tomohiro Nohara, Yusuke Ohno, Akio Kihara
Journal of dermatological science, 101, 1, 69, 71, 2021年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）

Production of branched-chain very-long-chain fatty acids by fatty acid elongases and their tissue distribution in mammals.
Honoka Tanno, Takayuki Sassa, Megumi Sawai, Akio Kihara
Biochimica et biophysica acta. Molecular and cell biology of lipids, 1866, 1, 158842, 158842, 2021年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Although most mammalian fatty acids (FAs) are straight-chain, there also exist branched-chain FAs such as iso- and anteiso-FAs, especially in the meibomian glands. Meibum lipids, which are secreted from the meibomian glands and are important for dry eye prevention, contain abundant branched-chain lipids, such as cholesteryl esters and wax esters with chain-lengths of ≥C21 (very-long-chain; VLC). However, the exact tissue distribution of branched-chain lipids or the enzymes involved in the production of branched-chain VLCFAs has remained poorly understood. Here, we revealed that FA elongases ELOVL1, ELOVL3, and ELOVL7, of the seven mammalian ELOVL isozymes, elongated saturated branched-chain acyl-CoAs. ELOVL3 was highly active toward iso-C17:0 and anteiso-C17:0 acyl-CoAs and elongated them up to iso-C23:0 and anteiso-C25:0 acyl-CoAs, respectively. ELOVL1 elongated both iso- and anteiso-C23:0 acyl-CoAs to C25:0 acyl-CoAs. By establishing a liquid chromatography-tandem mass spectrometry method capable of separating branched- and straight-chain lipids, we showed that essentially all of the cholesteryl esters and 88% of the wax esters in the mouse meibomian glands are branched. In Elovl1 mutant mice, the levels of ≥C24:0 branched-chain cholesteryl esters and ≥C25:0 branched-chain wax esters were decreased, indicating that ELOVL1 indeed elongates branched-chain VLC acyl-CoAs in vivo. In addition, substantial amounts of ceramides containing branched-chain FAs were present in the skin, meibomian glands, and liver. Our findings provide new insights into the molecular mechanisms that create FA and lipid diversity.

FTY720 Protects Against Ischemia-Reperfusion Injury by Preventing the Redistribution of Tight Junction Proteins and Decreases Inflammation in the Subacute Phase in an Experimental Stroke Model.
Zifeng Wang, Kei Higashikawa, Hironobu Yasui, Yuji Kuge, Yusuke Ohno, Akio Kihara, Yenari A Midori, Kiyohiro Houkin, Masahito Kawabori
Translational stroke research, 11, 5, 1103, 1116, 2020年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Injury due to brain ischemia followed by reperfusion (I/R) may be an important therapeutic target in the era of thrombectomy. FTY720, a widely known sphingosine-1-phosphate receptor agonist, exerts various neuroprotective effects. The aim of this study was to examine the protective effect of FTY720 with respect to I/R injury, especially focusing on blood-brain barrier (BBB) protection and anti-inflammatory effects. Male rats were subjected to transient ischemia and administered vehicle or 0.5 or 1.5 mg/kg of FTY720 immediately before reperfusion. Positron emission tomography (PET) with [18F]DPA-714 was performed 2 and 9 days after the insult to serially monitor neuroinflammation. Bovine and rat brain microvascular endothelial cells (MVECs) were also subjected to oxygen-glucose deprivation (OGD) and reperfusion, and administered FTY720, phosphorylated-FTY720 (FTY720-P), or their inhibitor. FTY720 dose-dependently reduced cell death, the infarct size, cell death including apoptosis, and inflammation. It also ameliorated BBB disruption and neurological deficits compared to in the vehicle group. PET indicated that FTY720 significantly inhibited the worsening of inflammation in later stages. FTY720-P significantly prevented the intracellular redistribution of tight junction proteins but did not increase their mRNA expression. These results suggest that FTY720 can ameliorate I/R injury by protecting the BBB and regulating neuroinflammation.

Catalytic residues, substrate specificity, and role in carbon starvation of the 2-hydroxy FA dioxygenase Mpo1 in yeast.
Keisuke Mori, Takashi Obara, Naoya Seki, Masatoshi Miyamoto, Tatsuro Naganuma, Takuya Kitamura, Akio Kihara
Journal of lipid research, 61, 7, 1104, 1114, 2020年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The yeast protein Mpo1 belongs to a protein family that is widely conserved in bacteria, fungi, protozoa, and plants, and is the only protein of this family whose function has so far been elucidated. Mpo1 is an Fe2+-dependent dioxygenase that catalyzes the α-oxidation reaction of 2-hydroxy (2-OH) long-chain FAs (LCFAs) produced in the degradation pathway of the long-chain base phytosphingosine. However, several biochemical characteristics of Mpo1, such as its catalytic residues, membrane topology, and substrate specificity, remain unclear. Here, we report that yeast Mpo1 contains two transmembrane domains and that both its N- and C-terminal regions are exposed to the cytosol. Mutational analyses revealed that three histidine residues conserved in the Mpo1 family are especially important for Mpo1 activity, suggesting that they may be responsible for the formation of coordinate bonds with Fe2+ We found that, in addition to activity toward 2-OH LCFAs, Mpo1 also exhibits activity toward 2-OH very-long-chain FAs derived from the FA moiety of sphingolipids. These results indicate that Mpo1 is involved in the metabolism of long-chain to very-long-chain 2-OH FAs produced in different pathways. We noted that the growth of mpo1Δ cells is delayed upon carbon deprivation, suggesting that the Mpo1-mediated conversion of 2-OH FAs to nonhydroxy FAs is important for utilizing 2-OH FAs as a carbon source under carbon starvation. Our findings help to elucidate the as yet unknown functions and activities of other Mpo1 family members.

Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS.
Momoko Kawana, Masatoshi Miyamoto, Yusuke Ohno, Akio Kihara
Journal of lipid research, 61, 6, 884, 895, 2020年06月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC/MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from human and mouse SC. Phytosphingosine- and 6-hydroxy sphingosine-type ceramides, which both contain an additional hydroxyl group, were abundant in the human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, phytosph-ingosine- and 6-hydroxy sphingosine-type ceramides were present at ∼1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ∼90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy or ω-hydroxy FA were abundant in mice. The hydroxylated β-carbon in β-hydroxy ceramides was in the (R) configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased β-hydroxy ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the ER, is a substrate for β-hydroxy ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Lipid polarity gradient formed by ω-hydroxy lipids in tear film prevents dry eye disease.
Masatoshi Miyamoto, Takayuki Sassa, Megumi Sawai, Akio Kihara
eLife, 9, 9e53582, 2020年04月07日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Meibum lipids form a lipid layer on the outermost side of the tear film and function to prevent water evaporation and reduce surface tension. (O-Acyl)-ω-hydroxy fatty acids (OAHFAs), a subclass of these lipids, are thought to be involved in connecting the lipid and aqueous layers in tears, although their actual function and synthesis pathway have to date remained unclear. Here, we reveal that the fatty acid ω-hydroxylase Cyp4f39 is involved in OAHFA production. Cyp4f39-deficient mice exhibited damaged corneal epithelium and shortening of tear film break-up time, both indicative of dry eye disease. In addition, tears accumulated on the lower eyelid side, indicating increased tear surface tension. In Cyp4f39-deficient mice, the production of wax diesters (type 1ω and 2ω) and cholesteryl OAHFAs was also impaired. These OAHFA derivatives show intermediate polarity among meibum lipids, suggesting that OAHFAs and their derivatives contribute to lipid polarity gradient formation for tear film stabilization.

Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid ω-O-acylceramide.
Haruka Yamamoto, Miku Hattori, Walee Chamulitrat, Yusuke Ohno, Akio Kihara
Proceedings of the National Academy of Sciences of the United States of America, 117, 6, 2914, 2922, 2020年02月11日, ［査読有り］, ［最終著者, 責任著者］, ［国際共著］, ［国際誌］
英語, The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to ∼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.

Biosynthesis of the anti-lipid-microdomain sphingoid base 4,14-sphingadiene by the ceramide desaturase FADS3.
Keisuke Jojima, Mai Edagawa, Megumi Sawai, Yusuke Ohno, Akio Kihara
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34, 2, 3318, 3335, 2020年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Sphingolipids are multifunctional lipids. Among the sphingolipid-component sphingoid bases, 4,14-sphingadiene (SPD) is unique such that it has a cis double bond with a bent structure. Although SPD was discovered half a century ago, its tissue distribution, biosynthesis, and degradation remain poorly understood. Here, we established a specific and quantitative method for SPD measurement and found that SPD exists in a wide range of mammalian tissues. SPD was especially abundant in kidney, where the amount of SPD was ~2/3 of sphingosine, the most abundant sphingoid base in mammals. Although SPD is metabolized to ceramides and SPD 1-phosphate with almost the same efficiency as sphingosine, it is less susceptible to degradation by a cleavage reaction, at least in vitro. We identified the fatty acid desaturase family protein FADS3 as a ceramide desaturase that produces SPD ceramides by desaturating ceramides containing sphingosine. SPD sphingolipids were preferentially localized outside lipid microdomains, suggesting that SPD has different functions compared to other sphingoid bases in the formation of lipid microdomains. In summary, we revealed the biosynthesis and degradation pathways of SPD and its characteristic membrane localization. Our findings contribute to the elucidation of the molecular mechanism underlying the generation of sphingolipid diversity.

Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration.
Kawaguchi M, Sassa T, Kidokoro H, Nakata T, Kato K, Muramatsu H, Okuno Y, Yamamoto H, Kaname T, Kihara A, Natsume J
Brain and development, 42, 2, 217, 221, 2020年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, FA2H encodes fatty acid 2-hydroxylase, which plays a significant role in maintaining the neuronal myelin sheath. Previous reports have revealed that a FA2H mutation leads to spastic paraplegia, leukodystrophy, and neurodegeneration with brain iron accumulation, collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). The disease severity of FAHN varies among individual patients and may be explained by the enzyme activity of FA2H mutant proteins. Here we report a 10-year-old Japanese boy with FAHN having novel heterozygous mutations in FA2H. The patient presented with a spastic gait since the age of 5 years and was unable to walk without a cane by the time he was 8 years old. Brain MRI demonstrated a partial thinning of the corpus callosum, slight reduction of cerebellar volume, and posterior dominant periventricular leukodystrophy. Whole exome sequencing revealed two novel missense mutations in FA2H with compound heterozygous inheritance (NM_024306, p.Val149Leu, and p.His260Gln mutations). The enzyme activities of the p.Val149Leu and p.His260Gln variants were 60%-80% and almost 0%, respectively. Our cell-based enzyme assay demonstrated partial functionality for one of the variants, indicating a milder phenotype. However, considered along with previous reports, there was no definite relationship between the disease severity and residual enzyme activity measured using a similar method. Further research is needed to precisely predict the phenotypic severity of this disorder.

Severe skin permeability barrier dysfunction in knockout mice deficient in a fatty acid ω-hydroxylase crucial to acylceramide production
Miyamoto M, Itoh N, Sawai M, Sassa T, Kihara A
Journal of Investigative Dermatology, 140, 2, 319, 326, Elsevier {BV}, 2020年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The skin permeability barrier is indispensable for maintaining water inside the body and preventing the invasion of pathogens and allergens; abnormalities lead to skin disorders such as atopic dermatitis and ichthyosis. Acylceramide is an essential lipid for skin barrier formation, and CYP4F22 is a fatty acid ω-hydroxylase involved in its synthesis. Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis, although the symptoms vary among mutation sites and types. Here, we generated knockout mice deficient in Cyp4f39, the mouse ortholog of human CYP4F22, to investigate the effects of completely abrogating the function of the fatty acid ω-hydroxylase involved in acylceramide production on skin barrier formation. Cyp4f39 knockout mice died within 8 hours of birth. Large increases in transepidermal water loss and penetration of a dye from outside the body were observed, indicating severe skin barrier dysfunction. Histologic analyses of the epidermis revealed impairment of lipid lamella formation, accumulation of corneodesmosomes in the stratum corneum, and persistence of periderm. In addition, lipid analyses by mass spectrometry showed almost complete loss of acylceramide and its precursor ω-hydroxy ceramide. In conclusion, our findings provide clues to the molecular mechanisms of skin barrier abnormalities and the pathogenesis of ichthyosis caused by Cyp4f39 and CYP4F22 by association.

N-glycosylation of Rim21 at an Unconventional Site Fine-tunes Its Behavior in the Plasma Membrane.
Keisuke Obara, Tetsuya Kotani, Hitoshi Nakatogawa, Akio Kihara, Takumi Kamura
Cell structure and function, 45, 1, 1, 8, 2020年01月11日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.

Reduced chain length in myelin sphingolipids and poorer motor coordination in mice deficient in the fatty acid elongase Elovl1.
Masashi Isokawa, Takayuki Sassa, Satoko Hattori, Tsuyoshi Miyakawa, Akio Kihara
FASEB bioAdvances, 1, 12, 747, 759, 2019年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Very-long-chain fatty acids, with a chain length of >C20, are abundant in myelin sphingolipids. Recently, a de novo mutation in the ELOVL1 gene, which encodes fatty acid elongase, was identified in patients with neurocutaneous disorders involving skin ichthyosis and multiple neurological abnormalities, including hypomyelination, spastic paraplegia, and high-frequency deafness. However, the consequences of ELOVL1 deficiency for lipid composition and detailed pathological changes in the brain remain unclear. Here, we analyzed Elovl1 mutant mice as a model of human ELOVL1 deficiency. The mice exhibited a decreased postnatal survival rate, and some died of startle epilepsy. The acyl chain length of sphingolipids such as galactosylceramides, sulfatides, sphingomyelins, and ceramides in the brains of these mice was markedly shortened. Moreover, the mice exhibited reduced levels of galactosylceramides, which are important for myelin formation and stability. Electron microscope analysis of the corpus callosum in Elovl1 mutant mice revealed modest hypomyelination, especially in large-diameter axons. Furthermore, behavioral testing of the mice revealed deficits such as poorer motor coordination and reduced acoustic startle response to high-intensity stimulus. These findings provide clues to the molecular mechanism of the neurological symptoms of patients with the ELOVL1 mutation.

The very-long-chain fatty acid elongase Elo2 rescues lethal defects associated with loss of the nuclear barrier function in fission yeast cells.
Kinugasa Y, Hirano Y, Sawai M, Ohno Y, Shindo T, Asakawa H, Chikashige Y, Shibata S, Kihara A, Haraguchi T, Hiraoka Y
Journal of cell science, 132, 10, 2019年05月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In eukaryotic cells, chromosomes are confined to the nucleus, which is compartmentalized by the nuclear membranes; these are continuous with the endoplasmic reticulum membranes. Maintaining the homeostasis of these membranes is an important cellular activity performed by lipid metabolic enzymes. However, how lipid metabolic enzymes affect nuclear membrane functions remains to be elucidated. We found that the very-long-chain fatty acid elongase Elo2 is located in the nuclear membrane and prevents lethal defects associated with nuclear membrane ruptures in mutants of the nuclear membrane proteins Lem2 and Bqt4 in the fission yeast Schizosaccharomyces pombe. Lipid composition analysis shows that t20:0/24:0 phytoceramide (a conjugate of C20:0 phytosphingosine and C24:0 fatty acid) is a major ceramide species in S. pombe The quantity of this ceramide is reduced in the absence of Lem2, and restored by increased expression of Elo2. Furthermore, loss of S. pombe Elo2 can be rescued by its human orthologs. These results suggest that the conserved very-long-chain fatty acid elongase producing the ceramide component is essential for nuclear membrane integrity and cell viability in eukaryotes.This article has an associated First Person interview with the first author of the paper.

Yeast Mpo1 is a novel dioxygenase that catalyzes the α-oxidation of a 2-hydroxy fatty acid in an Fe²⁺-dependent manner.
Seki N, Mori K, Kitamura T, Miyamoto M, Kihara A
Molecular and cellular biology, 39, 5, 2019年03月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Phytosphingosine (PHS) is the major long-chain base component of sphingolipids in Saccharomyces cerevisiae The PHS metabolic pathway includes a fatty acid (FA) α-oxidation reaction. Recently, we identified the novel protein Mpo1, which is involved in PHS metabolism. However, the details of the FA α-oxidation reaction and the role of Mpo1 in PHS metabolism remained unclear. In the present study, we revealed that Mpo1 is involved in the α-oxidation of 2-hydroxy (2-OH) palmitic acid (C16:0-COOH) in the PHS metabolic pathway. Our in vitro assay revealed that not only the Mpo1-containing membrane fraction but also the soluble fraction was required for the α-oxidation of 2-OH C16:0-COOH. The addition of Fe2+ eliminated the need for the soluble fraction. Purified Mpo1 converted 2-OH C16:0-COOH to C15:0-COOH in the presence of Fe2+, indicating that Mpo1 is the enzyme body responsible for catalyzing the FA α-oxidation reaction. This reaction was also found to require an oxygen molecule. Our findings indicate that Mpo1 catalyzes the FA α-oxidation reaction as 2-OH fatty acid dioxygenase, mediated by iron(IV) peroxide. Although numerous Mpo1 homologs exist in bacteria, fungi, protozoa, and plants, their functions had not yet been clarified. However, our findings suggest that these family members function as dioxygenases.

De novo mutation in ELOVL1 causes ichthyosis, acanthosis nigricans, hypomyelination, spastic paraplegia, high frequency deafness and optic atrophy.
Mueller N, Sassa T, Morales-Gonzalez S, Schneider J, Salchow DJ, Seelow D, Knierim E, Stenzel W, Kihara A, Schuelke M
Journal of medical genetics, 56, 3, 164, 175, 2019年03月, ［査読有り］, ［責任著者］, ［国際共著］, ［国際誌］
英語, BACKGROUND: Very long-chain fatty acids (VLCFAs) are essential for functioning of biological membranes. ELOVL fatty acid elongase 1 catalyses elongation of saturated and monounsaturated C22-C26-VLCFAs. We studied two patients with a dominant ELOVL1 mutation. Independently, Kutkowska-Kaźmierczak et al. had investigated the same patients and found the same mutation. We extended our study towards additional biochemical, functional, and therapeutic aspects. METHODS: We did mutation screening by whole exome sequencing. RNA-sequencing was performed in patient and control fibroblasts. Ceramide and sphingomyelin levels were measured by LC-MS/MS. ELOVL1 activity was determined by a stable isotope-labelled [13C]malonyl-CoA elongation assay. ELOVL1 expression patterns were investigated by immunofluorescence, in situ hybridisation and RT-qPCR. As treatment option, we investigated VLCFA loading of fibroblasts. RESULTS: Both patients carried an identical heterozygous de novo ELOVL1 mutation (c.494C>T, NM_001256399; p.S165F) not deriving from a founder allele. Patients suffered from epidermal hyperproliferation and increased keratinisation (ichthyosis). Hypomyelination of the central white matter explained spastic paraplegia and central nystagmus, while optic atrophy was causative for reduction of peripheral vision and visual acuity. The mutation abrogated ELOVL1 enzymatic activity and reduced ≥C24 ceramides and sphingomyelins in patient cells. Fibroblast loading with C22:0-VLCFAs increased C24:0-ceramides and sphingomyelins. We found competitive inhibition for ceramide and sphingomyelin synthesis between saturated and monounsaturated VLCFAs. Transcriptome analysis revealed upregulation of modules involved in epidermal development and keratinisation, and downregulation of genes for neurodevelopment, myelination, and synaptogenesis. Many regulated genes carried consensus proliferator-activated receptor (PPAR)α and PPARγ binding motifs in their 5'-regions. CONCLUSION: A dominant ELOVL1 mutation causes a neuro-ichthyotic disorder possibly amenable to treatment with PPAR-modulating drugs.

Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.
Kanetake T, Sassa T, Nojiri K, Sawai M, Hattori S, Miyakawa T, Kitamura T, Kihara A
FASEB journal, 33, 1, 928, 941, 2019年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Insulation by myelin lipids is essential to fast action potential conductivity: changes in their quality or amount can cause several neurologic disorders. Sjögren-Larsson syndrome (SLS) is one such disorder, which is caused by mutations in the fatty aldehyde dehydrogenase ALDH3A2. To date, the molecular mechanism underlying SLS pathology has remained unknown. In this study, we found that Aldh3a2 is expressed in oligodendrocytes and neurons in the mouse brain, and neurons of Aldh3a2 knockout (KO) mice exhibited impaired metabolism of the long-chain base, a component of sphingolipids. Aldh3a2 KO mice showed several abnormalities corresponding to SLS symptoms in behavioral tests, including increased paw slips on a balance beam and light-induced anxiety. In their brain tissue, 2-hydroxygalactosylceramide, an important lipid for myelin function and maintenance, was reduced by the inactivation of fatty acid 2-hydroxylase. Our findings provide important new insights into the molecular mechanisms responsible for neural pathogenesis caused by lipid metabolism abnormalities.-Kanetake, T., Sassa, T., Nojiri, K., Sawai, M., Hattori, S., Miyakawa, T., Kitamura, T., Kihara, A. Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.

Widespread tissue distribution and synthetic pathway of polyunsaturated C24:2 sphingolipids in mammals.
Edagawa M, Sawai M, Ohno Y, Kihara A
Biochimica et biophysica acta. Molecular and cell biology of lipids, 1863, 12, 1441, 1448, 2018年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Sphingolipids are multifunctional lipids and a major constituent of the cell membranes of eukaryotes. Although the fatty acid (FA) moiety of sphingolipids is usually a saturated or monounsaturated FA, polyunsaturated FA (PUFA)-containing species also exist in mammalian tissues. In the present study, we showed that C24:2 PUFA-containing ceramide is one of the seven major ceramide species in a wide range of tissues. C24:2 ceramide levels were especially high in spleen and small intestine; in the former, it was the fourth most abundant ceramide species. However, both the synthetic pathway and the physiological function of C24:2 ceramide had yet to be identified. Tracer analysis using deuterium-labeled linoleic acid (C18:2) revealed that C24:2 ceramide is produced via elongation of linoleic acid. We also found that the FA elongase ELOVL1 and the ceramide synthase CERS2 were involved in C24:2 ceramide production. Sphingolipids are known to form lipid microdomains in membranes; however, in a detergent-resistant membrane (DRM) assay, we observed a lower proportion of C24:2 sphingomyelin in the DRM fraction than of saturated sphingomyelins, suggesting that C24:2 sphingolipids may act to negatively regulate lipid microdomain formation. Our findings expand our knowledge of sphingolipid diversity, and provide insight into how different sphingolipid molecular species play different functions in biological membranes.

Molecular mechanism of the ichthyosis pathology of Chanarin-Dorfman syndrome: Stimulation of PNPLA1-catalyzed ω-O-acylceramide production by ABHD5.
Ohno Y, Nara A, Nakamichi S, Kihara A
Journal of dermatological science, 92, 3, 245, 253, 2018年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, BACKGROUND: ABHD5 mutations cause Chanarin-Dorfman syndrome accompanied by ichthyosis. ω-O-Acylceramide (acylceramide) is essential for skin permeability barrier formation. Acylceramide production is impaired in Abhd5 knockout mice. The transacylase PNPLA1 catalyzes the final step of acylceramide production: transfer of linoleic acid in triglyceride to ω-hydroxyceramide. OBJECTIVE: We aimed to elucidate the role of ABHD5 in acylceramide production and the molecular mechanism of the ichthyosis symptoms of Chanarin-Dorfman syndrome. METHODS: We investigated how ABHD5 influences acylceramide production using an acylceramide-producing cell system. The effects of ABHD5 and PNPLA1 expression on the morphology of lipid droplets were examined by indirect immunofluorescent microscopy and immunoelectron microscopy. RESULTS: When ABHD5 was expressed in the acylceramide-producing cell system, acylceramide synthesis by PNPLA1 was enhanced. Dispersed localization of PNPLA1 was observed by immunofluorescent microscopy in HeLa cells under lipid droplet-forming conditions. Co-expression with ABHD5 caused PNPLA1 to localize on the lipid droplet membranes or their periphery. This staining pattern was observed in cells where PNPLA1 and ABHD5 were expressed at low levels. In contrast, lipid droplets disappeared in cells where PNPLA1 and ABHD5 were highly expressed. Immunoelectron microscopic analyses suggested that lipid droplets underwent morphological changes, transforming into vesicles or becoming incorporated into the endoplasmic reticulum. ABHD5 mutations found in Chanarin-Dorfman syndrome patients reduced ABHD5's ability to promote PNPLA1-dependent acylceramide production. CONCLUSION: ABHD5 enhances PNPLA1-catalyzed acylceramide production. We speculate that ABHD5 retains triglycerides in the endoplasmic reticulum, and presents them to PNPLA1 to promote substrate recognition.

Structure-inspired design of a sphingolipid mimic sphingosine-1-phosphate receptor agonist from a naturally occurring sphingomyelin synthase inhibitor.
Swamy MMM, Murai Y, Ohno Y, Jojima K, Kihara A, Mitsutake S, Igarashi Y, Yu J, Yao M, Suga Y, Anetai M, Monde K
Chemical communications, 54, 90, 12758, 12761, 2018年11月08日, ［査読有り］, ［国際誌］
英語, Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P1) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P1.

Sphingosine 1-phosphate receptor modulator ONO-4641 stimulates CD11b⁺Gr-1⁺ cell expansion and inhibits lymphocyte infiltration in the lungs to ameliorate murine pulmonary emphysema.
Asakura T, Ishii M, Namkoong H, Suzuki S, Kagawa S, Yagi K, Komiya T, Hashimoto T, Okamori S, Kamata H, Tasaka S, Kihara A, Hegab AE, Hasegawa N, Betsuyaku T
Mucosal immunology, 11, 6, 1606, 1620, 2018年11月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Sphingolipids play a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, little is known about the precise roles of sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, and its receptor modulation in COPD. In this study, we demonstrated that the S1P receptor modulator ONO-4641 induced the expansion of lung CD11b+Gr-1+ cells and lymphocytopenia in naive mice. ONO-4641-expanded CD11b+Gr-1+ cells showed higher arginase-1 activity, decreased T cell proliferation, and lower IFN-γ production in CD3+ T cells, similar to the features of myeloid-derived suppressor cells. ONO-4641 treatment decreased airspace enlargement in elastase-induced and cigarette smoke-induced emphysema models and attenuated emphysema exacerbation induced by post-elastase pneumococcal infection, which was also associated with an increased number of lung CD11b+Gr-1+ cells. Adoptive transfer of ONO-4641-expanded CD11b+Gr-1+ cells protected against elastase-induced emphysema. Lymphocytopenia observed in these models likely contributed to beneficial ONO-4641 effects. Thus, ONO-4641 attenuated murine pulmonary emphysema by expanding lung CD11b+Gr-1+ cell populations and inducing lymphocytopenia. The S1P receptor might be a promising target for strategies aimed at ameliorating pulmonary emphysema progression.

Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice.
Sassa T, Tadaki M, Kiyonari H, Kihara A
FASEB journal, 32, 6, 2966, 2978, 2018年06月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Lipids secreted from the meibomian gland (meibum) form the superficial layer of the tear film and prevent water evaporation from the ocular surface and infection. Here, we identified the fatty acid (FA) elongases responsible for the synthesis of very long-chain FAs (VLCFAs) that constitute the meibum lipids. Elongation of VLCFAs (ELOVL)1 is primarily responsible for the production of saturated VLCFAs, whereas ELOVL1, ELOVL3, and ELOVL4 redundantly participate in the synthesis of monounsaturated VLCFAs. Gene disruption of Elovl1 in mice shortened acyl moieties in the 2 major meibum lipids: cholesteryl esters and wax esters. These changes were associated with dry eye phenotypes, including increases in eye-blink frequency and water evaporation from the ocular surface at younger ages. Aged Elovl1 mutant mice developed corneal opacity with vascular invasion, accompanied by epidermalization of the cornea. Thus, in addition to the well-known VLC ceramides (acylceramides) in the epidermis, VLC meibum lipids are barrier-forming lipids.-Sassa, T., Tadaki, M., Kiyonari, H., Kihara, A. Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice.

Decreased skin barrier lipid acylceramide and differentiation-dependent gene expression in ichthyosis gene Nipal4 -knockout mice
Yuichi Honda, Takuya Kitamura, Tatsuro Naganuma, Takaya Abe, Yusuke Ohno, Takayuki Sassa, Akio Kihara
Journal of Investigative Dermatology, 138, 4, 741, 749, Elsevier {BV}, 2018年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, NIPAL4 is one of the causative genes for autosomal recessive congenital ichthyosis. However, the role of NIPAL4 in skin barrier formation and the molecular mechanism of ichthyosis pathology caused by NIPAL4 mutations, have not yet been determined. Here, we found that Nipal4-knockout (KO) mice exhibited neonatal lethality due to skin barrier defects. Histological analyses showed several morphological abnormalities in the Nipal4-KO epidermis, including impairment of lipid multilayer structure formation, hyperkeratosis, immature keratohyalin granules, and developed heterochromatin structures. The levels of the skin barrier lipid acylceramide were decreased in Nipal4-KO mice. Expression of genes involved in skin barrier formation normally increases during keratinocyte differentiation, in which chromatin remodeling is involved. However, the induction of Krt1, Lor, Flg, Elovl1, and Dgat2 was impaired in Nipal4-KO mice. NIPAL4 is a putative Mg2+ transporter, and Mg2+ concentration in differentiated keratinocytes of Nipal4-KO mice was indeed lower than that of wild-type mice. Our results suggest that low Mg2+ concentration causes aberration in the proper chromatin remodeling process, which in turn leads to failure of differentiation-dependent gene induction in keratinocytes. Our findings provide insights into Mg2+-dependent regulation of gene expression and skin barrier formation during keratinocyte differentiation.

Sphingolipids activate the endoplasmic reticulum stress surveillance pathway
Francisco Piña, Fumi Yagisawa, Keisuke Obara, J.D. Gregerson, Akio Kihara, Maho Niwa
The Journal of Cell Biology, 217, 2, 495, 505, Rockefeller University Press, 2018年02月05日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Proper inheritance of functional organelles is vital to cell survival. In the budding yeast, Saccharomyces cerevisiae, the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway ensures that daughter cells inherit a functional ER. Here, we show that the ERSU pathway is activated by phytosphingosine (PHS), an early biosynthetic sphingolipid. Multiple lines of evidence support this: (1) Reducing PHS levels with myriocin diminishes the ability of cells to induce ERSU phenotypes. (2) Aureobasidin A treatment, which blocks conversion of early intermediates to downstream complex sphingolipids, induces ERSU. (3) orm1Δorm2Δ cells, which up-regulate PHS, show an ERSU response even in the absence of ER stress. (4) Lipid analyses confirm that PHS levels are indeed elevated in ER-stressed cells. (5) Lastly, the addition of exogenous PHS is sufficient to induce all ERSU phenotypes. We propose that ER stress elevates PHS, which in turn activates the ERSU pathway to ensure future daughter-cell viability.

Systematic analysis of Ca²⁺ homoeostasis in Saccharomyces cerevisiae based on chemical-genetic interaction profiles.
Ghanegolmohammadi F, Yoshida M, Ohnuki S, Sukegawa Y, Okada H, Obara K, Kihara A, Suzuki K, Kojima T, Yachie N, Hirata D, Ohya Y
Molecular biology of the cell, 28, 23, 3415, 3427, 2017年11月07日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We investigated the global landscape of Ca2+ homeostasis in budding yeast based on high-dimensional chemical-genetic interaction profiles. The morphological responses of 62 Ca2+-sensitive (cls) mutants were quantitatively analyzed with the image processing program CalMorph after exposure to a high concentration of Ca2+ After a generalized linear model was applied, an analysis of covariance model was used to detect significant Ca2+-cls interactions. We found that high-dimensional, morphological Ca2+-cls interactions were mixed with positive (86%) and negative (14%) chemical-genetic interactions, whereas one-dimensional fitness Ca2+-cls interactions were all negative in principle. Clustering analysis with the interaction profiles revealed nine distinct gene groups, six of which were functionally associated. In addition, characterization of Ca2+-cls interactions revealed that morphology-based negative interactions are unique signatures of sensitized cellular processes and pathways. Principal component analysis was used to discriminate between suppression and enhancement of the Ca2+-sensitive phenotypes triggered by inactivation of calcineurin, a Ca2+-dependent phosphatase. Finally, similarity of the interaction profiles was used to reveal a connected network among the Ca2+ homeostasis units acting in different cellular compartments. Our analyses of high-dimensional chemical-genetic interaction profiles provide novel insights into the intracellular network of yeast Ca2+ homeostasis.

Biallelic mutations in KDSR disrupt ceramide synthesis and result in a spectrum of keratinization disorders associated with thrombocytopenia.
Takeichi T, Torrelo A, Lee JYW, Ohno Y, Lozano ML, Kihara A, Liu L, Yasuda Y, Ishikawa J, Murase T, Rodrigo AB, Fernández-Crehuet P, Toi Y, Mellerio J, Rivera J, Vicente V, Kelsell DP, Nishimura Y, Okuno Y, Kojima D, Ogawa Y, Sugiura K, Simpson MA, McLean WHI, Akiyama M, McGrath JA
The Journal of investigative dermatology, 137, 11, 2344, 2353, 2017年11月, ［査読有り］, ［国際共著］, ［国際誌］
英語, Mutations in ceramide biosynthesis pathways have been implicated in a few Mendelian disorders of keratinization, although ceramides are known to have key roles in several biological processes in skin and other tissues. Using whole-exome sequencing in four probands with undiagnosed skin hyperkeratosis/ichthyosis, we identified compound heterozygosity for mutations in KDSR, encoding an enzyme in the de novo synthesis pathway of ceramides. Two individuals had hyperkeratosis confined to palms, soles, and anogenital skin, whereas the other two had more severe, generalized harlequin ichthyosis-like skin. Thrombocytopenia was present in all patients. The mutations in KDSR were associated with reduced ceramide levels in skin and impaired platelet function. KDSR enzymatic activity was variably reduced in all patients, resulting in defective acylceramide synthesis. Mutations in KDSR have recently been reported in inherited recessive forms of progressive symmetric erythrokeratoderma, but our study shows that biallelic mutations in KDSR are implicated in an extended spectrum of disorders of keratinization in which thrombocytopenia is also part of the phenotype. Mutations in KDSR cause defective ceramide biosynthesis, underscoring the importance of ceramide and sphingosine synthesis pathways in skin and platelet biology.

The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional redundancy and are active in a wide range of fatty acid elongation pathways.
Sawai M, Uchida Y, Ohno Y, Miyamoto M, Nishioka C, Itohara S, Sassa T, Kihara A
The Journal of biological chemistry, 292, 37, 15538, 15551, American Society for Biochemistry {\&} Molecular Biology ({ASBMB}), 2017年09月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs However, we observed only an ∼40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.

Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum
Takuya Kitamura, Naoya Seki, Akio Kihara
Proceedings of the National Academy of Sciences, 114, 13, E2616-E2623, E2623, Proceedings of the National Academy of Sciences, 2017年03月28日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

Decreases in 15-lipoxygenase metabolites in Olmsted syndrome model rats.
Wakabayashi M, Yoshioka T, Higashino K, Numata Y, Igarashi Y, Kihara A
Journal of dermatological science, 85, 3, 186, 196, 2017年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, BACKGROUND: Olmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. OBJECTIVE: We aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. METHODS: Comprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. RESULTS: Infiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by ≥16-fold in Ht rats compared with WT rats. Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of Il36b, Ccl20, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. CONCLUSION: The pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS.

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide
Ohno Y, Kamiyama N, Nakamichi S, Kihara A
Nature communications, 8, 14610, 14610, 2017年03月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Lipids are the primary components of the skin permeability barrier, which is the body's most powerful defensive mechanism against pathogens. Acylceramide (ω-O-acylceramide) is a specialized lipid essential for skin barrier formation. Here, we identify PNPLA1 as the long-sought gene involved in the final step of acylceramide synthesis, esterification of ω-hydroxyceramide with linoleic acid, by cell-based assays. We show that increasing triglyceride levels by overproduction of the diacylglycerol acyltransferase DGAT2 stimulates acylceramide production, suggesting that triglyceride may act as a linoleic acid donor. Indeed, the in vitro analyses confirm that PNPLA1 catalyses acylceramide synthesis using triglyceride as a substrate. Mutant forms of PNPLA1 found in patients with ichthyosis exhibit reduced or no enzyme activity in either cell-based or in vitro assays. Altogether, our results indicate that PNPLA1 is directly involved in acylceramide synthesis as a transacylase, and provide important insights into the molecular mechanisms of skin barrier formation and of ichthyosis pathogenesis.

Loop 5 region is important for the activity of the long-chain base transporter Rsb1.
Makuta H, Obara K, Kihara A
Journal of biochemistry, 161, 2, 207, 213, 2017年02月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Intracellular lipid amounts are regulated not only by metabolism but also by efflux. Yeast Rsb1 is the only known transporter/floppase of the sphingolipid components long-chain bases (LCBs). However, even fundamental knowledge about Rsb1, such as important amino acid residues for activity and substrate recognition, still remains unclear. Rsb1 belongs to the Rta1-like family. To date, it has not been determined whether all family members share a common ability to export LCBs. Here, we revealed that within the Rta1-like family, only Rsb1 suppressed the hypersensitivity of the mutant cells lacking LCB 1-phoshate-degrading enzymes, suggesting that LCB-exporting activity is specific to Rsb1. Rsb1 contains a characteristic region (loop 5), which does not exist in other proteins of the Rta1-like family. We found that deletion of this region caused loss of Rsb1 function. Further mutational analysis of loop 5 revealed that the charged amino acid residues E223, D225 and R236 were important for Rsb1 activity. In addition to LCBs, Rsb1 facilitated the export of 1-hexadecanol, but not palmitic acid, which suggests that Rsb1 recognizes the C1 hydroxyl group. Thus, our findings provide an important clue for understanding the molecular mechanism of LCB export.

The Rim101 pathway contributes to ER stress adaptation through sensing the state of plasma membrane.
Obara K, Kihara A
The Biochemical journal, 474, 1, 51, 63, 2017年01月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Yeast cells sense alterations in the plasma membrane (PM) lipid asymmetry and external alkalization by the sensor protein Rim21, which functions in the Rim101 pathway. Rim101 signaling is initiated at the PM by the recruitment of the Rim101 signaling complex. The PM physically associates with the cortical endoplasmic reticulum (ER) to form ER-PM contact sites, where several signaling events, lipid exchange, and ion transport take place. In the present study, we investigated the spatial relationship between ER-PM contact sites and the sites of Rim101 signaling. Rim101 signaling mostly proceeds outside ER-PM contact sites in the PM and did not require intact ER-PM contact for its activation. Rather, the Rim101 pathway was constitutively activated by ER-PM contact site disruption, which is known to cause ER stress. ER stress induced by tunicamycin treatment activated the Rim101 pathway. Furthermore, the sensitivity of cells to tunicamycin without ER-PM contact was considerably elevated by the deletion of RIM21. These results suggest that the Rim101 pathway is important for the adaptation to ER stress by compensating for alterations in PM lipid asymmetry induced by ER stress.

Mechanistic details of early steps in coenzyme Q biosynthesis pathway in yeast.
Payet LA, Leroux M, Willison JC, Kihara A, Pelosi L, Pierrel F
Cell chemical biology, 23, 10, 1241, 1250, 2016年10月20日, ［査読有り］, ［国際共著］, ［国際誌］
英語, Coenzyme Q (Q) is a redox lipid that is central for the energetic metabolism of eukaryotes. The biosynthesis of Q from the aromatic precursor 4-hydroxybenzoic acid (4-HB) is understood fairly well. However, biosynthetic details of how 4-HB is produced from tyrosine remain elusive. Here, we provide key insights into this long-standing biosynthetic problem by uncovering molecular details of the first and last reactions of the pathway in the yeast Saccharomyces cerevisiae, namely the deamination of tyrosine to 4-hydroxyphenylpyruvate by Aro8 and Aro9, and the oxidation of 4-hydroxybenzaldehyde to 4-HB by Hfd1. Inactivation of the HFD1 gene in yeast resulted in Q deficiency, which was rescued by the human enzyme ALDH3A1. This suggests that a similar pathway operates in animals, including humans, and led us to propose that patients with genetically unassigned Q deficiency should be screened for mutations in aldehyde dehydrogenase genes, especially ALDH3A1.

Disruption of the Sjögren-Larsson Syndrome Gene Aldh3a2 in Mice Increases Keratinocyte Growth and Retards Skin Barrier Recovery.
Naganuma T, Takagi S, Kanetake T, Kitamura T, Hattori S, Miyakawa T, Sassa T, Kihara A
The Journal of biological chemistry, 291, 22, 11676, 88, 2016年05月27日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjögren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were up-regulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2 As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.

Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases.
Narita T, Naganuma T, Sase Y, Kihara A
Scientific reports, 6, 25469, 25469, 2016年05月03日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Transport of dietary lipids into small-intestinal epithelial cells is pathologically and nutritionally important. However, lipid uptake remains an almost unexplored research area. Although we know that long-chain bases (LCBs), constituents of sphingolipids, can enter into cells efficiently, the molecular mechanism of LCB uptake is completely unclear. Here, we found that the yeast acyl-CoA synthetases (ACSs) Faa1 and Faa4 are redundantly involved in LCB uptake. In addition to fatty acid-activating activity, transporter activity toward long-chain fatty acids (LCFAs) has been suggested for ACSs. Both LCB and LCFA transports were largely impaired in faa1Δ faa4Δ cells. Furthermore, LCB and LCFA uptakes were mutually competitive. However, the energy dependency was different for their transports. Sodium azide/2-deoxy-D-glucose treatment inhibited import of LCFA but not that of LCB. Furthermore, the ATP-AMP motif mutation FAA1 S271A largely impaired the metabolic activity and LCFA uptake, while leaving LCB import unaffected. These results indicate that only LCFA transport requires ATP. Since ACSs do not metabolize LCBs as substrates, Faa1 and Faa4 are likely directly involved in LCB transport. Furthermore, we revealed that ACSs are also involved in LCB transport in mammalian cells. Thus, our findings provide strong support for the hypothesis that ACSs directly transport LCFAs.

Enzyme activities of the ceramide synthases CERS2-6 are regulated by phosphorylation in the C-terminal region.
Sassa T, Hirayama T, Kihara A
The Journal of biological chemistry, 291, 14, 7477, 87, 2016年04月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1-6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2-6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing itsVmaxvalue. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths.

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).
Sayaka Yamamoto, Yuta Yako, Yoichiro Fujioka, Mihoko Kajita, Takeshi Kameyama, Shunsuke Kon, Susumu Ishikawa, Yusuke Ohba, Yusuke Ohno, Akio Kihara, Yasuyuki Fujita
Molecular biology of the cell, 27, 3, 491, 9, 2016年02月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
Daniel J Klionsky, Kotb Abdelmohsen, Akihisa Abe, Md Joynal Abedin, Hagai Abeliovich, Abraham Acevedo Arozena, Hiroaki Adachi, Christopher M Adams, Peter D Adams, Khosrow Adeli, Peter J Adhihetty, Sharon G Adler, Galila Agam, Rajesh Agarwal, Manish K Aghi, Maria Agnello, Patrizia Agostinis, Patricia V Aguilar, Julio Aguirre-Ghiso, Edoardo M Airoldi, Slimane Ait-Si-Ali, Takahiko Akematsu, Emmanuel T Akporiaye, Mohamed Al-Rubeai, Guillermo M Albaiceta, Chris Albanese, Diego Albani, Matthew L Albert, Jesus Aldudo, Hana Algül, Mehrdad Alirezaei, Iraide Alloza, Alexandru Almasan, Maylin Almonte-Beceril, Emad S Alnemri, Covadonga Alonso, Nihal Altan-Bonnet, Dario C Altieri, Silvia Alvarez, Lydia Alvarez-Erviti, Sandro Alves, Giuseppina Amadoro, Atsuo Amano, Consuelo Amantini, Santiago Ambrosio, Ivano Amelio, Amal O Amer, Mohamed Amessou, Angelika Amon, Zhenyi An, Frank A Anania, Stig U Andersen, Usha P Andley, Catherine K Andreadi, Nathalie Andrieu-Abadie, Alberto Anel, David K Ann, Shailendra Anoopkumar-Dukie, Manuela Antonioli, Hiroshi Aoki, Nadezda Apostolova, Saveria Aquila, Katia Aquilano, Koichi Araki, Eli Arama, Agustin Aranda, Jun Araya, Alexandre Arcaro, Esperanza Arias, Hirokazu Arimoto, Aileen R Ariosa, Jane L Armstrong, Thierry Arnould, Ivica Arsov, Katsuhiko Asanuma, Valerie Askanas, Eric Asselin, Ryuichiro Atarashi, Sally S Atherton, Julie D Atkin, Laura D Attardi, Patrick Auberger, Georg Auburger, Laure Aurelian, Riccardo Autelli, Laura Avagliano, Maria Laura Avantaggiati, Limor Avrahami, Suresh Awale, Neelam Azad, Tiziana Bachetti, Jonathan M Backer, Dong-Hun Bae, Jae-Sung Bae, Ok-Nam Bae, Soo Han Bae, Eric H Baehrecke, Seung-Hoon Baek, Stephen Baghdiguian, Agnieszka Bagniewska-Zadworna, Hua Bai, Jie Bai, Xue-Yuan Bai, Yannick Bailly, Kithiganahalli Narayanaswamy Balaji, Walter Balduini, Andrea Ballabio, Rena Balzan, Rajkumar Banerjee, Gábor Bánhegyi, Haijun Bao, Benoit Barbeau, Maria D Barrachina, Esther Barreiro, Bonnie Bartel, Alberto Bartolomé, Diane C Bassham, Maria Teresa Bassi, Robert C Bast Jr, Alakananda Basu, Maria Teresa Batista, Henri Batoko, Maurizio Battino, Kyle Bauckman, Bradley L Baumgarner, K Ulrich Bayer, Rupert Beale, Jean-François Beaulieu, George R Beck Jr, Christoph Becker, J David Beckham, Pierre-André Bédard, Patrick J Bednarski, Thomas J Begley, Christian Behl, Christian Behrends, Georg Mn Behrens, Kevin E Behrns, Eloy Bejarano, Amine Belaid, Francesca Belleudi, Giovanni Bénard, Guy Berchem, Daniele Bergamaschi, Matteo Bergami, Ben Berkhout, Laura Berliocchi, Amélie Bernard, Monique Bernard, Francesca Bernassola, Anne Bertolotti, Amanda S Bess, Sébastien Besteiro, Saverio Bettuzzi, Savita Bhalla, Shalmoli Bhattacharyya, Sujit K Bhutia, Caroline Biagosch, Michele Wolfe Bianchi, Martine Biard-Piechaczyk, Viktor Billes, Claudia Bincoletto, Baris Bingol, Sara W Bird, Marc Bitoun, Ivana Bjedov, Craig Blackstone, Lionel Blanc, Guillermo A Blanco, Heidi Kiil Blomhoff, Emilio Boada-Romero, Stefan Böckler, Marianne Boes, Kathleen Boesze-Battaglia, Lawrence H Boise, Alessandra Bolino, Andrea Boman, Paolo Bonaldo, Matteo Bordi, Jürgen Bosch, Luis M Botana, Joelle Botti, German Bou, Marina Bouché, Marion Bouchecareilh, Marie-Josée Boucher, Michael E Boulton, Sebastien G Bouret, Patricia Boya, Michaël Boyer-Guittaut, Peter V Bozhkov, Nathan Brady, Vania Mm Braga, Claudio Brancolini, Gerhard H Braus, José M Bravo-San Pedro, Lisa A Brennan, Emery H Bresnick, Patrick Brest, Dave Bridges, Marie-Agnès Bringer, Marisa Brini, Glauber C Brito, Bertha Brodin, Paul S Brookes, Eric J Brown, Karen Brown, Hal E Broxmeyer, Alain Bruhat, Patricia Chakur Brum, John H Brumell, Nicola Brunetti-Pierri, Robert J Bryson-Richardson, Shilpa Buch, Alastair M Buchan, Hikmet Budak, Dmitry V Bulavin, Scott J Bultman, Geert Bultynck, Vladimir Bumbasirevic, Yan Burelle, Robert E Burke, Margit Burmeister, Peter Bütikofer, Laura Caberlotto, Ken Cadwell, Monika Cahova, Dongsheng Cai, Jingjing Cai, Qian Cai, Sara Calatayud, Nadine Camougrand, Michelangelo Campanella, Grant R Campbell, Matthew Campbell, Silvia Campello, Robin Candau, Isabella Caniggia, Lavinia Cantoni, Lizhi Cao, Allan B Caplan, Michele Caraglia, Claudio Cardinali, Sandra Morais Cardoso, Jennifer S Carew, Laura A Carleton, Cathleen R Carlin, Silvia Carloni, Sven R Carlsson, Didac Carmona-Gutierrez, Leticia Am Carneiro, Oliana Carnevali, Serena Carra, Alice Carrier, Bernadette Carroll, Caty Casas, Josefina Casas, Giuliana Cassinelli, Perrine Castets, Susana Castro-Obregon, Gabriella Cavallini, Isabella Ceccherini, Francesco Cecconi, Arthur I Cederbaum, Valentín Ceña, Simone Cenci, Claudia Cerella, Davide Cervia, Silvia Cetrullo, Hassan Chaachouay, Han-Jung Chae, Andrei S Chagin, Chee-Yin Chai, Gopal Chakrabarti, Georgios Chamilos, Edmond Yw Chan, Matthew Tv Chan, Dhyan Chandra, Pallavi Chandra, Chih-Peng Chang, Raymond Chuen-Chung Chang, Ta Yuan Chang, John C Chatham, Saurabh Chatterjee, Santosh Chauhan, Yongsheng Che, Michael E Cheetham, Rajkumar Cheluvappa, Chun-Jung Chen, Gang Chen, Guang-Chao Chen, Guoqiang Chen, Hongzhuan Chen, Jeff W Chen, Jian-Kang Chen, Min Chen, Mingzhou Chen, Peiwen Chen, Qi Chen, Quan Chen, Shang-Der Chen, Si Chen, Steve S-L Chen, Wei Chen, Wei-Jung Chen, Wen Qiang Chen, Wenli Chen, Xiangmei Chen, Yau-Hung Chen, Ye-Guang Chen, Yin Chen, Yingyu Chen, Yongshun Chen, Yu-Jen Chen, Yue-Qin Chen, Yujie Chen, Zhen Chen, Zhong Chen, Alan Cheng, Christopher Hk Cheng, Hua Cheng, Heesun Cheong, Sara Cherry, Jason Chesney, Chun Hei Antonio Cheung, Eric Chevet, Hsiang Cheng Chi, Sung-Gil Chi, Fulvio Chiacchiera, Hui-Ling Chiang, Roberto Chiarelli, Mario Chiariello, Marcello Chieppa, Lih-Shen Chin, Mario Chiong, Gigi Nc Chiu, Dong-Hyung Cho, Ssang-Goo Cho, William C Cho, Yong-Yeon Cho, Young-Seok Cho, Augustine Mk Choi, Eui-Ju Choi, Eun-Kyoung Choi, Jayoung Choi, Mary E Choi, Seung-Il Choi, Tsui-Fen Chou, Salem Chouaib, Divaker Choubey, Vinay Choubey, Kuan-Chih Chow, Kamal Chowdhury, Charleen T Chu, Tsung-Hsien Chuang, Taehoon Chun, Hyewon Chung, Taijoon Chung, Yuen-Li Chung, Yong-Joon Chwae, Valentina Cianfanelli, Roberto Ciarcia, Iwona A Ciechomska, Maria Rosa Ciriolo, Mara Cirone, Sofie Claerhout, Michael J Clague, Joan Clària, Peter Gh Clarke, Robert Clarke, Emilio Clementi, Cédric Cleyrat, Miriam Cnop, Eliana M Coccia, Tiziana Cocco, Patrice Codogno, Jörn Coers, Ezra Ew Cohen, David Colecchia, Luisa Coletto, Núria S Coll, Emma Colucci-Guyon, Sergio Comincini, Maria Condello, Katherine L Cook, Graham H Coombs, Cynthia D Cooper, J Mark Cooper, Isabelle Coppens, Maria Tiziana Corasaniti, Marco Corazzari, Ramon Corbalan, Elisabeth Corcelle-Termeau, Mario D Cordero, Cristina Corral-Ramos, Olga Corti, Andrea Cossarizza, Paola Costelli, Safia Costes, Susan L Cotman, Ana Coto-Montes, Sandra Cottet, Eduardo Couve, Lori R Covey, L Ashley Cowart, Jeffery S Cox, Fraser P Coxon, Carolyn B Coyne, Mark S Cragg, Rolf J Craven, Tiziana Crepaldi, Jose L Crespo, Alfredo Criollo, Valeria Crippa, Maria Teresa Cruz, Ana Maria Cuervo, Jose M Cuezva, Taixing Cui, Pedro R Cutillas, Mark J Czaja, Maria F Czyzyk-Krzeska, Ruben K Dagda, Uta Dahmen, Chunsun Dai, Wenjie Dai, Yun Dai, Kevin N Dalby, Luisa Dalla Valle, Guillaume Dalmasso, Marcello D'Amelio, Markus Damme, Arlette Darfeuille-Michaud, Catherine Dargemont, Victor M Darley-Usmar, Srinivasan Dasarathy, Biplab Dasgupta, Srikanta Dash, Crispin R Dass, Hazel Marie Davey, Lester M Davids, David Dávila, Roger J Davis, Ted M Dawson, Valina L Dawson, Paula Daza, Jackie de Belleroche, Paul de Figueiredo, Regina Celia Bressan Queiroz de Figueiredo, José de la Fuente, Luisa De Martino, Antonella De Matteis, Guido Ry De Meyer, Angelo De Milito, Mauro De Santi, Wanderley de Souza, Vincenzo De Tata, Daniela De Zio, Jayanta Debnath, Reinhard Dechant, Jean-Paul Decuypere, Shane Deegan, Benjamin Dehay, Barbara Del Bello, Dominic P Del Re, Régis Delage-Mourroux, Lea Md Delbridge, Louise Deldicque, Elizabeth Delorme-Axford, Yizhen Deng, Joern Dengjel, Melanie Denizot, Paul Dent, Channing J Der, Vojo Deretic, Benoît Derrien, Eric Deutsch, Timothy P Devarenne, Rodney J Devenish, Sabrina Di Bartolomeo, Nicola Di Daniele, Fabio Di Domenico, Alessia Di Nardo, Simone Di Paola, Antonio Di Pietro, Livia Di Renzo, Aaron DiAntonio, Guillermo Díaz-Araya, Ines Díaz-Laviada, Maria T Diaz-Meco, Javier Diaz-Nido, Chad A Dickey, Robert C Dickson, Marc Diederich, Paul Digard, Ivan Dikic, Savithrama P Dinesh-Kumar, Chan Ding, Wen-Xing Ding, Zufeng Ding, Luciana Dini, Jörg Hw Distler, Abhinav Diwan, Mojgan Djavaheri-Mergny, Kostyantyn Dmytruk, Renwick Cj Dobson, Volker Doetsch, Karol Dokladny, Svetlana Dokudovskaya, Massimo Donadelli, X Charlie Dong, Xiaonan Dong, Zheng Dong, Terrence M Donohue Jr, Kelly S Doran, Gabriella D'Orazi, Gerald W Dorn 2nd, Victor Dosenko, Sami Dridi, Liat Drucker, Jie Du, Li-Lin Du, Lihuan Du, André du Toit, Priyamvada Dua, Lei Duan, Pu Duann, Vikash Kumar Dubey, Michael R Duchen, Michel A Duchosal, Helene Duez, Isabelle Dugail, Verónica I Dumit, Mara C Duncan, Elaine A Dunlop, William A Dunn Jr, Nicolas Dupont, Luc Dupuis, Raúl V Durán, Thomas M Durcan, Stéphane Duvezin-Caubet, Umamaheswar Duvvuri, Vinay Eapen, Darius Ebrahimi-Fakhari, Arnaud Echard, Leopold Eckhart, Charles L Edelstein, Aimee L Edinger, Ludwig Eichinger, Tobias Eisenberg, Avital Eisenberg-Lerner, N Tony Eissa, Wafik S El-Deiry, Victoria El-Khoury, Zvulun Elazar, Hagit Eldar-Finkelman, Chris Jh Elliott, Enzo Emanuele, Urban Emmenegger, Nikolai Engedal, Anna-Mart Engelbrecht, Simone Engelender, Jorrit M Enserink, Ralf Erdmann, Jekaterina Erenpreisa, Rajaraman Eri, Jason L Eriksen, Andreja Erman, Ricardo Escalante, Eeva-Liisa Eskelinen, Lucile Espert, Lorena Esteban-Martínez, Thomas J Evans, Mario Fabri, Gemma Fabrias, Cinzia Fabrizi, Antonio Facchiano, Nils J Færgeman, Alberto Faggioni, W Douglas Fairlie, Chunhai Fan, Daping Fan, Jie Fan, Shengyun Fang, Manolis Fanto, Alessandro Fanzani, Thomas Farkas, Mathias Faure, Francois B Favier, Howard Fearnhead, Massimo Federici, Erkang Fei, Tania C Felizardo, Hua Feng, Yibin Feng, Yuchen Feng, Thomas A Ferguson, Álvaro F Fernández, Maite G Fernandez-Barrena, Jose C Fernandez-Checa, Arsenio Fernández-López, Martin E Fernandez-Zapico, Olivier Feron, Elisabetta Ferraro, Carmen Veríssima Ferreira-Halder, Laszlo Fesus, Ralph Feuer, Fabienne C Fiesel, Eduardo C Filippi-Chiela, Giuseppe Filomeni, Gian Maria Fimia, John H Fingert, Steven Finkbeiner, Toren Finkel, Filomena Fiorito, Paul B Fisher, Marc Flajolet, Flavio Flamigni, Oliver Florey, Salvatore Florio, R Andres Floto, Marco Folini, Carlo Follo, Edward A Fon, Francesco Fornai, Franco Fortunato, Alessandro Fraldi, Rodrigo Franco, Arnaud Francois, Aurélie François, Lisa B Frankel, Iain Dc Fraser, Norbert Frey, Damien G Freyssenet, Christian Frezza, Scott L Friedman, Daniel E Frigo, Dongxu Fu, José M Fuentes, Juan Fueyo, Yoshio Fujitani, Yuuki Fujiwara, Mikihiro Fujiya, Mitsunori Fukuda, Simone Fulda, Carmela Fusco, Bozena Gabryel, Matthias Gaestel, Philippe Gailly, Malgorzata Gajewska, Sehamuddin Galadari, Gad Galili, Inmaculada Galindo, Maria F Galindo, Giovanna Galliciotti, Lorenzo Galluzzi, Luca Galluzzi, Vincent Galy, Noor Gammoh, Sam Gandy, Anand K Ganesan, Swamynathan Ganesan, Ian G Ganley, Monique Gannagé, Fen-Biao Gao, Feng Gao, Jian-Xin Gao, Lorena García Nannig, Eleonora García Véscovi, Marina Garcia-Macía, Carmen Garcia-Ruiz, Abhishek D Garg, Pramod Kumar Garg, Ricardo Gargini, Nils Christian Gassen, Damián Gatica, Evelina Gatti, Julie Gavard, Evripidis Gavathiotis, Liang Ge, Pengfei Ge, Shengfang Ge, Po-Wu Gean, Vania Gelmetti, Armando A Genazzani, Jiefei Geng, Pascal Genschik, Lisa Gerner, Jason E Gestwicki, David A Gewirtz, Saeid Ghavami, Eric Ghigo, Debabrata Ghosh, Anna Maria Giammarioli, Francesca Giampieri, Claudia Giampietri, Alexandra Giatromanolaki, Derrick J Gibbings, Lara Gibellini, Spencer B Gibson, Vanessa Ginet, Antonio Giordano, Flaviano Giorgini, Elisa Giovannetti, Stephen E Girardin, Suzana Gispert, Sandy Giuliano, Candece L Gladson, Alvaro Glavic, Martin Gleave, Nelly Godefroy, Robert M Gogal Jr, Kuppan Gokulan, Gustavo H Goldman, Delia Goletti, Michael S Goligorsky, Aldrin V Gomes, Ligia C Gomes, Hernando Gomez, Candelaria Gomez-Manzano, Rubén Gómez-Sánchez, Dawit Ap Gonçalves, Ebru Goncu, Qingqiu Gong, Céline Gongora, Carlos B Gonzalez, Pedro Gonzalez-Alegre, Pilar Gonzalez-Cabo, Rosa Ana González-Polo, Ing Swie Goping, Carlos Gorbea, Nikolai V Gorbunov, Daphne R Goring, Adrienne M Gorman, Sharon M Gorski, Sandro Goruppi, Shino Goto-Yamada, Cecilia Gotor, Roberta A Gottlieb, Illana Gozes, Devrim Gozuacik, Yacine Graba, Martin Graef, Giovanna E Granato, Gary Dean Grant, Steven Grant, Giovanni Luca Gravina, Douglas R Green, Alexander Greenhough, Michael T Greenwood, Benedetto Grimaldi, Frédéric Gros, Charles Grose, Jean-Francois Groulx, Florian Gruber, Paolo Grumati, Tilman Grune, Jun-Lin Guan, Kun-Liang Guan, Barbara Guerra, Carlos Guillen, Kailash Gulshan, Jan Gunst, Chuanyong Guo, Lei Guo, Ming Guo, Wenjie Guo, Xu-Guang Guo, Andrea A Gust, Åsa B Gustafsson, Elaine Gutierrez, Maximiliano G Gutierrez, Ho-Shin Gwak, Albert Haas, James E Haber, Shinji Hadano, Monica Hagedorn, David R Hahn, Andrew J Halayko, Anne Hamacher-Brady, Kozo Hamada, Ahmed Hamai, Andrea Hamann, Maho Hamasaki, Isabelle Hamer, Qutayba Hamid, Ester M Hammond, Feng Han, Weidong Han, James T Handa, John A Hanover, Malene Hansen, Masaru Harada, Ljubica Harhaji-Trajkovic, J Wade Harper, Abdel Halim Harrath, Adrian L Harris, James Harris, Udo Hasler, Peter Hasselblatt, Kazuhisa Hasui, Robert G Hawley, Teresa S Hawley, Congcong He, Cynthia Y He, Fengtian He, Gu He, Rong-Rong He, Xian-Hui He, You-Wen He, Yu-Ying He, Joan K Heath, Marie-Josée Hébert, Robert A Heinzen, Gudmundur Vignir Helgason, Michael Hensel, Elizabeth P Henske, Chengtao Her, Paul K Herman, Agustín Hernández, Carlos Hernandez, Sonia Hernández-Tiedra, Claudio Hetz, P Robin Hiesinger, Katsumi Higaki, Sabine Hilfiker, Bradford G Hill, Joseph A Hill, William D Hill, Keisuke Hino, Daniel Hofius, Paul Hofman, Günter U Höglinger, Jörg Höhfeld, Marina K Holz, Yonggeun Hong, David A Hood, Jeroen Jm Hoozemans, Thorsten Hoppe, Chin Hsu, Chin-Yuan Hsu, Li-Chung Hsu, Dong Hu, Guochang Hu, Hong-Ming Hu, Hongbo Hu, Ming Chang Hu, Yu-Chen Hu, Zhuo-Wei Hu, Fang Hua, Ya Hua, Canhua Huang, Huey-Lan Huang, Kuo-How Huang, Kuo-Yang Huang, Shile Huang, Shiqian Huang, Wei-Pang Huang, Yi-Ran Huang, Yong Huang, Yunfei Huang, Tobias B Huber, Patricia Huebbe, Won-Ki Huh, Juha J Hulmi, Gang Min Hur, James H Hurley, Zvenyslava Husak, Sabah Na Hussain, Salik Hussain, Jung Jin Hwang, Seungmin Hwang, Thomas Is Hwang, Atsuhiro Ichihara, Yuzuru Imai, Carol Imbriano, Megumi Inomata, Takeshi Into, Valentina Iovane, Juan L Iovanna, Renato V Iozzo, Nancy Y Ip, Javier E Irazoqui, Pablo Iribarren, Yoshitaka Isaka, Aleksandra J Isakovic, Harry Ischiropoulos, Jeffrey S Isenberg, Mohammad Ishaq, Hiroyuki Ishida, Isao Ishii, Jane E Ishmael, Ciro Isidoro, Ken-Ichi Isobe, Erika Isono, Shohreh Issazadeh-Navikas, Koji Itahana, Eisuke Itakura, Andrei I Ivanov, Anand Krishnan V Iyer, José M Izquierdo, Yotaro Izumi, Valentina Izzo, Marja Jäättelä, Nadia Jaber, Daniel John Jackson, William T Jackson, Tony George Jacob, Thomas S Jacques, Chinnaswamy Jagannath, Ashish Jain, Nihar Ranjan Jana, Byoung Kuk Jang, Alkesh Jani, Bassam Janji, Paulo Roberto Jannig, Patric J Jansson, Steve Jean, Marina Jendrach, Ju-Hong Jeon, Niels Jessen, Eui-Bae Jeung, Kailiang Jia, Lijun Jia, Hong Jiang, Hongchi Jiang, Liwen Jiang, Teng Jiang, Xiaoyan Jiang, Xuejun Jiang, Xuejun Jiang, Ying Jiang, Yongjun Jiang, Alberto Jiménez, Cheng Jin, Hongchuan Jin, Lei Jin, Meiyan Jin, Shengkan Jin, Umesh Kumar Jinwal, Eun-Kyeong Jo, Terje Johansen, Daniel E Johnson, Gail Vw Johnson, James D Johnson, Eric Jonasch, Chris Jones, Leo Ab Joosten, Joaquin Jordan, Anna-Maria Joseph, Bertrand Joseph, Annie M Joubert, Dianwen Ju, Jingfang Ju, Hsueh-Fen Juan, Katrin Juenemann, Gábor Juhász, Hye Seung Jung, Jae U Jung, Yong-Keun Jung, Heinz Jungbluth, Matthew J Justice, Barry Jutten, Nadeem O Kaakoush, Kai Kaarniranta, Allen Kaasik, Tomohiro Kabuta, Bertrand Kaeffer, Katarina Kågedal, Alon Kahana, Shingo Kajimura, Or Kakhlon, Manjula Kalia, Dhan V Kalvakolanu, Yoshiaki Kamada, Konstantinos Kambas, Vitaliy O Kaminskyy, Harm H Kampinga, Mustapha Kandouz, Chanhee Kang, Rui Kang, Tae-Cheon Kang, Tomotake Kanki, Thirumala-Devi Kanneganti, Haruo Kanno, Anumantha G Kanthasamy, Marc Kantorow, Maria Kaparakis-Liaskos, Orsolya Kapuy, Vassiliki Karantza, Md Razaul Karim, Parimal Karmakar, Arthur Kaser, Susmita Kaushik, Thomas Kawula, A Murat Kaynar, Po-Yuan Ke, Zun-Ji Ke, John H Kehrl, Kate E Keller, Jongsook Kim Kemper, Anne K Kenworthy, Oliver Kepp, Andreas Kern, Santosh Kesari, David Kessel, Robin Ketteler, Isis do Carmo Kettelhut, Bilon Khambu, Muzamil Majid Khan, Vinoth Km Khandelwal, Sangeeta Khare, Juliann G Kiang, Amy A Kiger, Akio Kihara, Arianna L Kim, Cheol Hyeon Kim, Deok Ryong Kim, Do-Hyung Kim, Eung Kweon Kim, Hye Young Kim, Hyung-Ryong Kim, Jae-Sung Kim, Jeong Hun Kim, Jin Cheon Kim, Jin Hyoung Kim, Kwang Woon Kim, Michael D Kim, Moon-Moo Kim, Peter K Kim, Seong Who Kim, Soo-Youl Kim, Yong-Sun Kim, Yonghyun Kim, Adi Kimchi, Alec C Kimmelman, Tomonori Kimura, Jason S King, Karla Kirkegaard, Vladimir Kirkin, Lorrie A Kirshenbaum, Shuji Kishi, Yasuo Kitajima, Katsuhiko Kitamoto, Yasushi Kitaoka, Kaio Kitazato, Rudolf A Kley, Walter T Klimecki, Michael Klinkenberg, Jochen Klucken, Helene Knævelsrud, Erwin Knecht, Laura Knuppertz, Jiunn-Liang Ko, Satoru Kobayashi, Jan C Koch, Christelle Koechlin-Ramonatxo, Ulrich Koenig, Young Ho Koh, Katja Köhler, Sepp D Kohlwein, Masato Koike, Masaaki Komatsu, Eiki Kominami, Dexin Kong, Hee Jeong Kong, Eumorphia G Konstantakou, Benjamin T Kopp, Tamas Korcsmaros, Laura Korhonen, Viktor I Korolchuk, Nadya V Koshkina, Yanjun Kou, Michael I Koukourakis, Constantinos Koumenis, Attila L Kovács, Tibor Kovács, Werner J Kovacs, Daisuke Koya, Claudine Kraft, Dimitri Krainc, Helmut Kramer, Tamara Kravic-Stevovic, Wilhelm Krek, Carole Kretz-Remy, Roswitha Krick, Malathi Krishnamurthy, Janos Kriston-Vizi, Guido Kroemer, Michael C Kruer, Rejko Kruger, Nicholas T Ktistakis, Kazuyuki Kuchitsu, Christian Kuhn, Addanki Pratap Kumar, Anuj Kumar, Ashok Kumar, Deepak Kumar, Dhiraj Kumar, Rakesh Kumar, Sharad Kumar, Mondira Kundu, Hsing-Jien Kung, Atsushi Kuno, Sheng-Han Kuo, Jeff Kuret, Tino Kurz, Terry Kwok, Taeg Kyu Kwon, Yong Tae Kwon, Irene Kyrmizi, Albert R La Spada, Frank Lafont, Tim Lahm, Aparna Lakkaraju, Truong Lam, Trond Lamark, Steve Lancel, Terry H Landowski, Darius J R Lane, Jon D Lane, Cinzia Lanzi, Pierre Lapaquette, Louis R Lapierre, Jocelyn Laporte, Johanna Laukkarinen, Gordon W Laurie, Sergio Lavandero, Lena Lavie, Matthew J LaVoie, Betty Yuen Kwan Law, Helen Ka-Wai Law, Kelsey B Law, Robert Layfield, Pedro A Lazo, Laurent Le Cam, Karine G Le Roch, Hervé Le Stunff, Vijittra Leardkamolkarn, Marc Lecuit, Byung-Hoon Lee, Che-Hsin Lee, Erinna F Lee, Gyun Min Lee, He-Jin Lee, Hsinyu Lee, Jae Keun Lee, Jongdae Lee, Ju-Hyun Lee, Jun Hee Lee, Michael Lee, Myung-Shik Lee, Patty J Lee, Sam W Lee, Seung-Jae Lee, Shiow-Ju Lee, Stella Y Lee, Sug Hyung Lee, Sung Sik Lee, Sung-Joon Lee, Sunhee Lee, Ying-Ray Lee, Yong J Lee, Young H Lee, Christiaan Leeuwenburgh, Sylvain Lefort, Renaud Legouis, Jinzhi Lei, Qun-Ying Lei, David A Leib, Gil Leibowitz, Istvan Lekli, Stéphane D Lemaire, John J Lemasters, Marius K Lemberg, Antoinette Lemoine, Shuilong Leng, Guido Lenz, Paola Lenzi, Lilach O Lerman, Daniele Lettieri Barbato, Julia I-Ju Leu, Hing Y Leung, Beth Levine, Patrick A Lewis, Frank Lezoualc'h, Chi Li, Faqiang Li, Feng-Jun Li, Jun Li, Ke Li, Lian Li, Min Li, Min Li, Qiang Li, Rui Li, Sheng Li, Wei Li, Wei Li, Xiaotao Li, Yumin Li, Jiqin Lian, Chengyu Liang, Qiangrong Liang, Yulin Liao, Joana Liberal, Pawel P Liberski, Pearl Lie, Andrew P Lieberman, Hyunjung Jade Lim, Kah-Leong Lim, Kyu Lim, Raquel T Lima, Chang-Shen Lin, Chiou-Feng Lin, Fang Lin, Fangming Lin, Fu-Cheng Lin, Kui Lin, Kwang-Huei Lin, Pei-Hui Lin, Tianwei Lin, Wan-Wan Lin, Yee-Shin Lin, Yong Lin, Rafael Linden, Dan Lindholm, Lisa M Lindqvist, Paul Lingor, Andreas Linkermann, Lance A Liotta, Marta M Lipinski, Vitor A Lira, Michael P Lisanti, Paloma B Liton, Bo Liu, Chong Liu, Chun-Feng Liu, Fei Liu, Hung-Jen Liu, Jianxun Liu, Jing-Jing Liu, Jing-Lan Liu, Ke Liu, Leyuan Liu, Liang Liu, Quentin Liu, Rong-Yu Liu, Shiming Liu, Shuwen Liu, Wei Liu, Xian-De Liu, Xiangguo Liu, Xiao-Hong Liu, Xinfeng Liu, Xu Liu, Xueqin Liu, Yang Liu, Yule Liu, Zexian Liu, Zhe Liu, Juan P Liuzzi, Gérard Lizard, Mila Ljujic, Irfan J Lodhi, Susan E Logue, Bal L Lokeshwar, Yun Chau Long, Sagar Lonial, Benjamin Loos, Carlos López-Otín, Cristina López-Vicario, Mar Lorente, Philip L Lorenzi, Péter Lõrincz, Marek Los, Michael T Lotze, Penny E Lovat, Binfeng Lu, Bo Lu, Jiahong Lu, Qing Lu, She-Min Lu, Shuyan Lu, Yingying Lu, Frédéric Luciano, Shirley Luckhart, John Milton Lucocq, Paula Ludovico, Aurelia Lugea, Nicholas W Lukacs, Julian J Lum, Anders H Lund, Honglin Luo, Jia Luo, Shouqing Luo, Claudio Luparello, Timothy Lyons, Jianjie Ma, Yi Ma, Yong Ma, Zhenyi Ma, Juliano Machado, Glaucia M Machado-Santelli, Fernando Macian, Gustavo C MacIntosh, Jeffrey P MacKeigan, Kay F Macleod, John D MacMicking, Lee Ann MacMillan-Crow, Frank Madeo, Muniswamy Madesh, Julio Madrigal-Matute, Akiko Maeda, Tatsuya Maeda, Gustavo Maegawa, Emilia Maellaro, Hannelore Maes, Marta Magariños, Kenneth Maiese, Tapas K Maiti, Luigi Maiuri, Maria Chiara Maiuri, Carl G Maki, Roland Malli, Walter Malorni, Alina Maloyan, Fathia Mami-Chouaib, Na Man, Joseph D Mancias, Eva-Maria Mandelkow, Michael A Mandell, Angelo A Manfredi, Serge N Manié, Claudia Manzoni, Kai Mao, Zixu Mao, Zong-Wan Mao, Philippe Marambaud, Anna Maria Marconi, Zvonimir Marelja, Gabriella Marfe, Marta Margeta, Eva Margittai, Muriel Mari, Francesca V Mariani, Concepcio Marin, Sara Marinelli, Guillermo Mariño, Ivanka Markovic, Rebecca Marquez, Alberto M Martelli, Sascha Martens, Katie R Martin, Seamus J Martin, Shaun Martin, Miguel A Martin-Acebes, Paloma Martín-Sanz, Camille Martinand-Mari, Wim Martinet, Jennifer Martinez, Nuria Martinez-Lopez, Ubaldo Martinez-Outschoorn, Moisés Martínez-Velázquez, Marta Martinez-Vicente, Waleska Kerllen Martins, Hirosato Mashima, James A Mastrianni, Giuseppe Matarese, Paola Matarrese, Roberto Mateo, Satoaki Matoba, Naomichi Matsumoto, Takehiko Matsushita, Akira Matsuura, Takeshi Matsuzawa, Mark P Mattson, Soledad Matus, Norma Maugeri, Caroline Mauvezin, Andreas Mayer, Dusica Maysinger, Guillermo D Mazzolini, Mary Kate McBrayer, Kimberly McCall, Craig McCormick, Gerald M McInerney, Skye C McIver, Sharon McKenna, John J McMahon, Iain A McNeish, Fatima Mechta-Grigoriou, Jan Paul Medema, Diego L Medina, Klara Megyeri, Maryam Mehrpour, Jawahar L Mehta, Yide Mei, Ute-Christiane Meier, Alfred J Meijer, Alicia Meléndez, Gerry Melino, Sonia Melino, Edesio Jose Tenorio de Melo, Maria A Mena, Marc D Meneghini, Javier A Menendez, Regina Menezes, Liesu Meng, Ling-Hua Meng, Songshu Meng, Rossella Menghini, A Sue Menko, Rubem Fs Menna-Barreto, Manoj B Menon, Marco A Meraz-Ríos, Giuseppe Merla, Luciano Merlini, Angelica M Merlot, Andreas Meryk, Stefania Meschini, Joel N Meyer, Man-Tian Mi, Chao-Yu Miao, Lucia Micale, Simon Michaeli, Carine Michiels, Anna Rita Migliaccio, Anastasia Susie Mihailidou, Dalibor Mijaljica, Katsuhiko Mikoshiba, Enrico Milan, Leonor Miller-Fleming, Gordon B Mills, Ian G Mills, Georgia Minakaki, Berge A Minassian, Xiu-Fen Ming, Farida Minibayeva, Elena A Minina, Justine D Mintern, Saverio Minucci, Antonio Miranda-Vizuete, Claire H Mitchell, Shigeki Miyamoto, Keisuke Miyazawa, Noboru Mizushima, Katarzyna Mnich, Baharia Mograbi, Simin Mohseni, Luis Ferreira Moita, Marco Molinari, Maurizio Molinari, Andreas Buch Møller, Bertrand Mollereau, Faustino Mollinedo, Marco Mongillo, Martha M Monick, Serena Montagnaro, Craig Montell, Darren J Moore, Michael N Moore, Rodrigo Mora-Rodriguez, Paula I Moreira, Etienne Morel, Maria Beatrice Morelli, Sandra Moreno, Michael J Morgan, Arnaud Moris, Yuji Moriyasu, Janna L Morrison, Lynda A Morrison, Eugenia Morselli, Jorge Moscat, Pope L Moseley, Serge Mostowy, Elisa Motori, Denis Mottet, Jeremy C Mottram, Charbel E-H Moussa, Vassiliki E Mpakou, Hasan Mukhtar, Jean M Mulcahy Levy, Sylviane Muller, Raquel Muñoz-Moreno, Cristina Muñoz-Pinedo, Christian Münz, Maureen E Murphy, James T Murray, Aditya Murthy, Indira U Mysorekar, Ivan R Nabi, Massimo Nabissi, Gustavo A Nader, Yukitoshi Nagahara, Yoshitaka Nagai, Kazuhiro Nagata, Anika Nagelkerke, Péter Nagy, Samisubbu R Naidu, Sreejayan Nair, Hiroyasu Nakano, Hitoshi Nakatogawa, Meera Nanjundan, Gennaro Napolitano, Naweed I Naqvi, Roberta Nardacci, Derek P Narendra, Masashi Narita, Anna Chiara Nascimbeni, Ramesh Natarajan, Luiz C Navegantes, Steffan T Nawrocki, Taras Y Nazarko, Volodymyr Y Nazarko, Thomas Neill, Luca M Neri, Mihai G Netea, Romana T Netea-Maier, Bruno M Neves, Paul A Ney, Ioannis P Nezis, Hang Tt Nguyen, Huu Phuc Nguyen, Anne-Sophie Nicot, Hilde Nilsen, Per Nilsson, Mikio Nishimura, Ichizo Nishino, Mireia Niso-Santano, Hua Niu, Ralph A Nixon, Vincent Co Njar, Takeshi Noda, Angelika A Noegel, Elsie Magdalena Nolte, Erik Norberg, Koenraad K Norga, Sakineh Kazemi Noureini, Shoji Notomi, Lucia Notterpek, Karin Nowikovsky, Nobuyuki Nukina, Thorsten Nürnberger, Valerie B O'Donnell, Tracey O'Donovan, Peter J O'Dwyer, Ina Oehme, Clara L Oeste, Michinaga Ogawa, Besim Ogretmen, Yuji Ogura, Young J Oh, Masaki Ohmuraya, Takayuki Ohshima, Rani Ojha, Koji Okamoto, Toshiro Okazaki, F Javier Oliver, Karin Ollinger, Stefan Olsson, Daniel P Orban, Paulina Ordonez, Idil Orhon, Laszlo Orosz, Eyleen J O'Rourke, Helena Orozco, Angel L Ortega, Elena Ortona, Laura D Osellame, Junko Oshima, Shigeru Oshima, Heinz D Osiewacz, Takanobu Otomo, Kinya Otsu, Jing-Hsiung James Ou, Tiago F Outeiro, Dong-Yun Ouyang, Hongjiao Ouyang, Michael Overholtzer, Michelle A Ozbun, P Hande Ozdinler, Bulent Ozpolat, Consiglia Pacelli, Paolo Paganetti, Guylène Page, Gilles Pages, Ugo Pagnini, Beata Pajak, Stephen C Pak, Karolina Pakos-Zebrucka, Nazzy Pakpour, Zdena Palková, Francesca Palladino, Kathrin Pallauf, Nicolas Pallet, Marta Palmieri, Søren R Paludan, Camilla Palumbo, Silvia Palumbo, Olatz Pampliega, Hongming Pan, Wei Pan, Theocharis Panaretakis, Aseem Pandey, Areti Pantazopoulou, Zuzana Papackova, Daniela L Papademetrio, Issidora Papassideri, Alessio Papini, Nirmala Parajuli, Julian Pardo, Vrajesh V Parekh, Giancarlo Parenti, Jong-In Park, Junsoo Park, Ohkmae K Park, Roy Parker, Rosanna Parlato, Jan B Parys, Katherine R Parzych, Jean-Max Pasquet, Benoit Pasquier, Kishore Bs Pasumarthi, Daniel Patschan, Cam Patterson, Sophie Pattingre, Scott Pattison, Arnim Pause, Hermann Pavenstädt, Flaminia Pavone, Zully Pedrozo, Fernando J Peña, Miguel A Peñalva, Mario Pende, Jianxin Peng, Fabio Penna, Josef M Penninger, Anna Pensalfini, Salvatore Pepe, Gustavo Js Pereira, Paulo C Pereira, Verónica Pérez-de la Cruz, María Esther Pérez-Pérez, Diego Pérez-Rodríguez, Dolores Pérez-Sala, Celine Perier, Andras Perl, David H Perlmutter, Ida Perrotta, Shazib Pervaiz, Maija Pesonen, Jeffrey E Pessin, Godefridus J Peters, Morten Petersen, Irina Petrache, Basil J Petrof, Goran Petrovski, James M Phang, Mauro Piacentini, Marina Pierdominici, Philippe Pierre, Valérie Pierrefite-Carle, Federico Pietrocola, Felipe X Pimentel-Muiños, Mario Pinar, Benjamin Pineda, Ronit Pinkas-Kramarski, Marcello Pinti, Paolo Pinton, Bilal Piperdi, James M Piret, Leonidas C Platanias, Harald W Platta, Edward D Plowey, Stefanie Pöggeler, Marc Poirot, Peter Polčic, Angelo Poletti, Audrey H Poon, Hana Popelka, Blagovesta Popova, Izabela Poprawa, Shibu M Poulose, Joanna Poulton, Scott K Powers, Ted Powers, Mercedes Pozuelo-Rubio, Krisna Prak, Reinhild Prange, Mark Prescott, Muriel Priault, Sharon Prince, Richard L Proia, Tassula Proikas-Cezanne, Holger Prokisch, Vasilis J Promponas, Karin Przyklenk, Rosa Puertollano, Subbiah Pugazhenthi, Luigi Puglielli, Aurora Pujol, Julien Puyal, Dohun Pyeon, Xin Qi, Wen-Bin Qian, Zheng-Hong Qin, Yu Qiu, Ziwei Qu, Joe Quadrilatero, Frederick Quinn, Nina Raben, Hannah Rabinowich, Flavia Radogna, Michael J Ragusa, Mohamed Rahmani, Komal Raina, Sasanka Ramanadham, Rajagopal Ramesh, Abdelhaq Rami, Sarron Randall-Demllo, Felix Randow, Hai Rao, V Ashutosh Rao, Blake B Rasmussen, Tobias M Rasse, Edward A Ratovitski, Pierre-Emmanuel Rautou, Swapan K Ray, Babak Razani, Bruce H Reed, Fulvio Reggiori, Markus Rehm, Andreas S Reichert, Theo Rein, David J Reiner, Eric Reits, Jun Ren, Xingcong Ren, Maurizio Renna, Jane Eb Reusch, Jose L Revuelta, Leticia Reyes, Alireza R Rezaie, Robert I Richards, Des R Richardson, Clémence Richetta, Michael A Riehle, Bertrand H Rihn, Yasuko Rikihisa, Brigit E Riley, Gerald Rimbach, Maria Rita Rippo, Konstantinos Ritis, Federica Rizzi, Elizete Rizzo, Peter J Roach, Jeffrey Robbins, Michel Roberge, Gabriela Roca, Maria Carmela Roccheri, Sonia Rocha, Cecilia Mp Rodrigues, Clara I Rodríguez, Santiago Rodriguez de Cordoba, Natalia Rodriguez-Muela, Jeroen Roelofs, Vladimir V Rogov, Troy T Rohn, Bärbel Rohrer, Davide Romanelli, Luigina Romani, Patricia Silvia Romano, M Isabel G Roncero, Jose Luis Rosa, Alicia Rosello, Kirill V Rosen, Philip Rosenstiel, Magdalena Rost-Roszkowska, Kevin A Roth, Gael Roué, Mustapha Rouis, Kasper M Rouschop, Daniel T Ruan, Diego Ruano, David C Rubinsztein, Edmund B Rucker 3rd, Assaf Rudich, Emil Rudolf, Ruediger Rudolf, Markus A Ruegg, Carmen Ruiz-Roldan, Avnika Ashok Ruparelia, Paola Rusmini, David W Russ, Gian Luigi Russo, Giuseppe Russo, Rossella Russo, Tor Erik Rusten, Victoria Ryabovol, Kevin M Ryan, Stefan W Ryter, David M Sabatini, Michael Sacher, Carsten Sachse, Michael N Sack, Junichi Sadoshima, Paul Saftig, Ronit Sagi-Eisenberg, Sumit Sahni, Pothana Saikumar, Tsunenori Saito, Tatsuya Saitoh, Koichi Sakakura, Machiko Sakoh-Nakatogawa, Yasuhito Sakuraba, María Salazar-Roa, Paolo Salomoni, Ashok K Saluja, Paul M Salvaterra, Rosa Salvioli, Afshin Samali, Anthony Mj Sanchez, José A Sánchez-Alcázar, Ricardo Sanchez-Prieto, Marco Sandri, Miguel A Sanjuan, Stefano Santaguida, Laura Santambrogio, Giorgio Santoni, Claudia Nunes Dos Santos, Shweta Saran, Marco Sardiello, Graeme Sargent, Pallabi Sarkar, Sovan Sarkar, Maria Rosa Sarrias, Minnie M Sarwal, Chihiro Sasakawa, Motoko Sasaki, Miklos Sass, Ken Sato, Miyuki Sato, Joseph Satriano, Niramol Savaraj, Svetlana Saveljeva, Liliana Schaefer, Ulrich E Schaible, Michael Scharl, Hermann M Schatzl, Randy Schekman, Wiep Scheper, Alfonso Schiavi, Hyman M Schipper, Hana Schmeisser, Jens Schmidt, Ingo Schmitz, Bianca E Schneider, E Marion Schneider, Jaime L Schneider, Eric A Schon, Miriam J Schönenberger, Axel H Schönthal, Daniel F Schorderet, Bernd Schröder, Sebastian Schuck, Ryan J Schulze, Melanie Schwarten, Thomas L Schwarz, Sebastiano Sciarretta, Kathleen Scotto, A Ivana Scovassi, Robert A Screaton, Mark Screen, Hugo Seca, Simon Sedej, Laura Segatori, Nava Segev, Per O Seglen, Jose M Seguí-Simarro, Juan Segura-Aguilar, Ekihiro Seki, Christian Sell, Iban Seiliez, Clay F Semenkovich, Gregg L Semenza, Utpal Sen, Andreas L Serra, Ana Serrano-Puebla, Hiromi Sesaki, Takao Setoguchi, Carmine Settembre, John J Shacka, Ayesha N Shajahan-Haq, Irving M Shapiro, Shweta Sharma, Hua She, C-K James Shen, Chiung-Chyi Shen, Han-Ming Shen, Sanbing Shen, Weili Shen, Rui Sheng, Xianyong Sheng, Zu-Hang Sheng, Trevor G Shepherd, Junyan Shi, Qiang Shi, Qinghua Shi, Yuguang Shi, Shusaku Shibutani, Kenichi Shibuya, Yoshihiro Shidoji, Jeng-Jer Shieh, Chwen-Ming Shih, Yohta Shimada, Shigeomi Shimizu, Dong Wook Shin, Mari L Shinohara, Michiko Shintani, Takahiro Shintani, Tetsuo Shioi, Ken Shirabe, Ronit Shiri-Sverdlov, Orian Shirihai, Gordon C Shore, Chih-Wen Shu, Deepak Shukla, Andriy A Sibirny, Valentina Sica, Christina J Sigurdson, Einar M Sigurdsson, Puran Singh Sijwali, Beata Sikorska, Wilian A Silveira, Sandrine Silvente-Poirot, Gary A Silverman, Jan Simak, Thomas Simmet, Anna Katharina Simon, Hans-Uwe Simon, Cristiano Simone, Matias Simons, Anne Simonsen, Rajat Singh, Shivendra V Singh, Shrawan K Singh, Debasish Sinha, Sangita Sinha, Frank A Sinicrope, Agnieszka Sirko, Kapil Sirohi, Balindiwe Jn Sishi, Annie Sittler, Parco M Siu, Efthimios Sivridis, Anna Skwarska, Ruth Slack, Iva Slaninová, Nikolai Slavov, Soraya S Smaili, Keiran Sm Smalley, Duncan R Smith, Stefaan J Soenen, Scott A Soleimanpour, Anita Solhaug, Kumaravel Somasundaram, Jin H Son, Avinash Sonawane, Chunjuan Song, Fuyong Song, Hyun Kyu Song, Ju-Xian Song, Wei Song, Kai Y Soo, Anil K Sood, Tuck Wah Soong, Virawudh Soontornniyomkij, Maurizio Sorice, Federica Sotgia, David R Soto-Pantoja, Areechun Sotthibundhu, Maria João Sousa, Herman P Spaink, Paul N Span, Anne Spang, Janet D Sparks, Peter G Speck, Stephen A Spector, Claudia D Spies, Wolfdieter Springer, Daret St Clair, Alessandra Stacchiotti, Bart Staels, Michael T Stang, Daniel T Starczynowski, Petro Starokadomskyy, Clemens Steegborn, John W Steele, Leonidas Stefanis, Joan Steffan, Christine M Stellrecht, Harald Stenmark, Tomasz M Stepkowski, Stęphan T Stern, Craig Stevens, Brent R Stockwell, Veronika Stoka, Zuzana Storchova, Björn Stork, Vassilis Stratoulias, Dimitrios J Stravopodis, Pavel Strnad, Anne Marie Strohecker, Anna-Lena Ström, Per Stromhaug, Jiri Stulik, Yu-Xiong Su, Zhaoliang Su, Carlos S Subauste, Srinivasa Subramaniam, Carolyn M Sue, Sang Won Suh, Xinbing Sui, Supawadee Sukseree, David Sulzer, Fang-Lin Sun, Jiaren Sun, Jun Sun, Shi-Yong Sun, Yang Sun, Yi Sun, Yingjie Sun, Vinod Sundaramoorthy, Joseph Sung, Hidekazu Suzuki, Kuninori Suzuki, Naoki Suzuki, Tadashi Suzuki, Yuichiro J Suzuki, Michele S Swanson, Charles Swanton, Karl Swärd, Ghanshyam Swarup, Sean T Sweeney, Paul W Sylvester, Zsuzsanna Szatmari, Eva Szegezdi, Peter W Szlosarek, Heinrich Taegtmeyer, Marco Tafani, Emmanuel Taillebourg, Stephen Wg Tait, Krisztina Takacs-Vellai, Yoshinori Takahashi, Szabolcs Takáts, Genzou Takemura, Nagio Takigawa, Nicholas J Talbot, Elena Tamagno, Jerome Tamburini, Cai-Ping Tan, Lan Tan, Mei Lan Tan, Ming Tan, Yee-Joo Tan, Keiji Tanaka, Masaki Tanaka, Daolin Tang, Dingzhong Tang, Guomei Tang, Isei Tanida, Kunikazu Tanji, Bakhos A Tannous, Jose A Tapia, Inmaculada Tasset-Cuevas, Marc Tatar, Iman Tavassoly, Nektarios Tavernarakis, Allen Taylor, Graham S Taylor, Gregory A Taylor, J Paul Taylor, Mark J Taylor, Elena V Tchetina, Andrew R Tee, Fatima Teixeira-Clerc, Sucheta Telang, Tewin Tencomnao, Ba-Bie Teng, Ru-Jeng Teng, Faraj Terro, Gianluca Tettamanti, Arianne L Theiss, Anne E Theron, Kelly Jean Thomas, Marcos P Thomé, Paul G Thomes, Andrew Thorburn, Jeremy Thorner, Thomas Thum, Michael Thumm, Teresa Lm Thurston, Ling Tian, Andreas Till, Jenny Pan-Yun Ting, Vladimir I Titorenko, Lilach Toker, Stefano Toldo, Sharon A Tooze, Ivan Topisirovic, Maria Lyngaas Torgersen, Liliana Torosantucci, Alicia Torriglia, Maria Rosaria Torrisi, Cathy Tournier, Roberto Towns, Vladimir Trajkovic, Leonardo H Travassos, Gemma Triola, Durga Nand Tripathi, Daniela Trisciuoglio, Rodrigo Troncoso, Ioannis P Trougakos, Anita C Truttmann, Kuen-Jer Tsai, Mario P Tschan, Yi-Hsin Tseng, Takayuki Tsukuba, Allan Tsung, Andrey S Tsvetkov, Shuiping Tu, Hsing-Yu Tuan, Marco Tucci, David A Tumbarello, Boris Turk, Vito Turk, Robin Fb Turner, Anders A Tveita, Suresh C Tyagi, Makoto Ubukata, Yasuo Uchiyama, Andrej Udelnow, Takashi Ueno, Midori Umekawa, Rika Umemiya-Shirafuji, Benjamin R Underwood, Christian Ungermann, Rodrigo P Ureshino, Ryo Ushioda, Vladimir N Uversky, Néstor L Uzcátegui, Thomas Vaccari, Maria I Vaccaro, Libuše Váchová, Helin Vakifahmetoglu-Norberg, Rut Valdor, Enza Maria Valente, Francois Vallette, Angela M Valverde, Greet Van den Berghe, Ludo Van Den Bosch, Gijs R van den Brink, F Gisou van der Goot, Ida J van der Klei, Luc Jw van der Laan, Wouter G van Doorn, Marjolein van Egmond, Kenneth L van Golen, Luc Van Kaer, Menno van Lookeren Campagne, Peter Vandenabeele, Wim Vandenberghe, Ilse Vanhorebeek, Isabel Varela-Nieto, M Helena Vasconcelos, Radovan Vasko, Demetrios G Vavvas, Ignacio Vega-Naredo, Guillermo Velasco, Athanassios D Velentzas, Panagiotis D Velentzas, Tibor Vellai, Edo Vellenga, Mikkel Holm Vendelbo, Kartik Venkatachalam, Natascia Ventura, Salvador Ventura, Patrícia St Veras, Mireille Verdier, Beata G Vertessy, Andrea Viale, Michel Vidal, Helena L A Vieira, Richard D Vierstra, Nadarajah Vigneswaran, Neeraj Vij, Miquel Vila, Margarita Villar, Victor H Villar, Joan Villarroya, Cécile Vindis, Giampietro Viola, Maria Teresa Viscomi, Giovanni Vitale, Dan T Vogl, Olga V Voitsekhovskaja, Clarissa von Haefen, Karin von Schwarzenberg, Daniel E Voth, Valérie Vouret-Craviari, Kristina Vuori, Jatin M Vyas, Christian Waeber, Cheryl Lyn Walker, Mark J Walker, Jochen Walter, Lei Wan, Xiangbo Wan, Bo Wang, Caihong Wang, Chao-Yung Wang, Chengshu Wang, Chenran Wang, Chuangui Wang, Dong Wang, Fen Wang, Fuxin Wang, Guanghui Wang, Hai-Jie Wang, Haichao Wang, Hong-Gang Wang, Hongmin Wang, Horng-Dar Wang, Jing Wang, Junjun Wang, Mei Wang, Mei-Qing Wang, Pei-Yu Wang, Peng Wang, Richard C Wang, Shuo Wang, Ting-Fang Wang, Xian Wang, Xiao-Jia Wang, Xiao-Wei Wang, Xin Wang, Xuejun Wang, Yan Wang, Yanming Wang, Ying Wang, Ying-Jan Wang, Yipeng Wang, Yu Wang, Yu Tian Wang, Yuqing Wang, Zhi-Nong Wang, Pablo Wappner, Carl Ward, Diane McVey Ward, Gary Warnes, Hirotaka Watada, Yoshihisa Watanabe, Kei Watase, Timothy E Weaver, Colin D Weekes, Jiwu Wei, Thomas Weide, Conrad C Weihl, Günther Weindl, Simone Nardin Weis, Longping Wen, Xin Wen, Yunfei Wen, Benedikt Westermann, Cornelia M Weyand, Anthony R White, Eileen White, J Lindsay Whitton, Alexander J Whitworth, Joëlle Wiels, Franziska Wild, Manon E Wildenberg, Tom Wileman, Deepti Srinivas Wilkinson, Simon Wilkinson, Dieter Willbold, Chris Williams, Katherine Williams, Peter R Williamson, Konstanze F Winklhofer, Steven S Witkin, Stephanie E Wohlgemuth, Thomas Wollert, Ernst J Wolvetang, Esther Wong, G William Wong, Richard W Wong, Vincent Kam Wai Wong, Elizabeth A Woodcock, Karen L Wright, Chunlai Wu, Defeng Wu, Gen Sheng Wu, Jian Wu, Junfang Wu, Mian Wu, Min Wu, Shengzhou Wu, William Kk Wu, Yaohua Wu, Zhenlong Wu, Cristina Pr Xavier, Ramnik J Xavier, Gui-Xian Xia, Tian Xia, Weiliang Xia, Yong Xia, Hengyi Xiao, Jian Xiao, Shi Xiao, Wuhan Xiao, Chuan-Ming Xie, Zhiping Xie, Zhonglin Xie, Maria Xilouri, Yuyan Xiong, Chuanshan Xu, Congfeng Xu, Feng Xu, Haoxing Xu, Hongwei Xu, Jian Xu, Jianzhen Xu, Jinxian Xu, Liang Xu, Xiaolei Xu, Yangqing Xu, Ye Xu, Zhi-Xiang Xu, Ziheng Xu, Yu Xue, Takahiro Yamada, Ai Yamamoto, Koji Yamanaka, Shunhei Yamashina, Shigeko Yamashiro, Bing Yan, Bo Yan, Xianghua Yan, Zhen Yan, Yasuo Yanagi, Dun-Sheng Yang, Jin-Ming Yang, Liu Yang, Minghua Yang, Pei-Ming Yang, Peixin Yang, Qian Yang, Wannian Yang, Wei Yuan Yang, Xuesong Yang, Yi Yang, Ying Yang, Zhifen Yang, Zhihong Yang, Meng-Chao Yao, Pamela J Yao, Xiaofeng Yao, Zhenyu Yao, Zhiyuan Yao, Linda S Yasui, Mingxiang Ye, Barry Yedvobnick, Behzad Yeganeh, Elizabeth S Yeh, Patricia L Yeyati, Fan Yi, Long Yi, Xiao-Ming Yin, Calvin K Yip, Yeong-Min Yoo, Young Hyun Yoo, Seung-Yong Yoon, Ken-Ichi Yoshida, Tamotsu Yoshimori, Ken H Young, Huixin Yu, Jane J Yu, Jin-Tai Yu, Jun Yu, Li Yu, W Haung Yu, Xiao-Fang Yu, Zhengping Yu, Junying Yuan, Zhi-Min Yuan, Beatrice Yjt Yue, Jianbo Yue, Zhenyu Yue, David N Zacks, Eldad Zacksenhaus, Nadia Zaffaroni, Tania Zaglia, Zahra Zakeri, Vincent Zecchini, Jinsheng Zeng, Min Zeng, Qi Zeng, Antonis S Zervos, Donna D Zhang, Fan Zhang, Guo Zhang, Guo-Chang Zhang, Hao Zhang, Hong Zhang, Hong Zhang, Hongbing Zhang, Jian Zhang, Jian Zhang, Jiangwei Zhang, Jianhua Zhang, Jing-Pu Zhang, Li Zhang, Lin Zhang, Lin Zhang, Long Zhang, Ming-Yong Zhang, Xiangnan Zhang, Xu Dong Zhang, Yan Zhang, Yang Zhang, Yanjin Zhang, Yingmei Zhang, Yunjiao Zhang, Mei Zhao, Wei-Li Zhao, Xiaonan Zhao, Yan G Zhao, Ying Zhao, Yongchao Zhao, Yu-Xia Zhao, Zhendong Zhao, Zhizhuang J Zhao, Dexian Zheng, Xi-Long Zheng, Xiaoxiang Zheng, Boris Zhivotovsky, Qing Zhong, Guang-Zhou Zhou, Guofei Zhou, Huiping Zhou, Shu-Feng Zhou, Xu-Jie Zhou, Hongxin Zhu, Hua Zhu, Wei-Guo Zhu, Wenhua Zhu, Xiao-Feng Zhu, Yuhua Zhu, Shi-Mei Zhuang, Xiaohong Zhuang, Elio Ziparo, Christos E Zois, Teresa Zoladek, Wei-Xing Zong, Antonio Zorzano, Susu M Zughaier
Autophagy, 12, 1, 1, 222, 2016年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.
Yoshinaga T, Uwabe K, Naito S, Higashino K, Nakano T, Numata Y, Kihara A
PloS one, 11, 12, e0167848, Public Library of Science ({PLoS}), 2016年, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition: INSIGHTS INTO SENSING MECHANISMS FOR PLASMA MEMBRANE LIPID ASYMMETRY.
Nishino K, Obara K, Kihara A
The Journal of biological chemistry, 290, 52, 30797, 805, 2015年12月25日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an "antenna hypothesis," in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.

Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains.
Sugimoto M, Shimizu Y, Yoshioka T, Wakabayashi M, Tanaka Y, Higashino K, Numata Y, Sakai S, Kihara A, Igarashi Y, Kuge Y
Biochimica et biophysica acta, 1851, 12, 1554, 65, 2015年12月, ［査読有り］, ［国際誌］
英語, Sphingomyelin (SM) is synthesized by SM synthase (SMS) from ceramide (Cer). SM regulates signaling pathways and maintains organ structure. SM comprises a sphingoid base and differing lengths of acyl-chains, but the importance of its various forms and regulatory synthases is not known. It has been reported that Cer synthase (CerS) has restricted substrate specificity, whereas SMS has no specificity for different lengths of acyl-chains. We hypothesized that the distribution of each SM molecular species was regulated by expression of the CerS family. Thus, we compared the distribution of SM species and CerS mRNA expression using molecular imaging. Spatial distribution of each SM molecular species was investigated using ultra-high-resolution imaging mass spectrometry (IMS). IMS revealed that distribution of SM molecular species varied according to the lengths of acyl-chains found in each brain section. Furthermore, a combination study using in situ hybridization and IMS revealed the spatial expression of CerS1 to be associated with the localization of SM (d18:1/18:0) in cell body-rich gray matter, and CerS2 to be associated with SM (d18:1/24:1) in myelin-rich white matter. Our study is the first comparison of spatial distribution between SM molecular species and CerS isoforms, and revealed their distinct association in the brain. These observations were demonstrated by suppression of CerS2 using siRNA in HepG2 cells; that is, siRNA for CerS2 specifically decreased C22 very long-chain fatty acid (VLCFA)- and C24 VLCFA-containing SMs. Thus, histological analyses of SM species by IMS could be a useful approach to consider their molecular function and regulative mechanism.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.
Blondelle J, Ohno Y, Gache V, Guyot S, Storck S, Blanchard-Gutton N, Barthélémy I, Walmsley G, Rahier A, Gadin S, Maurer M, Guillaud L, Prola A, Ferry A, Aubin-Houzelstein G, Demarquoy J, Relaix F, Piercy RJ, Blot S, Kihara A, Tiret L, Pilot-Storck F
Journal of molecular cell biology, 7, 5, 429, 40, 2015年10月, ［査読有り］, ［国際誌］
英語, The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide, the key lipid for skin permeability barrier formation.
Ohno Y, Nakamichi S, Ohkuni A, Kamiyama N, Naoe A, Tsujimura H, Yokose U, Sugiura K, Ishikawa J, Akiyama M, Kihara A
Proceedings of the National Academy of Sciences of the United States of America, 112, 25, 7707, 12, 2015年06月23日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.

Mouse aldehyde dehydrogenase ALDH3B2 is localized to lipid droplets via two C-terminal tryptophan residues and lipid modification.
Kitamura T, Takagi S, Naganuma T, Kihara A
The Biochemical journal, 465, 1, 79, 87, 2015年01月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Aldehyde dehydrogenases (ALDHs) catalyse the conversion of toxic aldehydes into non-toxic carboxylic acids. Of the 21 ALDHs in mice, it is the ALDH3 family members (ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2 and ALDH3B3) that are responsible for the removal of lipid-derived aldehydes. However, ALDH3B2 and ALDH3B3 have yet to be characterized. In the present study, we examined the enzyme activity, tissue distribution and subcellular localization of ALDH3B2 and ALDH3B3. Both were found to exhibit broad substrate preferences from medium- to long-chain aldehydes, resembling ALDH3A2 and ALDH3B1. Although ALDH3B2 and ALDH3B3 share extremely high sequence similarity, their localizations differ, with ALDH3B2 found in lipid droplets and ALDH3B3 localized to the plasma membrane. Both were modified by prenylation at their C-termini; this modification greatly influenced their membrane localization and enzymatic activity towards hexadecanal. We found that their C-terminal regions, particularly the two tryptophan residues (Trp462 and Trp469) of ALDH3B2 and the two arginine residues (Arg462 and Arg463) of ALDH3B3, were important for the determination of their specific localization. Abnormal quantity and perhaps quality of lipid droplets are implicated in several metabolic diseases. We speculate that ALDH3B2 acts to remove lipid-derived aldehydes in lipid droplets generated via oxidative stress as a quality control mechanism.

A novel factor OPT2 mediates exposure of phospholipids during cellular adaptation to altered lipid asymmetry.
Yamauchi S, Obara K, Uchibori K, Kamimura A, Azumi K, Kihara A
Journal of cell science, 128, 1, 61, 9, 2015年01月01日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Plasma membrane lipid asymmetry is important for various membrane-associated functions and is regulated by membrane proteins termed flippases and floppases. The Rim101 pathway senses altered lipid asymmetry in the yeast plasma membrane. The mutant lem3Δ cells, in which lipid asymmetry is disturbed owing to the inactivation of the plasma membrane flippases, showed a severe growth defect when the Rim101 pathway was impaired. To identify factors involved in the Rim101-pathway-dependent adaptation to altered lipid asymmetry, we performed DNA microarray analysis and found that Opt2 induced by the Rim101 pathway plays an important role in the adaptation to altered lipid asymmetry. Biochemical investigation of Opt2 revealed its localization to the plasma membrane and the Golgi, and provided several lines of evidence for the Opt2-mediated exposure of phospholipids. In addition, Opt2 was found to be required for the maintenance of vacuolar morphology and polarized cell growth. These results suggest that Opt2 is a novel factor involved in cell homeostasis by regulating lipid asymmetry.

Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids.
Kondo N, Ohno Y, Yamagata M, Obara T, Seki N, Kitamura T, Naganuma T, Kihara A
Nature communications, 5, 5338, 5338, 2014年10月27日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The long-chain base phytosphingosine is a component of sphingolipids and exists in yeast, plants and some mammalian tissues. Phytosphingosine is unique in that it possesses an additional hydroxyl group compared with other long-chain bases. However, its metabolism is unknown. Here we show that phytosphingosine is metabolized to odd-numbered fatty acids and is incorporated into glycerophospholipids both in yeast and mammalian cells. Disruption of the yeast gene encoding long-chain base 1-phosphate lyase, which catalyzes the committed step in the metabolism of phytosphingosine to glycerophospholipids, causes an ~40% reduction in the level of phosphatidylcholines that contain a C15 fatty acid. We also find that 2-hydroxypalmitic acid is an intermediate of the phytosphingosine metabolic pathway. Furthermore, we show that the yeast MPO1 gene, whose product belongs to a large, conserved protein family of unknown function, is involved in phytosphingosine metabolism. Our findings provide insights into fatty acid diversity and identify a pathway by which hydroxyl group-containing lipids are metabolized.

Signaling events of the Rim101 pathway occur at the plasma membrane in a ubiquitination-dependent manner.
Obara K, Kihara A
Molecular and cellular biology, 34, 18, 3525, 34, 18, 2014年09月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, In yeast, external alkalization and alteration in plasma membrane lipid asymmetry are sensed by the Rim101 pathway. It is currently under debate whether the signal elicited by external alkalization is transduced to downstream molecules at the plasma membrane or via endocytosis of the Rim21 sensor protein at the late endosome. We found that the downstream molecules, including arrestin-related protein Rim8, calpain-like protein Rim13, and scaffold protein Rim20, accumulated at the plasma membrane upon external alkalization and that the accumulation was dependent on Rim21. Snf7, an endosomal sorting complex required for transport (ESCRT) III subunit also essential for the Rim101 pathway, localized to the plasma membrane, in addition to the late endosome, under alkaline conditions. Snf7 at the plasma membrane but not at the late endosome was shown to be involved in Rim101 signaling. In addition, the Rim101 pathway was normally activated, even when endocytosis was severely impaired. Considering this information as a whole, we propose that Rim101 signaling proceeds at the plasma membrane. We also found that activity of the Rsp5 ubiquitin ligase was required for recruiting the downstream molecules to the plasma membrane, suggesting that ubiquitination mediates Rim101 signaling at the plasma membrane.

Dual functions of the trans-2-enoyl-CoA reductase TER in the sphingosine 1-phosphate metabolic pathway and in fatty acid elongation.
Wakashima T, Abe K, Kihara A
The Journal of biological chemistry, 289, 36, 24736, 48, 36, 2014年09月05日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P.

Lorenzo's oil inhibits ELOVL1 and lowers the level of sphingomyelin with a saturated very long-chain fatty acid.
Sassa T, Wakashima T, Ohno Y, Kihara A
Journal of lipid research, 55, 3, 524, 30, 3, 2014年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by impaired degradation of very long-chain fatty acids (VLCFAs) due to mutations in the ABCD1 gene responsible for VLCFA transport into peroxisomes. Lorenzo's oil, a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, has been used to reduce the saturated VLCFA level in the plasma of X-ALD patients; however, the mechanism by which this occurs remains elusive. We report the biochemical characterization of Lorenzo's oil activity toward elongation of very long-chain fatty acid (ELOVL) 1, the primary enzyme responsible for the synthesis of saturated and monounsaturated VLCFAs. Oleic and erucic acids inhibited ELOVL1, and, moreover, their 4:1 mixture (the FA composition of Lorenzo's oil) exhibited the most potent inhibitory activity. The kinetics analysis revealed that this was a mixed (not a competitive) inhibition. At the cellular level, treatment with the 4:1 mixture reduced the level of SM with a saturated VLCFA accompanied by an increased level of SM with a monounsaturated VLCFA, probably due to the incorporation of erucic acid into the FA elongation cycle. These results suggest that inhibition of ELOVL1 may be an underlying mechanism by which Lorenzo's oil exerts its action.

Integrin α9 on lymphatic endothelial cells regulates lymphocyte egress.
Ito K, Morimoto J, Kihara A, Matsui Y, Kurotaki D, Kanayama M, Simmons S, Ishii M, Sheppard D, Takaoka A, Uede T
Proceedings of the National Academy of Sciences of the United States of America, 111, 8, 3080, 5, 8, 2014年02月25日, ［査読有り］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) plays a role in lymphocyte egress from lymphoid organs. However, it remains unclear how S1P production and secretion are regulated. We show that under inflammatory conditions, α9 integrin, which is closely associated with activated β1 integrin, and its ligand, tenascin-C, colocalize on medullary and cortical sinuses of draining lymph nodes (dLNs), which is a gate for lymphocyte exit, and that inhibition of lymphocyte egress is evident by blockade of α9 integrin-mediated signaling at dLNs. Based on in vitro analysis using lymphatic endothelial cells obtained from mice embryos, we suggested the possibility that stimulation of lymphatic endothelial cells by tenascin-C enhances S1P secretion in an α9 integrin-dependent manner without affecting S1P synthesis and/or degradation. Blockade of α9 integrin-mediated signaling reduced lymphocyte egress from dLNs in several models, including experimental autoimmune encephalomyelitis, where it improved clinical scores and pathology. Therefore, manipulating α9 integrin function may offer a therapeutic strategy for treating various inflammatory disorders.

Two modes of regulation of the fatty acid elongase ELOVL6 by the 3-ketoacyl-CoA reductase KAR in the fatty acid elongation cycle.
Naganuma T, Kihara A
PloS one, 9, 7, e101823, 7, 2014年, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Fatty acids (FAs) are diverse molecules, and such diversity is important for lipids to exert their functions under several environmental conditions. FA elongation occurs at the endoplasmic reticulum and produces a variety of FA species; the FA elongation cycle consists of four distinct enzyme reactions. For this cycle to be driven efficiently, there must exist coordinated regulation of protein components of the FA elongation machinery. However, such regulation is poorly understood. In the present study, we performed biochemical analyses using the FA elongase ELOVL6 and the 3-ketoacyl-CoA reductase KAR, which catalyze the first and second steps of the FA elongation cycle, respectively. In vitro FA elongation assays using membrane fractions demonstrated that ELOVL6 activity was enhanced ∼10-fold in the presence of NADPH, although ELOVL6 itself did not require NADPH for its catalysis. On the other hand, KAR does use NADPH as a reductant in its enzyme reaction. Activity of purified ELOVL6 was enhanced by ∼3-fold in the presence of KAR. This effect was KAR enzyme activity-independent, since it was observed in the absence of NADPH and in the KAR mutant. However, ELOVL6 enzyme activity was further enhanced in a KAR enzyme activity-dependent manner. Therefore, KAR regulates ELOVL6 via two modes. In the first mode, KAR may induce conformational changes in ELOVL6 to become structure that can undergo catalysis. In the second mode, conversion of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA by KAR may facilitate release of the product from the presumed ELOVL6-KAR complex.

Phs1 and the synthesis of very long chain fatty acids are required for ballistospore formation.
Ianiri G, Abhyankar R, Kihara A, Idnurm A
PloS one, 9, 8, e105147, 8, 2014年, ［査読有り］, ［国際誌］
英語, The production and dissemination of spores by members of the fungal kingdom is a major reason for the success of this eukaryotic lineage in colonizing most terrestrial ecosystems. Ballistospores are a type of spore produced by basidiomycete fungi, such as the mushrooms and plant pathogenic rusts. These spores are forcefully discharged through a unique liquid-drop fusion mechanism, enabling the aerosolization of these particles that can contribute to plant disease and human allergies. The genes responsible for this process are unknown due to technical challenges in studying many of the fungi that produce ballistospores. Here, we applied newly-developed techniques in a forward genetic screen to identify genes required for ballistospore formation or function in a tractable red yeast, a species of Sporobolomyces. One strain bearing a mutation in the PHS1 gene was identified as a mirror mutant. PHS1 encodes 3-hydroxyacyl-CoA dehydratase required for the third step in very long chain fatty acid biosynthesis. The Sporobolomyces PHS1 gene complements the essential functions of a S. cerevisiae phs1 mutant. The Sporobolomyces phs1 mutant strain has less dehydratase activity and a reduction in very long chain fatty acids compared to wild type. The mutant strain also exhibits sensitivity to cell wall stress agents and loss of shooting due to a delay in ballistospore formation, indicating that the role of Phs1 in spore dissemination may be primarily in cellular integrity.

Congenital myopathy is caused by mutation of HACD1.
Muhammad E, Reish O, Ohno Y, Scheetz T, Deluca A, Searby C, Regev M, Benyamini L, Fellig Y, Kihara A, Sheffield VC, Parvari R
Human molecular genetics, 22, 25, 5229, 36, 25, 2013年12月20日, ［査読有り］, ［国際誌］
英語, Congenital myopathies are heterogeneous inherited diseases of muscle characterized by a range of distinctive histologic abnormalities. We have studied a consanguineous family with congenital myopathy. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous non-sense mutation in 3-hydroxyacyl-CoA dehydratase 1 (HACD1) in affected individuals. The mutation results in non-sense mediated decay of the HACD1 mRNA to 31% of control levels in patient muscle and completely abrogates the enzymatic activity of dehydration of 3-hydroxyacyl-CoA, the third step in the elongation of very long-chain fatty acids (VLCFAs). We describe clinical findings correlated with a deleterious mutation in a gene not previously known to be associated with congenital myopathy in humans. We suggest that the mutation in the HACD1 gene causes a reduction in the synthesis of VLCFAs, which are components of membrane lipids and participants in physiological processes, leading to congenital myopathy. These data indicate that HACD1 is necessary for muscle function.

Mutation for nonsyndromic mental retardation in the trans-2-enoyl-CoA reductase TER gene involved in fatty acid elongation impairs the enzyme activity and stability, leading to change in sphingolipid profile.
Abe K, Ohno Y, Sassa T, Taguchi R, Çalışkan M, Ober C, Kihara A
The Journal of biological chemistry, 288, 51, 36741, 9, 51, 2013年12月20日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TER(P182L/P182L) B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TER(P182L/P182L) B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.

Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway.
Ohkuni A, Ohno Y, Kihara A
Biochemical and biophysical research communications, 442, 3-4, 195, 201, 3-4, 2013年12月13日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in S1P metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where S1P metabolism takes place. We previously proposed the entire S1P metabolic pathway from results obtained using yeast cells, i.e., S1P is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as S1P is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that S1P is metabolized by a common pathway in eukaryotes.

Unperverted synthesis of complex sphingolipids is essential for cell survival under nitrogen starvation.
Yamagata M, Obara K, Kihara A
Genes to cells, 18, 8, 650, 9, 8, 2013年08月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Changes in membrane dynamics are known to occur in cells faced with starvation. However, the functions of the major lipid components of biological membranes, sphingolipids, during the starvation response remain unclear. In this study, we found that yeast cells lacking genes encoding mannosylinositol phosphorylceramide (MIPC) synthases (csg1Δ csh1Δ) underwent rapid cell death upon nitrogen starvation, but not upon carbon starvation or carbon and nitrogen starvation. Addition of NaN3 prevented the nitrogen starvation-induced cell death of the csg1Δ csh1Δ cells, indicating that energy production is required for this rapid cell death. The cell death was caused by an accumulation of inositol phosphorylceramide (IPC) species containing phytosphingosine. Removing Ca(2+) by treating the cells with a calcium chelator or by changing the medium to a Ca(2+) -free medium before nitrogen starvation rescued the cells from death. Approximately half of the cells died shortly after collapse of the vacuole, whereas in the other half, morphological changes in the cytoplasm preceded vacuole disruption. Because the vacuole is the major Ca(2+) storage organelle, we suggest that the vacuole is involved in the cell death either directly or indirectly. We report here that normal synthesis of complex sphingolipids is important for cell survival in nitrogen-starved medium.

Substrate specificity, plasma membrane localization, and lipid modification of the aldehyde dehydrogenase ALDH3B1.
Kitamura T, Naganuma T, Abe K, Nakahara K, Ohno Y, Kihara A
Biochimica et biophysica acta, 1831, 8, 1395, 401, 8, 2013年08月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum.

Impaired epidermal permeability barrier in mice lacking Elovl1, the gene responsible for very-long-chain fatty acid production.
Sassa T, Ohno Y, Suzuki S, Nomura T, Nishioka C, Kashiwagi T, Hirayama T, Akiyama M, Taguchi R, Shimizu H, Itohara S, Kihara A
Molecular and cellular biology, 33, 14, 2787, 96, 14, 2013年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C20 to C28 FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C26 FAs were decreased, while those of Cers with ≤C24 FAs were increased. In contrast, the levels of C24 sphingomyelin were reduced, accompanied by an increase in C20 sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.

Identification of residues important for the catalysis, structure maintenance, and substrate specificity of yeast 3-hydroxyacyl-CoA dehydratase Phs1.
Yazawa T, Naganuma T, Yamagata M, Kihara A
FEBS letters, 587, 6, 804, 9, 6, 2013年03月18日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Yeast Phs1 is a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation. In the present study, we biochemically characterized Phs1 mutants with Ala-substitution at each of seven highly conserved amino-acid residues. All mutants exhibited reduced Phs1 activity. The E60A, Q79A, and R141A mutants were sensitive to digitonin, indicative of their reduced structural integrity. The fatty acid elongation cycle was greatly inhibited in the R83A, R141A, and G152A mutant membranes. The enzyme kinetics study implicated the direct involvement of the Arg83 and Gly152 residues in the catalytic process. The E60A mutation was found to affect the substrate specificity.

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids.
Obara K, Kojima R, Kihara A
Journal of lipid research, 54, 3, 831, 42, 3, 2013年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.

Cooperative synthesis of ultra long-chain fatty acid and ceramide during keratinocyte differentiation.
Mizutani Y, Sun H, Ohno Y, Sassa T, Wakashima T, Obara M, Yuyama K, Kihara A, Igarashi Y
PloS one, 8, 6, e67317, 6, 2013年, ［査読有り］, ［責任著者］, ［国際誌］
英語, The lipid lamellae in the stratum corneum is important for the epidermal permeability barrier. The lipid lamellae component ceramide (CER), comprising an ultra long-chain (ULC) fatty acid (FA) of ≥26 carbons (ULC CER), plays an essential role in barrier formation. ULC acyl-CoAs, produced by the FA elongase ELOVL4, are converted to ULC CERs by the CER synthase CERS3. In the presented study, we observed that ELOVL4 and CERS3 mRNAs increased during keratinocyte differentiation in vivo and in vitro. We also determined that peroxisome proliferator-activated receptor β/δ is involved in the up-regulation of the mRNAs. Knockdown of CERS3 caused a reduction in the elongase activities toward ULC acyl-CoAs, suggesting that CERS3 positively regulates ULCFA. Thus, we reveal that the two key players in ULC CER production in epidermis, CERS3 and ELOVL4, are coordinately regulated at both the transcriptional and enzymatic levels.

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system.
Ohno Y, Kashio A, Ogata R, Ishitomi A, Yamazaki Y, Kihara A
Molecular Biology of the Cell, 23, 23, 4543, 51, 23, 2012年12月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins.

Membrane protein Rim21 plays a central role in sensing ambient pH in Saccharomyces cerevisiae.
Obara K, Yamamoto H, Kihara A
The Journal of Biological Chemistry, 287, 46, 38473, 81, 46, 2012年11月09日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, External alkalization activates the Rim101 pathway in Saccharomyces cerevisiae. In this pathway, three integral membrane proteins, Rim21, Dfg16, and Rim9, are considered to be the components of the pH sensor machinery. However, how these proteins are involved in pH sensing is totally unknown. In this work, we investigated the localization, physical interaction, and interrelationship of Rim21, Dfg16, and Rim9. These proteins were found to form a complex and to localize to the plasma membrane in a patchy and mutually dependent manner. Their cellular level was also mutually dependent. In particular, the Rim21 level was significantly decreased in dfg16Δ and rim9Δ cells. Upon external alkalization, the proteins were internalized and degraded. We also demonstrate that the transient degradation of Rim21 completely suppressed the Rim101 pathway but that the degradation of Dfg16 or Rim9 did not. This finding strongly suggests that Rim21 is the pH sensor protein and that Dfg16 and Rim9 play auxiliary functions through maintaining the level of Rim21 and assisting in its plasma membrane localization. Even without external alkalization, the Rim101 pathway was activated in a Rim21-dependent manner by either protonophore treatment or depletion of phosphatidylserine in the inner leaflet of the plasma membrane, both of which caused plasma membrane depolarization like the external alkalization. Therefore, plasma membrane depolarization seems to be one of the key signals for the pH sensor molecule Rim21.

A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells.
Sassa T, Suto S, Okayasu Y, Kihara A
Biochimica et Biophysica Acta, 1821, 7, 1031, 7, 7, 2012年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.

Sphingolipids regulate the yeast high-osmolarity glycerol response pathway.
Tanigawa M, Kihara A, Terashima M, Takahara T, Maeda T
Molecular and Cellular Biology, 32, 14, 2861, 70, 14, 2012年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.

The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway.
Nakahara K, Ohkuni A, Kitamura T, Abe K, Naganuma T, Ohno Y, Zoeller RA, Kihara A
Molecular Cell, 46, 4, 461, 71, 4, 2012年05月25日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS.

Degradation of long-chain base 1-phosphate (LCBP) in Arabidopsis: functional characterization of LCBP phosphatase involved in the dehydration stress response.
Nakagawa N, Kato M, Takahashi Y, Shimazaki K, Tamura K, Tokuji Y, Kihara A, Imai H
Journal of Plant Research, 125, 3, 439, 49, 3, 2012年05月, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Sphingolipid metabolites, long-chain base 1-phosphates (LCBPs), are involved in ABA signaling pathways. The LCBPs synthesized by long-chain base kinase are dephosphorylated by LCBP phosphatase or degraded by LCBP lyase. Here we show that the At3g58490 gene encodes AtSPP1, a functional LCBP phosphatase. Transient expression of green fluorescent protein fusion in suspension-cultured Arabidopsis cells showed that AtSPP1 is localized in the endoplasmic reticulum. The level of dihydrosphingosine 1-phosphate was increased in loss-of-function mutants (spp1) compared with wild-type (WT) plants, suggesting a role of AtSPP1 in regulating LCBP levels. The rate of decrease in fresh weight of detached aerial parts was significantly slower in spp1 mutants than in WT plants. A stomatal closure bioassay showed that the stomata of spp1 mutants were more sensitive than the WT to ABA, suggesting that AtSPP1 is involved in guard cell signaling. However, spp1 mutants showed decreased sensitivity to ABA with respect to primary root growth but not to seed germination. The response to fumonisin B(1) did not differ between the WT and spp1 mutant. A significant decrease in AtDPL1 (LCBP lyase) transcripts in spp1 mutants was observed. We conclude that AtSPP1 is a functional LCBP phosphatase that may play a role in stomatal responses through LCBP-mediated ABA signaling.

Palmitoylated calnexin is a key component of the ribosome-translocon complex.
Lakkaraju AK, Abrami L, Lemmin T, Blaskovic S, Kunz B, Kihara A, Dal Peraro M, van der Goot FG
The EMBO Journal, 31, 7, 1823, 35, 7, 2012年04月04日, ［査読有り］, ［国際誌］
英語, A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.

Guidelines for the use and interpretation of assays for monitoring autophagy.
Daniel J Klionsky, Fabio C Abdalla, Hagai Abeliovich, Robert T Abraham, Abraham Acevedo-Arozena, Khosrow Adeli, Lotta Agholme, Maria Agnello, Patrizia Agostinis, Julio A Aguirre-Ghiso, Hyung Jun Ahn, Ouardia Ait-Mohamed, Slimane Ait-Si-Ali, Takahiko Akematsu, Shizuo Akira, Hesham M Al-Younes, Munir A Al-Zeer, Matthew L Albert, Roger L Albin, Javier Alegre-Abarrategui, Maria Francesca Aleo, Mehrdad Alirezaei, Alexandru Almasan, Maylin Almonte-Becerril, Atsuo Amano, Ravi Amaravadi, Shoba Amarnath, Amal O Amer, Nathalie Andrieu-Abadie, Vellareddy Anantharam, David K Ann, Shailendra Anoopkumar-Dukie, Hiroshi Aoki, Nadezda Apostolova, Giuseppe Arancia, John P Aris, Katsuhiko Asanuma, Nana Y O Asare, Hisashi Ashida, Valerie Askanas, David S Askew, Patrick Auberger, Misuzu Baba, Steven K Backues, Eric H Baehrecke, Ben A Bahr, Xue-Yuan Bai, Yannick Bailly, Robert Baiocchi, Giulia Baldini, Walter Balduini, Andrea Ballabio, Bruce A Bamber, Edward T W Bampton, Gábor Bánhegyi, Clinton R Bartholomew, Diane C Bassham, Robert C Bast Jr, Henri Batoko, Boon-Huat Bay, Isabelle Beau, Daniel M Béchet, Thomas J Begley, Christian Behl, Christian Behrends, Soumeya Bekri, Bryan Bellaire, Linda J Bendall, Luca Benetti, Laura Berliocchi, Henri Bernardi, Francesca Bernassola, Sébastien Besteiro, Ingrid Bhatia-Kissova, Xiaoning Bi, Martine Biard-Piechaczyk, Janice S Blum, Lawrence H Boise, Paolo Bonaldo, David L Boone, Beat C Bornhauser, Karina R Bortoluci, Ioannis Bossis, Frédéric Bost, Jean-Pierre Bourquin, Patricia Boya, Michaël Boyer-Guittaut, Peter V Bozhkov, Nathan R Brady, Claudio Brancolini, Andreas Brech, Jay E Brenman, Ana Brennand, Emery H Bresnick, Patrick Brest, Dave Bridges, Molly L Bristol, Paul S Brookes, Eric J Brown, John H Brumell, Nicola Brunetti-Pierri, Ulf T Brunk, Dennis E Bulman, Scott J Bultman, Geert Bultynck, Lena F Burbulla, Wilfried Bursch, Jonathan P Butchar, Wanda Buzgariu, Sergio P Bydlowski, Ken Cadwell, Monika Cahová, Dongsheng Cai, Jiyang Cai, Qian Cai, Bruno Calabretta, Javier Calvo-Garrido, Nadine Camougrand, Michelangelo Campanella, Jenny Campos-Salinas, Eleonora Candi, Lizhi Cao, Allan B Caplan, Simon R Carding, Sandra M Cardoso, Jennifer S Carew, Cathleen R Carlin, Virginie Carmignac, Leticia A M Carneiro, Serena Carra, Rosario A Caruso, Giorgio Casari, Caty Casas, Roberta Castino, Eduardo Cebollero, Francesco Cecconi, Jean Celli, Hassan Chaachouay, Han-Jung Chae, Chee-Yin Chai, David C Chan, Edmond Y Chan, Raymond Chuen-Chung Chang, Chi-Ming Che, Ching-Chow Chen, Guang-Chao Chen, Guo-Qiang Chen, Min Chen, Quan Chen, Steve S-L Chen, WenLi Chen, Xi Chen, Xiangmei Chen, Xiequn Chen, Ye-Guang Chen, Yingyu Chen, Yongqiang Chen, Yu-Jen Chen, Zhixiang Chen, Alan Cheng, Christopher H K Cheng, Yan Cheng, Heesun Cheong, Jae-Ho Cheong, Sara Cherry, Russ Chess-Williams, Zelda H Cheung, Eric Chevet, Hui-Ling Chiang, Roberto Chiarelli, Tomoki Chiba, Lih-Shen Chin, Shih-Hwa Chiou, Francis V Chisari, Chi Hin Cho, Dong-Hyung Cho, Augustine M K Choi, DooSeok Choi, Kyeong Sook Choi, Mary E Choi, Salem Chouaib, Divaker Choubey, Vinay Choubey, Charleen T Chu, Tsung-Hsien Chuang, Sheau-Huei Chueh, Taehoon Chun, Yong-Joon Chwae, Mee-Len Chye, Roberto Ciarcia, Maria R Ciriolo, Michael J Clague, Robert S B Clark, Peter G H Clarke, Robert Clarke, Patrice Codogno, Hilary A Coller, María I Colombo, Sergio Comincini, Maria Condello, Fabrizio Condorelli, Mark R Cookson, Graham H Coombs, Isabelle Coppens, Ramon Corbalan, Pascale Cossart, Paola Costelli, Safia Costes, Ana Coto-Montes, Eduardo Couve, Fraser P Coxon, James M Cregg, José L Crespo, Marianne J Cronjé, Ana Maria Cuervo, Joseph J Cullen, Mark J Czaja, Marcello D'Amelio, Arlette Darfeuille-Michaud, Lester M Davids, Faith E Davies, Massimo De Felici, John F de Groot, Cornelis A M de Haan, Luisa De Martino, Angelo De Milito, Vincenzo De Tata, Jayanta Debnath, Alexei Degterev, Benjamin Dehay, Lea M D Delbridge, Francesca Demarchi, Yi Zhen Deng, Jörn Dengjel, Paul Dent, Donna Denton, Vojo Deretic, Shyamal D Desai, Rodney J Devenish, Mario Di Gioacchino, Gilbert Di Paolo, Chiara Di Pietro, Guillermo Díaz-Araya, Inés Díaz-Laviada, Maria T Diaz-Meco, Javier Diaz-Nido, Ivan Dikic, Savithramma P Dinesh-Kumar, Wen-Xing Ding, Clark W Distelhorst, Abhinav Diwan, Mojgan Djavaheri-Mergny, Svetlana Dokudovskaya, Zheng Dong, Frank C Dorsey, Victor Dosenko, James J Dowling, Stephen Doxsey, Marlène Dreux, Mark E Drew, Qiuhong Duan, Michel A Duchosal, Karen Duff, Isabelle Dugail, Madeleine Durbeej, Michael Duszenko, Charles L Edelstein, Aimee L Edinger, Gustavo Egea, Ludwig Eichinger, N Tony Eissa, Suhendan Ekmekcioglu, Wafik S El-Deiry, Zvulun Elazar, Mohamed Elgendy, Lisa M Ellerby, Kai Er Eng, Anna-Mart Engelbrecht, Simone Engelender, Jekaterina Erenpreisa, Ricardo Escalante, Audrey Esclatine, Eeva-Liisa Eskelinen, Lucile Espert, Virginia Espina, Huizhou Fan, Jia Fan, Qi-Wen Fan, Zhen Fan, Shengyun Fang, Yongqi Fang, Manolis Fanto, Alessandro Fanzani, Thomas Farkas, Jean-Claude Farré, Mathias Faure, Marcus Fechheimer, Carl G Feng, Jian Feng, Qili Feng, Youji Feng, László Fésüs, Ralph Feuer, Maria E Figueiredo-Pereira, Gian Maria Fimia, Diane C Fingar, Steven Finkbeiner, Toren Finkel, Kim D Finley, Filomena Fiorito, Edward A Fisher, Paul B Fisher, Marc Flajolet, Maria L Florez-McClure, Salvatore Florio, Edward A Fon, Francesco Fornai, Franco Fortunato, Rati Fotedar, Daniel H Fowler, Howard S Fox, Rodrigo Franco, Lisa B Frankel, Marc Fransen, José M Fuentes, Juan Fueyo, Jun Fujii, Kozo Fujisaki, Eriko Fujita, Mitsunori Fukuda, Ruth H Furukawa, Matthias Gaestel, Philippe Gailly, Malgorzata Gajewska, Brigitte Galliot, Vincent Galy, Subramaniam Ganesh, Barry Ganetzky, Ian G Ganley, Fen-Biao Gao, George F Gao, Jinming Gao, Lorena Garcia, Guillermo Garcia-Manero, Mikel Garcia-Marcos, Marjan Garmyn, Andrei L Gartel, Evelina Gatti, Mathias Gautel, Thomas R Gawriluk, Matthew E Gegg, Jiefei Geng, Marc Germain, Jason E Gestwicki, David A Gewirtz, Saeid Ghavami, Pradipta Ghosh, Anna M Giammarioli, Alexandra N Giatromanolaki, Spencer B Gibson, Robert W Gilkerson, Michael L Ginger, Henry N Ginsberg, Jakub Golab, Michael S Goligorsky, Pierre Golstein, Candelaria Gomez-Manzano, Ebru Goncu, Céline Gongora, Claudio D Gonzalez, Ramon Gonzalez, Cristina González-Estévez, Rosa Ana González-Polo, Elena Gonzalez-Rey, Nikolai V Gorbunov, Sharon Gorski, Sandro Goruppi, Roberta A Gottlieb, Devrim Gozuacik, Giovanna Elvira Granato, Gary D Grant, Kim N Green, Aleš Gregorc, Frédéric Gros, Charles Grose, Thomas W Grunt, Philippe Gual, Jun-Lin Guan, Kun-Liang Guan, Sylvie M Guichard, Anna S Gukovskaya, Ilya Gukovsky, Jan Gunst, Asa B Gustafsson, Andrew J Halayko, Amber N Hale, Sandra K Halonen, Maho Hamasaki, Feng Han, Ting Han, Michael K Hancock, Malene Hansen, Hisashi Harada, Masaru Harada, Stefan E Hardt, J Wade Harper, Adrian L Harris, James Harris, Steven D Harris, Makoto Hashimoto, Jeffrey A Haspel, Shin-ichiro Hayashi, Lori A Hazelhurst, Congcong He, You-Wen He, Marie-Joseé Hébert, Kim A Heidenreich, Miep H Helfrich, Gudmundur V Helgason, Elizabeth P Henske, Brian Herman, Paul K Herman, Claudio Hetz, Sabine Hilfiker, Joseph A Hill, Lynne J Hocking, Paul Hofman, Thomas G Hofmann, Jörg Höhfeld, Tessa L Holyoake, Ming-Huang Hong, David A Hood, Gökhan S Hotamisligil, Ewout J Houwerzijl, Maria Høyer-Hansen, Bingren Hu, Chien-An A Hu, Hong-Ming Hu, Ya Hua, Canhua Huang, Ju Huang, Shengbing Huang, Wei-Pang Huang, Tobias B Huber, Won-Ki Huh, Tai-Ho Hung, Ted R Hupp, Gang Min Hur, James B Hurley, Sabah N A Hussain, Patrick J Hussey, Jung Jin Hwang, Seungmin Hwang, Atsuhiro Ichihara, Shirin Ilkhanizadeh, Ken Inoki, Takeshi Into, Valentina Iovane, Juan L Iovanna, Nancy Y Ip, Yoshitaka Isaka, Hiroyuki Ishida, Ciro Isidoro, Ken-ichi Isobe, Akiko Iwasaki, Marta Izquierdo, Yotaro Izumi, Panu M Jaakkola, Marja Jäättelä, George R Jackson, William T Jackson, Bassam Janji, Marina Jendrach, Ju-Hong Jeon, Eui-Bae Jeung, Hong Jiang, Hongchi Jiang, Jean X Jiang, Ming Jiang, Qing Jiang, Xuejun Jiang, Xuejun Jiang, Alberto Jiménez, Meiyan Jin, Shengkan Jin, Cheol O Joe, Terje Johansen, Daniel E Johnson, Gail V W Johnson, Nicola L Jones, Bertrand Joseph, Suresh K Joseph, Annie M Joubert, Gábor Juhász, Lucienne Juillerat-Jeanneret, Chang Hwa Jung, Yong-Keun Jung, Kai Kaarniranta, Allen Kaasik, Tomohiro Kabuta, Motoni Kadowaki, Katarina Kagedal, Yoshiaki Kamada, Vitaliy O Kaminskyy, Harm H Kampinga, Hiromitsu Kanamori, Chanhee Kang, Khong Bee Kang, Kwang Il Kang, Rui Kang, Yoon-A Kang, Tomotake Kanki, Thirumala-Devi Kanneganti, Haruo Kanno, Anumantha G Kanthasamy, Arthi Kanthasamy, Vassiliki Karantza, Gur P Kaushal, Susmita Kaushik, Yoshinori Kawazoe, Po-Yuan Ke, John H Kehrl, Ameeta Kelekar, Claus Kerkhoff, David H Kessel, Hany Khalil, Jan A K W Kiel, Amy A Kiger, Akio Kihara, Deok Ryong Kim, Do-Hyung Kim, Dong-Hou Kim, Eun-Kyoung Kim, Hyung-Ryong Kim, Jae-Sung Kim, Jeong Hun Kim, Jin Cheon Kim, John K Kim, Peter K Kim, Seong Who Kim, Yong-Sun Kim, Yonghyun Kim, Adi Kimchi, Alec C Kimmelman, Jason S King, Timothy J Kinsella, Vladimir Kirkin, Lorrie A Kirshenbaum, Katsuhiko Kitamoto, Kaio Kitazato, Ludger Klein, Walter T Klimecki, Jochen Klucken, Erwin Knecht, Ben C B Ko, Jan C Koch, Hiroshi Koga, Jae-Young Koh, Young Ho Koh, Masato Koike, Masaaki Komatsu, Eiki Kominami, Hee Jeong Kong, Wei-Jia Kong, Viktor I Korolchuk, Yaichiro Kotake, Michael I Koukourakis, Juan B Kouri Flores, Attila L Kovács, Claudine Kraft, Dimitri Krainc, Helmut Krämer, Carole Kretz-Remy, Anna M Krichevsky, Guido Kroemer, Rejko Krüger, Oleg Krut, Nicholas T Ktistakis, Chia-Yi Kuan, Roza Kucharczyk, Ashok Kumar, Raj Kumar, Sharad Kumar, Mondira Kundu, Hsing-Jien Kung, Tino Kurz, Ho Jeong Kwon, Albert R La Spada, Frank Lafont, Trond Lamark, Jacques Landry, Jon D Lane, Pierre Lapaquette, Jocelyn F Laporte, Lajos László, Sergio Lavandero, Josée N Lavoie, Robert Layfield, Pedro A Lazo, Weidong Le, Laurent Le Cam, Daniel J Ledbetter, Alvin J X Lee, Byung-Wan Lee, Gyun Min Lee, Jongdae Lee, Ju-Hyun Lee, Michael Lee, Myung-Shik Lee, Sug Hyung Lee, Christiaan Leeuwenburgh, Patrick Legembre, Renaud Legouis, Michael Lehmann, Huan-Yao Lei, Qun-Ying Lei, David A Leib, José Leiro, John J Lemasters, Antoinette Lemoine, Maciej S Lesniak, Dina Lev, Victor V Levenson, Beth Levine, Efrat Levy, Faqiang Li, Jun-Lin Li, Lian Li, Sheng Li, Weijie Li, Xue-Jun Li, Yan-bo Li, Yi-Ping Li, Chengyu Liang, Qiangrong Liang, Yung-Feng Liao, Pawel P Liberski, Andrew Lieberman, Hyunjung J Lim, Kah-Leong Lim, Kyu Lim, Chiou-Feng Lin, Fu-Cheng Lin, Jian Lin, Jiandie D Lin, Kui Lin, Wan-Wan Lin, Weei-Chin Lin, Yi-Ling Lin, Rafael Linden, Paul Lingor, Jennifer Lippincott-Schwartz, Michael P Lisanti, Paloma B Liton, Bo Liu, Chun-Feng Liu, Kaiyu Liu, Leyuan Liu, Qiong A Liu, Wei Liu, Young-Chau Liu, Yule Liu, Richard A Lockshin, Chun-Nam Lok, Sagar Lonial, Benjamin Loos, Gabriel Lopez-Berestein, Carlos López-Otín, Laura Lossi, Michael T Lotze, Peter Lőw, Binfeng Lu, Bingwei Lu, Bo Lu, Zhen Lu, Frédéric Luciano, Nicholas W Lukacs, Anders H Lund, Melinda A Lynch-Day, Yong Ma, Fernando Macian, Jeff P MacKeigan, Kay F Macleod, Frank Madeo, Luigi Maiuri, Maria Chiara Maiuri, Davide Malagoli, May Christine V Malicdan, Walter Malorni, Na Man, Eva-Maria Mandelkow, Stéphen Manon, Irena Manov, Kai Mao, Xiang Mao, Zixu Mao, Philippe Marambaud, Daniela Marazziti, Yves L Marcel, Katie Marchbank, Piero Marchetti, Stefan J Marciniak, Mateus Marcondes, Mohsen Mardi, Gabriella Marfe, Guillermo Mariño, Maria Markaki, Mark R Marten, Seamus J Martin, Camille Martinand-Mari, Wim Martinet, Marta Martinez-Vicente, Matilde Masini, Paola Matarrese, Saburo Matsuo, Raffaele Matteoni, Andreas Mayer, Nathalie M Mazure, David J McConkey, Melanie J McConnell, Catherine McDermott, Christine McDonald, Gerald M McInerney, Sharon L McKenna, BethAnn McLaughlin, Pamela J McLean, Christopher R McMaster, G Angus McQuibban, Alfred J Meijer, Miriam H Meisler, Alicia Meléndez, Thomas J Melia, Gerry Melino, Maria A Mena, Javier A Menendez, Rubem F S Menna-Barreto, Manoj B Menon, Fiona M Menzies, Carol A Mercer, Adalberto Merighi, Diane E Merry, Stefania Meschini, Christian G Meyer, Thomas F Meyer, Chao-Yu Miao, Jun-Ying Miao, Paul A M Michels, Carine Michiels, Dalibor Mijaljica, Ana Milojkovic, Saverio Minucci, Clelia Miracco, Cindy K Miranti, Ioannis Mitroulis, Keisuke Miyazawa, Noboru Mizushima, Baharia Mograbi, Simin Mohseni, Xavier Molero, Bertrand Mollereau, Faustino Mollinedo, Takashi Momoi, Iryna Monastyrska, Martha M Monick, Mervyn J Monteiro, Michael N Moore, Rodrigo Mora, Kevin Moreau, Paula I Moreira, Yuji Moriyasu, Jorge Moscat, Serge Mostowy, Jeremy C Mottram, Tomasz Motyl, Charbel E-H Moussa, Sylke Müller, Sylviane Muller, Karl Münger, Christian Münz, Leon O Murphy, Maureen E Murphy, Antonio Musarò, Indira Mysorekar, Eiichiro Nagata, Kazuhiro Nagata, Aimable Nahimana, Usha Nair, Toshiyuki Nakagawa, Kiichi Nakahira, Hiroyasu Nakano, Hitoshi Nakatogawa, Meera Nanjundan, Naweed I Naqvi, Derek P Narendra, Masashi Narita, Miguel Navarro, Steffan T Nawrocki, Taras Y Nazarko, Andriy Nemchenko, Mihai G Netea, Thomas P Neufeld, Paul A Ney, Ioannis P Nezis, Huu Phuc Nguyen, Daotai Nie, Ichizo Nishino, Corey Nislow, Ralph A Nixon, Takeshi Noda, Angelika A Noegel, Anna Nogalska, Satoru Noguchi, Lucia Notterpek, Ivana Novak, Tomoyoshi Nozaki, Nobuyuki Nukina, Thorsten Nürnberger, Beat Nyfeler, Keisuke Obara, Terry D Oberley, Salvatore Oddo, Michinaga Ogawa, Toya Ohashi, Koji Okamoto, Nancy L Oleinick, F Javier Oliver, Laura J Olsen, Stefan Olsson, Onya Opota, Timothy F Osborne, Gary K Ostrander, Kinya Otsu, Jing-hsiung James Ou, Mireille Ouimet, Michael Overholtzer, Bulent Ozpolat, Paolo Paganetti, Ugo Pagnini, Nicolas Pallet, Glen E Palmer, Camilla Palumbo, Tianhong Pan, Theocharis Panaretakis, Udai Bhan Pandey, Zuzana Papackova, Issidora Papassideri, Irmgard Paris, Junsoo Park, Ohkmae K Park, Jan B Parys, Katherine R Parzych, Susann Patschan, Cam Patterson, Sophie Pattingre, John M Pawelek, Jianxin Peng, David H Perlmutter, Ida Perrotta, George Perry, Shazib Pervaiz, Matthias Peter, Godefridus J Peters, Morten Petersen, Goran Petrovski, James M Phang, Mauro Piacentini, Philippe Pierre, Valérie Pierrefite-Carle, Gérard Pierron, Ronit Pinkas-Kramarski, Antonio Piras, Natik Piri, Leonidas C Platanias, Stefanie Pöggeler, Marc Poirot, Angelo Poletti, Christian Poüs, Mercedes Pozuelo-Rubio, Mette Prætorius-Ibba, Anil Prasad, Mark Prescott, Muriel Priault, Nathalie Produit-Zengaffinen, Ann Progulske-Fox, Tassula Proikas-Cezanne, Serge Przedborski, Karin Przyklenk, Rosa Puertollano, Julien Puyal, Shu-Bing Qian, Liang Qin, Zheng-Hong Qin, Susan E Quaggin, Nina Raben, Hannah Rabinowich, Simon W Rabkin, Irfan Rahman, Abdelhaq Rami, Georg Ramm, Glenn Randall, Felix Randow, V Ashutosh Rao, Jeffrey C Rathmell, Brinda Ravikumar, Swapan K Ray, Bruce H Reed, John C Reed, Fulvio Reggiori, Anne Régnier-Vigouroux, Andreas S Reichert, John J Reiners Jr, Russel J Reiter, Jun Ren, José L Revuelta, Christopher J Rhodes, Konstantinos Ritis, Elizete Rizzo, Jeffrey Robbins, Michel Roberge, Hernan Roca, Maria C Roccheri, Stephane Rocchi, H Peter Rodemann, Santiago Rodríguez de Córdoba, Bärbel Rohrer, Igor B Roninson, Kirill Rosen, Magdalena M Rost-Roszkowska, Mustapha Rouis, Kasper M A Rouschop, Francesca Rovetta, Brian P Rubin, David C Rubinsztein, Klaus Ruckdeschel, Edmund B Rucker 3rd, Assaf Rudich, Emil Rudolf, Nelson Ruiz-Opazo, Rossella Russo, Tor Erik Rusten, Kevin M Ryan, Stefan W Ryter, David M Sabatini, Junichi Sadoshima, Tapas Saha, Tatsuya Saitoh, Hiroshi Sakagami, Yasuyoshi Sakai, Ghasem Hoseini Salekdeh, Paolo Salomoni, Paul M Salvaterra, Guy Salvesen, Rosa Salvioli, Anthony M J Sanchez, José A Sánchez-Alcázar, Ricardo Sánchez-Prieto, Marco Sandri, Uma Sankar, Poonam Sansanwal, Laura Santambrogio, Shweta Saran, Sovan Sarkar, Minnie Sarwal, Chihiro Sasakawa, Ausra Sasnauskiene, Miklós Sass, Ken Sato, Miyuki Sato, Anthony H V Schapira, Michael Scharl, Hermann M Schätzl, Wiep Scheper, Stefano Schiaffino, Claudio Schneider, Marion E Schneider, Regine Schneider-Stock, Patricia V Schoenlein, Daniel F Schorderet, Christoph Schüller, Gary K Schwartz, Luca Scorrano, Linda Sealy, Per O Seglen, Juan Segura-Aguilar, Iban Seiliez, Oleksandr Seleverstov, Christian Sell, Jong Bok Seo, Duska Separovic, Vijayasaradhi Setaluri, Takao Setoguchi, Carmine Settembre, John J Shacka, Mala Shanmugam, Irving M Shapiro, Eitan Shaulian, Reuben J Shaw, James H Shelhamer, Han-Ming Shen, Wei-Chiang Shen, Zu-Hang Sheng, Yang Shi, Kenichi Shibuya, Yoshihiro Shidoji, Jeng-Jer Shieh, Chwen-Ming Shih, Yohta Shimada, Shigeomi Shimizu, Takahiro Shintani, Orian S Shirihai, Gordon C Shore, Andriy A Sibirny, Stan B Sidhu, Beata Sikorska, Elaine C M Silva-Zacarin, Alison Simmons, Anna Katharina Simon, Hans-Uwe Simon, Cristiano Simone, Anne Simonsen, David A Sinclair, Rajat Singh, Debasish Sinha, Frank A Sinicrope, Agnieszka Sirko, Parco M Siu, Efthimios Sivridis, Vojtech Skop, Vladimir P Skulachev, Ruth S Slack, Soraya S Smaili, Duncan R Smith, Maria S Soengas, Thierry Soldati, Xueqin Song, Anil K Sood, Tuck Wah Soong, Federica Sotgia, Stephen A Spector, Claudia D Spies, Wolfdieter Springer, Srinivasa M Srinivasula, Leonidas Stefanis, Joan S Steffan, Ruediger Stendel, Harald Stenmark, Anastasis Stephanou, Stephan T Stern, Cinthya Sternberg, Björn Stork, Peter Strålfors, Carlos S Subauste, Xinbing Sui, David Sulzer, Jiaren Sun, Shi-Yong Sun, Zhi-Jun Sun, Joseph J Y Sung, Kuninori Suzuki, Toshihiko Suzuki, Michele S Swanson, Charles Swanton, Sean T Sweeney, Lai-King Sy, Gyorgy Szabadkai, Ira Tabas, Heinrich Taegtmeyer, Marco Tafani, Krisztina Takács-Vellai, Yoshitaka Takano, Kaoru Takegawa, Genzou Takemura, Fumihiko Takeshita, Nicholas J Talbot, Kevin S W Tan, Keiji Tanaka, Kozo Tanaka, Daolin Tang, Dingzhong Tang, Isei Tanida, Bakhos A Tannous, Nektarios Tavernarakis, Graham S Taylor, Gregory A Taylor, J Paul Taylor, Lance S Terada, Alexei Terman, Gianluca Tettamanti, Karin Thevissen, Craig B Thompson, Andrew Thorburn, Michael Thumm, FengFeng Tian, Yuan Tian, Glauco Tocchini-Valentini, Aviva M Tolkovsky, Yasuhiko Tomino, Lars Tönges, Sharon A Tooze, Cathy Tournier, John Tower, Roberto Towns, Vladimir Trajkovic, Leonardo H Travassos, Ting-Fen Tsai, Mario P Tschan, Takeshi Tsubata, Allan Tsung, Boris Turk, Lorianne S Turner, Suresh C Tyagi, Yasuo Uchiyama, Takashi Ueno, Midori Umekawa, Rika Umemiya-Shirafuji, Vivek K Unni, Maria I Vaccaro, Enza Maria Valente, Greet Van den Berghe, Ida J van der Klei, Wouter van Doorn, Linda F van Dyk, Marjolein van Egmond, Leo A van Grunsven, Peter Vandenabeele, Wim P Vandenberghe, Ilse Vanhorebeek, Eva C Vaquero, Guillermo Velasco, Tibor Vellai, Jose Miguel Vicencio, Richard D Vierstra, Miquel Vila, Cécile Vindis, Giampietro Viola, Maria Teresa Viscomi, Olga V Voitsekhovskaja, Clarissa von Haefen, Marcela Votruba, Keiji Wada, Richard Wade-Martins, Cheryl L Walker, Craig M Walsh, Jochen Walter, Xiang-Bo Wan, Aimin Wang, Chenguang Wang, Dawei Wang, Fan Wang, Fen Wang, Guanghui Wang, Haichao Wang, Hong-Gang Wang, Horng-Dar Wang, Jin Wang, Ke Wang, Mei Wang, Richard C Wang, Xinglong Wang, Xuejun Wang, Ying-Jan Wang, Yipeng Wang, Zhen Wang, Zhigang Charles Wang, Zhinong Wang, Derick G Wansink, Diane M Ward, Hirotaka Watada, Sarah L Waters, Paul Webster, Lixin Wei, Conrad C Weihl, William A Weiss, Scott M Welford, Long-Ping Wen, Caroline A Whitehouse, J Lindsay Whitton, Alexander J Whitworth, Tom Wileman, John W Wiley, Simon Wilkinson, Dieter Willbold, Roger L Williams, Peter R Williamson, Bradly G Wouters, Chenghan Wu, Dao-Cheng Wu, William K K Wu, Andreas Wyttenbach, Ramnik J Xavier, Zhijun Xi, Pu Xia, Gengfu Xiao, Zhiping Xie, Zhonglin Xie, Da-zhi Xu, Jianzhen Xu, Liang Xu, Xiaolei Xu, Ai Yamamoto, Akitsugu Yamamoto, Shunhei Yamashina, Michiaki Yamashita, Xianghua Yan, Mitsuhiro Yanagida, Dun-Sheng Yang, Elizabeth Yang, Jin-Ming Yang, Shi Yu Yang, Wannian Yang, Wei Yuan Yang, Zhifen Yang, Meng-Chao Yao, Tso-Pang Yao, Behzad Yeganeh, Wei-Lien Yen, Jia-jing Yin, Xiao-Ming Yin, Ook-Joon Yoo, Gyesoon Yoon, Seung-Yong Yoon, Tomohiro Yorimitsu, Yuko Yoshikawa, Tamotsu Yoshimori, Kohki Yoshimoto, Ho Jin You, Richard J Youle, Anas Younes, Li Yu, Long Yu, Seong-Woon Yu, Wai Haung Yu, Zhi-Min Yuan, Zhenyu Yue, Cheol-Heui Yun, Michisuke Yuzaki, Olga Zabirnyk, Elaine Silva-Zacarin, David Zacks, Eldad Zacksenhaus, Nadia Zaffaroni, Zahra Zakeri, Herbert J Zeh 3rd, Scott O Zeitlin, Hong Zhang, Hui-Ling Zhang, Jianhua Zhang, Jing-Pu Zhang, Lin Zhang, Long Zhang, Ming-Yong Zhang, Xu Dong Zhang, Mantong Zhao, Yi-Fang Zhao, Ying Zhao, Zhizhuang J Zhao, Xiaoxiang Zheng, Boris Zhivotovsky, Qing Zhong, Cong-Zhao Zhou, Changlian Zhu, Wei-Guo Zhu, Xiao-Feng Zhu, Xiongwei Zhu, Yuangang Zhu, Teresa Zoladek, Wei-Xing Zong, Antonio Zorzano, Jürgen Zschocke, Brian Zuckerbraun
Autophagy, 8, 4, 445, 544, 2012年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Biochemical characterization of the very long-chain fatty acid elongase ELOVL7.
Naganuma T, Sato Y, Sassa T, Ohno Y, Kihara A
FEBS Letters, 585, 20, 3337, 41, 20, 2011年10月20日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated K(m) values toward C18:3(n-3)-CoA and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity.

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae.
Yamagata M, Obara K, Kihara A
Biochemical and Biophysical Research Communications, 410, 4, 786, 91, 4, 2011年07月15日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.

ケラチノサイトにおけるセラミド生合成とPPAR活性化剤によるその制御(Ceramide biosynthesis in keratinocyte and its regulation by PPAR activators)　　　　　　　　　　　　　　　
水谷 有紀子, 木原 章雄, 五十嵐 靖之
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 1P, 0040, (公社)日本生化学会, 2010年12月
英語

Hetero-oligomeric interactions of an ELOVL4 mutant protein: implications in the molecular mechanism of Stargardt-3 macular dystrophy.
Okuda A, Naganuma T, Ohno Y, Abe K, Yamagata M, Igarashi Y, Kihara A
Molecular Vision, 16, 2438, 45, 2010年11月18日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, PURPOSE: Stargardt disease 3 (STGD3) is a juvenile macular dystrophy caused by mutations in the elongase of very long-chain fatty acids-like 4 (ELOVL4) gene, which encodes an elongase involved in the production of extremely long-chain fatty acids. The STGD3-related mutations cause production of C-terminally truncated proteins (ELOVL4ΔC). STGD3 is transmitted in an autosomal dominant manner. To date, molecular mechanisms of this pathology have been proposed based solely on the interaction between wild-type ELOVL4 and ELOVL4ΔC. However, analyses of Elovl4ΔC knockin mice revealed reduced levels of not only ELOVL4 substrates, but also of fatty acids with a broad spectrum of chain lengths. Therefore, we investigated the molecular mechanisms responsible for ELOVL4ΔC affecting the entire very long-chain fatty acid (VLCFA) elongation pathway. METHODS: The ELOVL4ΔC protein was expressed in HEK 293T cells, and its effect on elongase activities toward several acyl-CoAs were examined. We also investigated the homo- and hetero-oligomerization of ELOVL4ΔC with other elongases (ELOVL1-7) or with other enzymes involved in VLCFA elongation using coimmunoprecipitation experiments. RESULTS: We found that ELOVL4ΔC forms a homo-oligomer more strongly than wild-type ELOVL4. ELOVL4ΔC also interacts strongly with other elongases, although similar interactions for wild-type ELOVL4 were observed as only weak. In addition, ELOVL4ΔC is able to form an elongase complex by interacting with other components of the VLCFA elongation machinery, similar to wild-type ELOVL4. CONCLUSIONS: We propose that not only the ELOVL4-ELOVL4ΔC homo-oligomeric interaction, but also several hetero-oligomeric interactions, may contribute to the pathology of STGD3.

Characterization of HACD1 K64Q mutant found in arrhythmogenic right ventricular dysplasia patients.
Konishi H, Okuda A, Ohno Y, Kihara A
Journal of Biochemistry, 148, 5, 617, 22, 5, 2010年11月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Arrhythmogenic right ventricular dysplasia (ARVD) is an autosomal dominant heart disease. A K64Q mutation was found in ARVD-affected individuals in the HACD1 gene, which encodes an enzyme involved in very long-chain fatty acid (VLCFA) elongation, although any relationship between mutation and pathology remained unclear. Here, we demonstrate that HACD1 (K64Q) exhibits normal enzyme activity, intracellular localization and interaction with other VLCFA enzymes, with no dominant negative effect on VLCFA elongation. Thus, it appears unlikely that this mutation is ARVD-causative. Moreover, through these analyses we found that HACD1 interacts with KAR and TER, the reductase enzymes involved in the second and fourth VLCFA elongation cycle, respectively. This finding indicates that the enzymes responsible for the VLCFA elongation cycle form an elongase complex.

ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis.
Ohno Y, Suto S, Yamanaka M, Mizutani Y, Mitsutake S, Igarashi Y, Sassa T, Kihara A
Proceedings of the National Academy of Sciences of the United States of America, 107, 43, 18439, 44, 43, 2010年10月26日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Very long-chain fatty acids (VLCFAs) exert a variety of cellular functions and are associated with numerous diseases. However, the precise pathway behind their elongation has remained elusive. Moreover, few regulatory mechanisms for VLCFAs synthesis have been identified. Elongases catalyze the first of four steps in the VLCFA elongation cycle; mammals have seven elongases (ELOVL1-7). In the present study, we determined the precise substrate specificities of all the ELOVLs by in vitro analyses. Particularly notable was the high activity exhibited by ELOVL1 toward saturated and monounsaturated C20- and C22-CoAs, and that it was essential for the production of C24 sphingolipids, which are unique in their capacity to interdigitate within the membrane as a result of their long chain length. We further established that ELOVL1 activity is regulated with the ceramide synthase CERS2, an enzyme essential for C24 sphingolipid synthesis. This regulation may ensure that the production of C24-CoA by elongation is coordinated with its utilization. Finally, knockdown of ELOVL1 caused a reduction in the activity of the Src kinase LYN, confirming that C24-sphingolipids are particularly important in membrane microdomain function.

Palmitoylation of the sphingosine 1-phosphate receptor S1P is involved in its signaling functions and internalization.
Ohno Y, Ito A, Ogata R, Hiraga Y, Igarashi Y, Kihara A
Genes to Cells, 14, 8, 911, 23, 8, 2009年08月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The lipid mediator sphingosine 1-phosphate (S1P) regulates several cellular processes through binding to its receptors (S1P(1)-S1P(5)), which are heterotrimeric G protein-coupled receptors. Here, we report that all S1P receptors are palmitoylated. In S1P(1), three Cys residues in the cytoplasmic tail are palmitoylated. We examined the roles of palmitoylation of S1P(1) using model cells in which wild-type S1P(1) or a non-palmitoylated mutant S1P(1) was overproduced. Compared with wild-type S1P(1), the non-palmitoylated S1P(1) exhibited binding affinity similar to the natural ligand S1P but lower to the synthetic ligand FTY720 phosphate (FTY720-P), the active form of the immunomodulator FTY720. However, downstream signaling of non-palmitoylated S1P(1) was similarly affected by S1P and FTY720-P stimulation. Moreover, upon stimulation with S1P, internalization of the mutant non-palmitoylated S1P(1) was retarded, compared with that of the wild-type protein. This effect was much more pronounced with FTY720-P stimulation. Similar differences were observed for the phosphorylation of S1P(1) and its mutant. These findings may provide insights into the molecular mechanisms of the pharmacological effects of FTY720. Finally, palmitoylation of wild-type S1P(1) increased upon treatment with S1P, suggesting that S1P(1) undergoes a palmitoylation/depalmitoylation cycle after stimulation by its ligands.

Lysophosphatidic acid 2 receptor-mediated supramolecular complex formation regulates its antiapoptotic effect.
E S, Lai YJ, Tsukahara R, Chen CS, Fujiwara Y, Yue J, Yu JH, Guo H, Kihara A, Tigyi G, Lin FT
The Journal of Biological Chemistry, 284, 21, 14558, 71, 21, 2009年05月22日, ［査読有り］, ［国際誌］
英語, The G protein-coupled lysophosphatidic acid 2 (LPA(2)) receptor elicits prosurvival responses to prevent and rescue cells from apoptosis. However, G protein-coupled signals are not sufficient for the full protective effect of LPA(2). LPA(2) differs from other LPA receptor subtypes in the C-terminal tail, where it contains a zinc finger-binding motif for the interactions with LIM domain-containing TRIP6 and proapoptotic Siva-1, and a PDZ-binding motif through which it complexes with the NHERF2 scaffold protein. In this report, we identify a unique CXXC motif of LPA(2) responsible for the binding to TRIP6 and Siva-1, and demonstrate that disruption of these macromolecular complexes or knockdown of TRIP6 or NHERF2 expression attenuates LPA(2)-mediated protection from chemotherapeutic agent-induced apoptosis. In contrast, knockdown of Siva-1 expression enhances this effect. Furthermore, a PDZ-mediated direct interaction between TRIP6 and NHERF2 facilitates their interaction with LPA(2). Together, these results suggest that in addition to G protein-activated signals, the cooperation embedded in the LPA(2)-TRIP6-NHERF2 ternary complex provides a novel ligand-dependent signal amplification mechanism that is required for LPA(2)-mediated full activation of antiapoptotic signaling.

Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane.
Balan L, Foltyn VN, Zehl M, Dumin E, Dikopoltsev E, Knoh D, Ohno Y, Kihara A, Jensen ON, Radzishevsky IS, Wolosker H
Proceedings of the National Academy of Sciences of the United States of America, 106, 18, 7589, 94, 18, 2009年05月05日, ［査読有り］, ［国際誌］
英語, D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [ (3)H]palmitate or [ (3)H]octanoic acid as precursors. In contrast to classical S-palmitoylation of cysteines, acylation of SR occurs through the formation of an oxyester bond with serine or threonine residues. In addition, we show that phosphorylation of Thr-227 is also required for steady-state binding of SR to the membrane under basal, nonstimulated condition. We propose that the inhibition of D-serine synthesis caused by translocation of SR to the membrane provides a fail-safe mechanism to prevent NMDAR overactivation in vicinal cells or synapses.

2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length.
Mizutani Y, Kihara A, Chiba H, Tojo H, Igarashi Y
Journal of Lipid Research, 49, 11, 2356, 64, 11, 2008年11月, ［査読有り］, ［国際誌］
英語, Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. Ceramides in epidermis also comprise an unusual variety, including 2-hydroxy (alpha-hydroxy)-ceramide. Six mammalian ceramide synthase/longevity assurance homologue (CerS/LASS) family members have been identified as synthases responsible for ceramide (CER) production. We reveal here that of the six, CerS3/LASS3 mRNA is the most predominantly expressed in keratinocytes. Moreover, its expression is increased upon differentiation. CerS family members have known substrate specificities for fatty acyl-CoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In the present study, we demonstrate that all CerS members can produce 2-hydroxy-CER when overproduced in HEK 293T cells. Each produced a 2-hydroxy-CER with a chain length similar to that of the respective nonhydroxy-CER produced. In HeLa cells overproducing the FA 2-hydroxylase FA2H, knock-down of CerS2 resulted in a reduction in total long-chain 2-hydroxy-CERs, confirming enzyme substrate specificity for chain length. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate. These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acyl-CoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.

A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate.
Yamanaka M, Anada Y, Igarashi Y, Kihara A
Biochemical and Biophysical Research Communications, 375, 4, 675, 9, 4, 2008年10月31日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.

A sphingosine kinase activity assay using direct infusion electrospray ionization tandem mass spectrometry.
Jin YX, Shi LH, Yoo HS, Lee YM, Kihara A, Igarashi Y, So HY, Yim YH
Analytical Biochemistry, 380, 1, 35, 40, 1, 2008年09月01日, ［査読有り］, ［国際誌］
英語, D-erythro-Sphingosine is known to be phosphorylated by sphingosine kinase to yield sphingosine-1-phosphate. With the importance of sphingosine-1-phosphate in biological functions being made evident by recent research, a selective and convenient method of assay to measure sphingosine kinase activity is required. Here we developed a new sphingosine kinase assay using murine teratocarcinoma mutant F9-12 cells and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with direct infusion. Sphingosine-1-phosphate in the crude extract of enzyme reaction mixture was selectively characterized and quantitated using precursor ion scanning for [PO(3)](-) in the negative electrospray ionization mode. The method was successfully validated for an activator and an inhibitor of sphingosine kinase. Direct quantitation of S1P without the use of radioactive reagents, chemical derivatization, and extensive chromatographic separation enables simplified assay for sphingosine kinase activity at the cellular system level, and the use of a structural analog as an internal standard provides robustness to the assay.

Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis.
Ikeda M, Kanao Y, Yamanaka M, Sakuraba H, Mizutani Y, Igarashi Y, Kihara A
FEBS Letters, 582, 16, 2435, 40, 16, 2008年07月09日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, Very long-chain fatty acids are produced through a four-step cycle. However, the 3-hydroxyacyl-CoA dehydratase catalyzing the third step in mammals has remained unidentified. Mammals have four candidates, HACD1-4, based on sequence similarities to the recently identified yeast Phs1, although HACD3 and HACD4 share relatively weak similarity. We demonstrate that all four of these human proteins are indeed 3-hydroxyacyl-CoA dehydratases, in growth suppression experiments using a PHS1-shut off yeast strain and/or in vitro 3-hydroxypalmitoyl-CoA dehydratase assays. HACD proteins exhibit distinct tissue-expression patterns. We also establish that HACD proteins interact with the condensation enzymes ELOVL1-7, with some preferences.

The Rim101 pathway is involved in Rsb1 expression induced by altered lipid asymmetry.
Ikeda M, Kihara A, Denpoh A, Igarashi Y
Molecular Biology of the Cell, 19, 5, 1922, 31, 5, 2008年05月, ［査読有り］, ［責任著者］, ［国際誌］
英語, Biological membranes consist of lipid bilayers. The lipid compositions between the two leaflets of the plasma membrane differ, generating lipid asymmetry. Maintenance of proper lipid asymmetry is physiologically quite important, and its collapse induces several cellular responses including apoptosis and platelet coagulation. Thus, a change in lipid asymmetry must be restored to maintain "lipid asymmetry homeostasis." However, to date no lipid asymmetry-sensing proteins or any related downstream signaling pathways have been identified. We recently demonstrated that expression of the putative yeast sphingoid long-chain base transporter/translocase Rsb1 is induced when glycerophospholipid asymmetry is altered. Using mutant screening, we determined that the pH-responsive Rim101 pathway, the protein kinase Mck1, and the transcription factor Mot3 all act in lipid asymmetry signaling, and that the Rim101 pathway was activated in response to a change in lipid asymmetry. The activated transcription factor Rim101 induces Rsb1 expression via repression of another transcription repressor, Nrg1. Changes in lipid asymmetry are accompanied by cell surface exposure of negatively charged phospholipids; we speculate that the Rim101 pathway recognizes the surface charges.

Membrane topology and essential amino acid residues of Phs1, a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation.
Kihara A, Sakuraba H, Ikeda M, Denpoh A, Igarashi Y
The Journal of Biological Chemistry, 283, 17, 11199, 209, 17, 2008年04月25日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

Sphingosine kinase 2 inhibitor accelerates Fas-mediated cell death progression in A20/2J cells.　　　　　　　　　　　　　　　
Hara-Yokoyama M, Terasawa K, Kihara A, Kim JW, Par CS, Hirabayashi Y, Igarashi Y, Yanagishita M
The Open Bioactive Compounds Journal, 1, 22, 27, 2008年, ［査読有り］

Intracellular trafficking pathway of yeast long-chain base kinase Lcb4, from its synthesis to its degradation.
Iwaki S, Sano T, Takagi T, Osumi M, Kihara A, Igarashi Y
The Journal of Biological Chemistry, 282, 39, 28485, 92, 39, 2007年09月28日, ［査読有り］, ［責任著者］, ［国際誌］
英語, Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G(1) cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact.

The immunomodulator FTY720 is phosphorylated and released from platelets.
Anada Y, Igarashi Y, Kihara A
European Journal of Pharmacology, 568, 1-3, 106, 11, 1-3, 2007年07月30日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, The novel immunomodulator FTY720 causes lymphocytes from peripheral blood to accumulate in lymphoid tissues. In vivo, FTY720 is phosphorylated to FTY720-P, which binds to the sphingosine 1-phosphate receptor S1P(1). So far, it has been unclear where FTY720-P is produced. We demonstrate that platelets efficiently convert FTY720 to FTY720-P and release it into the extracellular space. This release is mostly independent of platelet activation, but is slightly increased upon thrombin stimulation. These results suggest that platelets are a major source of plasma FTY720-P, and that FTY720-P release is mediated by two different transporters.

Rapid trafficking of c-Src, a non-palmitoylated Src-family kinase, between the plasma membrane and late endosomes/lysosomes.
Kasahara K, Nakayama Y, Kihara A, Matsuda D, Ikeda K, Kuga T, Fukumoto Y, Igarashi Y, Yamaguchi N
Experimental Cell Research, 313, 12, 2651, 66, 12, 2007年07月15日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Src-family kinases (SFKs) are co-expressed with multiple combinations of each member in a single cell and involved in various signalings. Recently, we showed by sucrose-density gradient fractionation that the subcellular distribution of c-Src is distinct from that of Lyn. However, little is known about the trafficking of c-Src in living cells. Here, we show by time-lapse monitoring combined with photobleaching techniques that c-Src, a non-palmitoylated SFK, is rapidly exchanged between the plasma membrane and intracellular organelles representing late endosomes/lysosomes possibly through its cytosolic release. Although Lyn, a palmitoylated SFK, is exocytosed to the plasma membrane via the Golgi apparatus along the secretory pathway, lack of palmitoylation directs Lyn away from the exocytotic transport to the c-Src-type trafficking between the plasma membrane and late endosomes/lysosomes. Intriguingly, c-Src and a non-palmitoylated Lyn mutant are efficiently delivered and immobilized to focal adhesions when their SH2 domains are able to mediate protein-protein interactions in place of intramolecular bindings. However, palmitoylation of Lyn inhibits its recruitment to focal adhesions. These results suggest that palmitoylation of SFKs is critical for SFK localization and trafficking and implicate that two distinct trafficking pathways for SFKs may be involved in SFKs' specific functions.

Lack of sphingosine 1-phosphate-degrading enzymes in erythrocytes.
Ito K, Anada Y, Tani M, Ikeda M, Sano T, Kihara A, Igarashi Y
Biochemical and Biophysical Research Communications, 357, 1, 212, 7, 1, 2007年05月25日, ［査読有り］, ［責任著者］, ［国際誌］
英語, Platelets are known to store a large amount of the bioactive lipid molecule sphingosine 1-phosphate (S1P) and to release it into the plasma in a stimuli-dependent manner. Erythrocytes can also release S1P, independently from any stimuli. We measured the S1P and sphingosine (Sph) levels in erythrocytes by HPLC and found that the contribution of erythrocyte S1P to whole blood S1P levels is actually higher than that of platelets. In vitro assays demonstrated that erythrocytes possess much weaker Sph kinase activity compared to platelets but lack the S1P-degrading activities of either S1P lyase or S1P phosphohydrolase. This combination may enable erythrocytes to maintain a high S1P content relative to Sph. The absence of both S1P-degrading enzymes has not been reported for other cell types. Thus, erythrocytes may be specialized cells for storing and supplying plasma S1P.

Regulation of the transport and protein levels of the inositol phosphorylceramide mannosyltransferases Csg1 and Csh1 by the Ca2+-binding protein Csg2.
Uemura S, Kihara A, Iwaki S, Inokuchi J, Igarashi Y
The Journal of Biological Chemistry, 282, 12, 8613, 21, 12, 2007年03月23日, ［査読有り］, ［責任著者］, ［国際誌］
英語, Complex sphingolipids in yeast are known to function in cellular adaptation to environmental changes. One of the yeast complex sphingolipids, mannosylinositol phosphorylceramide (MIPC), is produced by the redundant inositol phosphorylceramide (IPC) mannosyltransferases Csg1 and Csh1. The Ca2+-binding protein Csg2 can form a complex with either Csg1 or Csh1 and is considered to act as a regulatory subunit. However, the role of Csg2 in MIPC synthesis has remained unclear. In this study, we found that Csg1 and Csh1 are N-glycosylated with core-type and mannan-type structures, respectively. Further identification of the glycosylated residues suggests that both Csg1 and Csh1 exhibit membrane topology with their C termini in the cytosol and their mannosyltransferase domains in the lumen. After complexing with Csg2, both Csg1 and Csh1 function in the Golgi, and then are delivered to the vacuole for degradation. However, uncomplexed Csh1 cannot exit from the endoplasmic reticulum. We also demonstrated that Ca2+ stimulates IPC-to-MIPC conversion, because of a Csg2-dependent increase in Csg1 levels. Thus, Csg2 has several regulatory functions for Csg1 and Csh1, including stability, transport, and gene expression.

Sphingosine kinase assay system with fluorescent detection in high performance liquid chromatography.
Jin YX, Yoo HS, Kihara A, Choi CH, Oh S, Moon DC, Igarashi Y, Lee YM
Archives of Pharmacal Research, 29, 11, 1049, 54, 11, 2006年11月, ［査読有り］, ［国際誌］
英語, Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; 100 microM of C17-Sph and 30 microg protein of F9-12 cells lysate in 20 min. Sphingosine analog C17-Sph was efficiently phosphorylated by Sphk activity (Km:67.08 microM, Vmax :1507.5 pmol/min/mg). New product C17,S1P was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while 20 microM of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.

LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity.
Mizutani Y, Kihara A, Igarashi Y
The Biochemical Journal, 398, 3, 531, 8, 3, 2006年09月15日, ［査読有り］, ［国際誌］
英語, The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells.
Hiraga Y, Kihara A, Sano T, Igarashi Y
Biochemical and Biophysical Research Communications, 344, 3, 852, 8, 3, 2006年06月09日, ［査読有り］, ［責任著者］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).

Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins.
Ohno Y, Kihara A, Sano T, Igarashi Y
Biochimica et Biophysica Acta, 1761, 4, 474, 83, 4, 2006年04月, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein.

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner.
Kobayashi N, Nishi T, Hirata T, Kihara A, Sano T, Igarashi Y, Yamaguchi A
Journal of Lipid Research, 47, 3, 614, 21, 3, 2006年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca(2+). Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca(2+)-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca(2+) also stimulated S1P release from ATX platelets without ATP, whereas the Ca(2+)-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca(2+).

Mouse sphingosine kinase isoforms SPHK1a and SPHK1b differ in enzymatic traits including stability, localization, modification, and oligomerization.
Kihara A, Anada Y, Igarashi Y
The Journal of Biological Chemistry, 281, 7, 4532, 9, 7, 2006年02月17日, ［査読有り］, ［筆頭著者, 責任著者］, ［国際誌］
英語, Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different.

Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis.
Tani M, Kihara A, Igarashi Y
The Biochemical Journal, 394, Pt 1, 237, 42, Pt 1, 2006年02月15日, ［査読有り］, ［国際誌］
英語, In the yeast Saccharomyces cerevisiae, sphingolipids are essential for cell growth. Inactivation of sphingolipid biosynthesis, such as by disrupting the serine palmitoyltransferase gene (LCB2), is lethal, but cells can be rescued by supplying an exogenous LCB (long-chain base) like PHS (phytosphingosine) or DHS (dihydrosphingosine). In the present study, supplying SPH (sphingosine), an unnatural LCB for yeast, similarly rescued the Deltalcb2 cells, but only when SPH 1-phosphate production was inhibited by deleting the LCB kinase gene LCB4. Exogenously added SPH was adequately converted into phosphoinositol-containing complex sphingolipids. Interestingly, cells carrying SPH-based sphingolipids exhibited a defect in the association of Pma1p with Triton X-100-insoluble membrane fractions, and displayed sensitivities to both Ca2+ and hygromycin B. These results suggest that the SPH-based sphingolipids in these cells have properties that differ from those of the PHS- or DHS-based sphingolipids in regard to lipid microdomain formation, leading to abnormal sensitivities towards certain environmental stresses. The present paper is the first report showing that in sphingolipid-deficient S. cerevisiae, the requirement for LCB can be fulfilled by exogenous SPH, although this supplement results in failure of lipid microdomain formation.

Regulation of the sphingoid long-chain base kinase Lcb4p by ergosterol and heme: studies in phytosphingosine-resistant mutants.
Sano T, Kihara A, Kurotsu F, Iwaki S, Igarashi Y
The Journal of Biological Chemistry, 280, 44, 36674, 82, 44, 2005年11月04日, ［査読有り］, ［責任著者］, ［国際誌］
英語, Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca(2+) mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes and screened for phytosphingosine-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance. Of these eight, four are ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We also demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in Deltahem14 and Deltahmg1 cells, likely as a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (Deltaerg2, Deltaerg3, and Deltaerg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in Deltaerg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p because of the altered localization. The other genes isolated (PBP1, UFD4, TPS1, and WHI2) may be involved in LCBP signaling.

Long-chain base kinase Lcb4 Is anchored to the membrane through its palmitoylation by Akr1.
Kihara A, Kurotsu F, Sano T, Iwaki S, Igarashi Y
Molecular and Cellular Biology, 25, 21, 9189, 97, 21, 2005年11月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the long-chain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation.

Mammalian Lass6 and its related family members regulate synthesis of specific ceramides.
Mizutani Y, Kihara A, Igarashi Y
The Biochemical Journal, 390, Pt 1, 263, 71, Pt 1, 2005年08月15日, ［査読有り］, ［国際誌］
英語, The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.

Products by the sphingosine kinase/sphingosine 1-phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis.
Kariya Y, Kihara A, Ikeda M, Kikuchi F, Nakamura S, Hashimoto S, Choi CH, Lee YM, Igarashi Y
Genes to Cells, 10, 6, 605, 15, 6, 2005年06月, ［査読有り］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) functions as a ligand for the S1P/EDG family receptors. For years, intracellular signaling roles for S1P have also been suggested, especially in cell proliferation. Now, we have generated several mouse F9 embryonic carcinoma cell lines varying in expression of the S1P-degrading enzyme, S1P lyase (SPL) and/or sphingosine kinase (SPHK1). All these cell lines accumulated S1P compared to the wild-type F9 cells, but the amounts varied. We investigated the ability of these cells to proliferate under low serum conditions, as measured by a thymidine uptake assay. Although F9 cells over-expressing SPHK1 did exhibit enhanced DNA synthesis, other S1P-accumulating cells (SPL-null cells and SPL-null cells over-expressing SPHK1) did not. The overproduction of both SPL and SPHK1 resulted in the most striking mitogenic effect. Moreover, nM concentrations of sphingosine (or dihydrosphingosine) stimulated DNA synthesis in an SPL-dependent manner. These results indicate that products by the SPL pathway, not S1P itself, function in mitogenesis.

Sphingolipid-to-glycerophospholipid conversion in SPL-null cells implies the existence of an alternative isozyme.
Ikeda M, Kihara A, Kariya Y, Lee YM, Igarashi Y
Biochemical and Biophysical Research Communications, 329, 2, 474, 9, 2, 2005年04月08日, ［査読有り］, ［国際誌］
英語, Sphingosine-1-phosphate (S1P) lyase catalyzes the cleavage of the bioactive lipid molecule S1P to phosphoethanolamine and hexadecenal, both of which are utilized as glycerophospholipid precursors. Until now, only one gene, SPL, has been identified as encoding a S1P lyase. In the present study, SPL-null F9 cells were able to convert radiolabeled dihydrosphingosine to glycerophospholipids, albeit at much lower efficiency than parent cells. Lysates prepared from the SPL-null cells exhibited weak but significant dihydrosphingosine-1-phosphate lyase activity in vitro. These results provide evidence of the existence of an alternative S1P lyase.

Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase.
Iwaki S, Kihara A, Sano T, Igarashi Y
The Journal of Biological Chemistry, 280, 8, 6520, 7, 8, 2005年02月25日, ［査読有り］, ［国際誌］
英語, Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells. In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p. Until now, the regulatory mechanism for Lcb4p has been unclear. In the present study, we found that Lcb4p is post-translationally modified by phosphorylation. Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation. Pho85p functions in a number of cellular processes, especially in response to environmental changes. Two of 10 Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p. Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser(451) and Ser(455). Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway. The protein was stabilized in Deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation. Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process. Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body. Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.

Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5'-phosphate binding domain exposed to the cytosol.
Ikeda M, Kihara A, Igarashi Y
Biochemical and Biophysical Research Communications, 325, 1, 338, 43, 1, 2004年12月03日, ［査読有り］, ［国際誌］
英語, Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S1P is degraded either by S1P lyase or by S1P phosphohydrolase. The gene encoding mammalian S1P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-1-phosphate (DHS1P) lyase activity in most tissues. However, liver and heart exhibited high DHS1P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S1P phosphohydrolase, indicating that the degradation of S1P by two S1P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.

Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition.
Yokota S, Taniguchi Y, Kihara A, Mitsutake S, Igarashi Y
FEBS Letters, 578, 1-2, 106, 10, 1-2, 2004年12月03日, ［査読有り］, ［国際誌］
英語, Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177-->Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1-->108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.

FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane.
Kihara A, Igarashi Y
The Journal of Biological Chemistry, 279, 47, 49243, 50, 47, 2004年11月19日, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of l-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.

Cross talk between sphingolipids and glycerophospholipids in the establishment of plasma membrane asymmetry.
Kihara A, Igarashi Y
Molecular Biology of the Cell, 15, 11, 4949, 59, 11, 2004年11月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, Glycerophospholipids and sphingolipids are distributed asymmetrically between the two leaflets of the lipid bilayer. Recent studies revealed that certain P-type ATPases and ATP-binding cassette (ABC) transporters are involved in the inward movement (flip) and outward movement (flop) of glycerophospholipids, respectively. In this study of phytosphingosine (PHS)-resistant yeast mutants, we isolated mutants for PDR5, an ABC transporter involved in drug efflux as well as in the flop of phosphatidylethanolamine. The pdr5 mutants exhibited an increase in the efflux of sphingoid long-chain bases (LCBs). Genetic analysis revealed that the PHS-resistant phenotypes exhibited by the pdr5 mutants were dependent on Rsb1p, a putative LCB-specific transporter/translocase. We found that the expression of Rsb1p was increased in the pdr5 mutants. We also demonstrated that expression of RSB1 is under the control of the transcriptional factor Pdr1p. Expression of Rsb1p also was enhanced in mutants for the genes involved in the flip of glycerophospholipids, including ROS3, DNF1, and DNF2. These results suggest that altered glycerophospholipid asymmetry induces the expression of Rsb1p. Conversely, overexpression of Rsb1p resulted in increased flip and decreased flop of fluorescence-labeled glycerophospholipids. Thus, there seems to be cross talk between sphingolipids and glycerophospholipids in maintaining the functional lipid asymmetry of the plasma membrane.

Identification of the human sphingolipid C4-hydroxylase, hDES2, and its up-regulation during keratinocyte differentiation.
Mizutani Y, Kihara A, Igarashi Y
FEBS Letters, 563, 1-3, 93, 7, 1-3, 2004年04月09日, ［査読有り］, ［国際誌］
英語, The C4-hydroxylation of dihydrosphingosine or dihydroceramide is a key reaction in the biosynthesis of phytosphingolipids, both in yeasts and in mammalian cells. Mouse DES2 (mDES2) was recently cloned and shown to work as a Delta4-desaturase/C4-hydroxylase, when expressed in yeast cells. Here, we cloned a human homologue of mDES2, hDES2, by homology search utilizing a BLAST program. When expressed in HEK 293 cells, hDES2 exhibited hydroxylase activity for dihydroceramide. Northern blot analyses of hDES2 revealed high expression in skin, intestines, and kidney, sites reportedly possessing high levels of phytosphingolipids. Furthermore, up-regulation of hDES2 mRNA expression and subsequent phytoceramide production were observed during vitamin C/serum-induced differentiation of human keratinocytes. These results suggest that the newly cloned hDES2 plays an essential role in phytosphingolipid synthesis in human skin and other phytosphingolipid-containing tissues.

Csg1p and newly identified Csh1p function in mannosylinositol phosphorylceramide synthesis by interacting with Csg2p.
Uemura S, Kihara A, Inokuchi J, Igarashi Y
The Journal of Biological Chemistry, 278, 46, 45049, 55, 46, 2003年11月14日, ［査読有り］, ［国際誌］
英語, Csg1p and Csg2p have been shown to be involved in the synthesis of mannosylinositol phosphorylceramide (MIPC) from inositol phosphorylceramide. YBR161w, termed CSH1 here, encodes a protein that exhibits a strong similarity to Csg1p. To examine whether Csh1p also functions in MIPC synthesis, we performed a [3H]dihydrosphingosine labeling experiment. Deltacsg1 cells exhibited only a reduction in the synthesis of mannosylated sphingolipids compared with wild-type cells, whereas the Deltacsg1 Deltacsh1 double deletion mutant exhibited a total loss. These results indicated that Csg1p and Csh1p have redundant functions in MIPC synthesis. Analyses using Deltacsg1 and Deltacsh1 cells in the Deltaipt1, Deltasur2, or Deltascs7 genetic background demonstrated that Csh1p has a different substrate specificity from Csg1p. We also revealed that Csg2p interacts with both Csg1p and Csh1p. Deletion of the CSG2 gene reduced the Csg1p activity and abolished the Csh1p activity. These results suggested that two distinct inositol phosphorylceramide mannosyltransferase complexes, Csg1p-Csg2p and Csh1p-Csg2p, exist.

Distribution of sphingosine kinase activity in mouse tissues: contribution of SPHK1.
Fukuda Y, Kihara A, Igarashi Y
Biochemical and Biophysical Research Communications, 309, 1, 155, 60, 1, 2003年09月12日, ［査読有り］, ［国際誌］
英語, Sphingosine kinase (SPHK) phosphorylates sphingosine to form a bioactive lipid mediator, sphingosine 1-phosphate (S1P). S1P mediates such diverse biological processes as regulation of cell differentiation, motility, and apoptosis both extracellularly, via S1P (Edg) family receptors, and intracellularly, through unidentified targets. In cells S1P is short-lived, so the synthetic process catalyzed by sphingosine kinase may be important in maintaining the cellular levels of the compound. Thus far, two sphingosine kinases have been reported, with SPHK1 exhibiting the higher activity. However, several studies suggest the existence of unidentified sphingosine kinases. Therefore, to further elucidate the role of SPHK1 in the formation of S1P, we investigated its contribution to the total sphingosine kinase activity in mouse tissues. We found that SPHK1 is a major sphingosine kinase in many tissues, especially in brain, heart, and colon. However, some tissues such as spleen, small intestine, and lung contained sphingosine kinase activity that was not attributable to SPHK1 or SPHK2, as determined by immunodepletion assays. Furthermore, the presence of other sphingosine kinases with different properties, i.e., higher activity toward phytosphingosine and a different subcellular distribution, is suggested.

Transmembrane topology of sphingoid long-chain base-1-phosphate phosphatase, Lcb3p.
Kihara A, Sano T, Iwaki S, Igarashi Y
Genes to Cells, 8, 6, 525, 35, 6, 2003年06月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, BACKGROUND: Sphingoid long-chain base-1-phosphates (LCBPs) are thought to act as intracellular signalling molecules in yeast. Lcb3p is a member of the LCBPs-specific phosphatase family (SPP family). Other yeast phosphatases, Lpp1p and Dpp1p, are members of a different lipid phosphatase family (LPP family) known to exhibit broader substrate specificities. Until now, only the membrane topology of mammalian LPP family members has been reported, whereas that of the SPP family has remained unclear. RESULTS: In our in vitro system, Lcb3p displayed major phosphatase activity against dihydrosphingosine-1-phosphate, while Dpp1p and Lpp1p also exhibited activities. Here, we determined that Lpp1p and Dpp1p exhibit the topology common to the LPP family. Moreover, we examined the transmembrane topology of Lcb3p using a C-terminal reporter approach. From our results we deduced a structural model illustrating that Lcb3p has eight membrane-spanning domains with its highly conserved phosphatase motifs positioned within the endoplasmic reticulum (ER) lumen. Consistent with this result, Lcb3p collected in low speed pellet fractions was highly resistant to exogenous proteinase K unless the membrane was disrupted. CONCLUSION: Our results suggest that the active site of Lcb3p is located in the ER lumen and, thus, the phosphate group of the LCBP is hydrolysed on the lumenal side.

Sphingosine-1-phosphate lyase is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm.
Kihara A, Ikeda M, Kariya Y, Lee EY, Lee YM, Igarashi Y
The Journal of Biological Chemistry, 278, 16, 14578, 85, 16, 2003年04月18日, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase, hSPP2.
Ogawa C, Kihara A, Gokoh M, Igarashi Y
The Journal of Biological Chemistry, 278, 2, 1268, 72, 2, 2003年01月10日, ［査読有り］, ［国際誌］
英語, Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.

Identification and characterization of a Saccharomyces cerevisiae gene, RSB1, involved in sphingoid long-chain base release.
Kihara A, Igarashi Y
The Journal of Biological Chemistry, 277, 33, 30048, 54, 33, 2002年08月16日, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1 encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.

Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion
N Ishihara, M Hamasaki, S Yokota, K Suzuki, Y Kamada, A Kihara, T Yoshimori, T Noda, Y Ohsumi
MOLECULAR BIOLOGY OF THE CELL, 12, 11, 3690, 3702, AMER SOC CELL BIOLOGY, 2001年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg, proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles,, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.

Revisiting the lysogenization control of bacteriophage lambda - Identification and characterization of a new host component, HflD
A Kihara, Y Akiyama, K Ito
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 17, 13695, 13700, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Upon infection to the Escherichia coli cell, the genome of bacteriophage Lambda either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization). The lambda CII protein is a key determinant in the lysis-lysogeny decision. It is a short-lived transcription activator for the Lambda genes essential for lysogeny establishment. In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E. coli, using a new selection for enhancement of CII-stimulated transcription. The gene affected was termed hflD, which encodes a protein of 213 amino acids, An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization, HflD is associated peripherally with the cytoplasmic membrane. Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system. Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components. We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network
A Kihara, Y Kabeya, Y Ohsumi, T Yoshimori
EMBO REPORTS, 2, 4, 330, 335, OXFORD UNIV PRESS, 2001年04月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co-immunoprecipitated with phosphatidylinositol (PtdIns) 3-kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3-kinase complex. Quantitative analyses using a cross-linker showed that all Beclin forms a complex with PtdIns 3-kinase, whereas similar to 50% of PtdIns 3-kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3-kinase localize to the trans-Golgi network (TGN). Some PtdIns 3-kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3-kinase control autophagy as a complex at the TGN.

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae
A Kihara, T Noda, N Ishihara, Y Ohsumi
JOURNAL OF CELL BIOLOGY, 152, 3, 519, 530, ROCKEFELLER UNIV PRESS, 2001年02月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p acid Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p. Apg14p, and Vps15p. an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively. whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

Dislocation of membrane proteins in FtsH-mediated proteolysis
A Kihara, Y Akiyama, K Ito
EMBO JOURNAL, 18, 11, 2970, 2981, OXFORD UNIV PRESS, 1999年06月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Escherichia coli FtsH degrades several integral membrane proteins, including YccA, having seven transmembrane segments, a cytosolic N-terminus and a periplasmic C-terminus, Evidence indicates that FtsH initiates proteolysis at the N-terminal cytosolic domain. SecY: having 10 transmembrane segments, is also a substrate of FtsH, We studied whether and how the FtsH-catalyzed proteolysis on the cytosolic side continues into the transmembrane and periplasmic regions using chimeric proteins, YccA-(P3)-PhoA-His(6)-Myc and SecY-(P5)-PhoA, with the alkaline phosphatase (PhoA) mature sequence in a periplasmic domain. The PhoA domain that was present within the fusion protein was rapidly degraded by FtsH when it lacked the DsbA-dependent folding. In contrast, both PhoA itself and the TM9-PhoA region of SecY-(P5)-PhoA were stable when expressed as independent polypeptides. In the presence of DsbA, the FtsH-dependent degradation stopped at a site near to the N-terminus of the PhoA moiety, leaving the PhoA domain land its C-terminal region) undigested. The efficiency of this degradation stop correlated well with the rapidity of the folding of the PhoA domain. Thus, both transmembrane and periplasmic domains are degraded by the processive proteolysis by FtsH, provided they are not tightly folded. We propose that FtsH dislocates the extracytoplasmic domain of a substrate, probably using its ATPase activity.

Three-dimensional structure of phosphoenolpyruvate carboxylase: A proposed mechanism for allosteric inhibition
Y Kai, H Matsumura, T Inoue, K Terada, Y Nagara, T Yoshinaga, A Kihara, K Tsumura, K Izui
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 96, 3, 823, 828, NATL ACAD SCIENCES, 1999年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has been determined by x-ray diffraction methods at 2.8-Angstrom resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs, The four subunits are arranged in a "dimer-of-dimers" form with respect to subunit contact, resulting in an overall square arrangement. The contents of alpha-helices and beta-strands are 65% and 5%, respectively. All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-barrel, Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers, The location of the catalytic site is likely to be near the C-terminal side of the P-barrel, The binding site for L-aspartate is located about 20 Angstrom away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate.

Translocation, folding, and stability of the HflKC complex with signal anchor topogenic sequences
A Kihara, K Ito
JOURNAL OF BIOLOGICAL CHEMISTRY, 273, 45, 29770, 29775, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1998年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, HflK and HflC are plasma membrane proteins of Escherichia coli, each having a large C-terminal domain exposed to the periplasmic space and an N-terminally located transmembrane segment, which should act as a signal anchor sequence for their biogenesis. They form a complex, HflKC. We studied in vivo processes of biogenesis of this pair of membrane proteins. Translocation of the C-terminal domains across the membrane, as assessed by their accessibility to externally added protease, was completed within 1 min after the synthesis in wild-type cells as web as in the secB mutant cells or in the FtsY-depleted cells. In contrast, translocation of these domains was retarded markedly when sodium azide was added to inhibit SecA ATPase and blocked almost completely in secY- or secD-defective mutant cells. Thus, although targeting of these membrane proteins depends neither on the SecB chaperone nor on the SRP pathway, their translocation occurs exclusively via the Sec translocase complex. Translocated HflK molecules were then folded into a partially protease-resistant conformation, taking a few minutes, and this folding was induced upon association with HflC. Singly expressed HflK and HflC were unstable in vivo and periplasmic proteases DegP and Pre were involved in the degradation of the HflK subunit. We characterized several hflA alleles isolated in early studies; they alter the HflK or the HflC sequence and destabilize the HflKC complex.

Roles of the periplasmic domain of Escherichia coli FtsH (HflB) in protein interactions and activity modulation
Y Akiyama, A Kihara, H Mori, T Ogura, K Ito
JOURNAL OF BIOLOGICAL CHEMISTRY, 273, 35, 22326, 22333, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1998年08月, ［査読有り］
英語, 研究論文（学術雑誌）, FtsH is a membrane-bound and ATP-dependent protease of Escherichia coli, known to degrade SecY, a membrane protein for protein translocation, and CII, a soluble transcription factor for lysis/lysogeny decision of phage lambda. FtsH forms a homo-oligomeric complex as well as a hetero-oligomeric complex with HflKC, a putative modulator of FtsH. Although FtsH has a small periplasmic region, HflKC is mostly exposed to the periplasmic space. We studied the roles of the periplasmic region of FtsH by engineering mutations in this protein. FtsH Delta 236, lacking most of the periplasmic region, retained the in vivo ability to degrade SecY but not CII, resulting in high frequency lysogenization of lambda. Several insertion mutations in the periplasmic region of FtsH also differentially affected the proteolytic activities of FtsH. interestingly purified and detergent-solubilized FtsH Delta 236 was as active as the wild-type enzyme in degrading SecY and CII, although its ATPase activity was lowered 5-fold. Affinity chromatography using histidine-tagged derivatives showed that the periplasmic domain-deleted FtsH no longer interacted with FtsH or HflKC. Although FtsH Delta 236-His(6)-Myc lost the static FtsH-FtsH interaction, it retained the ability to change its conformation in an ATP-dependent manner at 37 degrees C, leading to a limited oligomerization. These results suggest that the periplasmic region of FtsH has crucial roles in the protein-protein interactions of this complex and in the modulation of its proteolytic functions against different substrates.

Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: An implication from the interference by a mutant form of a new substrate protein, YccA
A Kihara, Y Akiyama, K Ito
JOURNAL OF MOLECULAR BIOLOGY, 279, 1, 175, 188, ACADEMIC PRESS LTD, 1998年05月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here,we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F-0 segment as well, but not the CII protein of bacteriophage lambda or the sigma(32) protein. From these results we suggest that there are at least two pathways for FtsH-deyendent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate. (C) 1998 Academic Press Limited.

Polypeptide binding of Escherichia coli FtsH (HflB)
Y Akiyama, M Ehrmann, A Kihara, K Ito
MOLECULAR MICROBIOLOGY, 28, 4, 803, 812, WILEY-BLACKWELL, 1998年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATP gamma S prevented self-aggregation of detergent-solubilized FtsH-His(6)-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His(6)-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His(6)-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.

Host regulation of lysogenic decision in bacteriophage lambda: Transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA)
A Kihara, Y Akiyama, K Ito
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 94, 11, 5544, 5549, NATL ACAD SCIENCES, 1997年05月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, The cII gene product of bacteriophage A is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome, One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein, However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control, Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane, In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation, In vivo, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action, These results, together with our previous finding that FtsH and HBlKC form a complex, suggest that FtsH is the cII degrading protease and HPlKC is a modulator of the FtsH function, We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein, This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins
Y Akiyama, A Kihara, H Tokuda, K Ito
JOURNAL OF BIOLOGICAL CHEMISTRY, 271, 49, 31196, 31201, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1996年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins, SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein), We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH, We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini, Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (Al) is essential for ATPase activity and in two functioning of FtsH, Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation, These results demonstrate that FtsH binds to and degrades SecY, its Al motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.

Subunit a of proton ATPase F-0 sector is a substrate of the FtsH protease in Escherichia coli
Y Akiyama, A Kihara, K Ito
FEBS LETTERS, 399, 1-2, 26, 28, ELSEVIER SCIENCE BV, 1996年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Escherichia coli FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase. We now report that subunit alpha of the membrane-embedded F-0 part of H+-ATPase is another substrate of FtsH. Pulse-chase experiments shelved that subunit alpha is unstable when it alone (without F-0 subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants. Selective and ATP-dependent degradation of subunit alpha by purified FtsH protein was demonstrated in vitro. These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit alpha in the membrane.

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY
A Kihara, Y Akiyama, K Ito
EMBO JOURNAL, 15, 22, 6122, 6131, OXFORD UNIV PRESS UNITED KINGDOM, 1996年11月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit, We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex, Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY, Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY, These results raise the possibility that even the wildtype HflKC protein acts to antagonize FtsH, Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein, Furthermore crosslinking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro, Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro, These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

INTRACELLULAR STABILITY OF ALPHA-FRAGMENTS OF BETA-GALACTOSIDASE - EFFECTS OF AMINO-TERMINALLY FUSED POLYPEPTIDES
T HOMMA, T YOSHIHISA, A KIHARA, Y AKIYAMA, K ITO
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 215, 2, 452, 458, ACADEMIC PRESS INC ELSEVIER SCIENCE, 1995年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galaclosidase, which is unique in that it is largely unstructured but can be ''active'' in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it. (C) 1995 Academic Press, Inc.

PHOSPHOENOLPYRUVATE CARBOXYLASE - ALTERATION OF CATALYTIC AND REGULATORY PROPERTIES BY SITE-DIRECTED MUTAGENESIS AND ISOLATION OF THE GENE FROM AN EXTREME THERMOPHILE
K IZUI, K TERADA, M YANO, T NAKAMURA, K ABE, A KIHARA, YOSHIOKA, I, M TAKAHASHI
ENERGY CONVERSION AND MANAGEMENT, 36, 6-9, 751, 754, PERGAMON-ELSEVIER SCIENCE LTD, 1995年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The roles of several conserved amino acid residues in phosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) of E.coli were studied by site-directed mutagenesis. Mutant enzymes H138N (His138 replaced by Asn) and R587S lost the original catalytic activity but revealed the weak activity of HCO3- -dependent hydrolysis of PEP to yield pyruvate. By the use of H138N the formation of carboxyphosphate, a postulated reaction intermediate, was demonstrated for the first time. K620S and R438C were almost insensitive to an allosteric feedback inhibitor, aspartate, and the latter showed a tendency to dissociate to dimer. Furthermore, the gene for extremely thermostable PEPC was cloned and expressed in E. coli.

FTSH IS REQUIRED FOR PROTEOLYTIC ELIMINATION OF UNCOMPLEXED FORMS OF SECY, AN ESSENTIAL PROTEIN TRANSLOCASE SUBUNIT
A KIHARA, Y AKIYAMA, K ITO
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 92, 10, 4532, 4536, NATL ACAD SCIENCES, 1995年05月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, When secY is overexpressed over secE or secE is underexpressed, a Fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined, We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.

PRODUCT OF A NEW GENE, SYD, FUNCTIONALLY INTERACTS WITH SECY WHEN OVERPRODUCED IN ESCHERICHIA-COLI
T SHIMOIKE, T TAURA, A KIHARA, T YOSHIHISA, Y AKIYAMA, K CANNON, K ITO
JOURNAL OF BIOLOGICAL CHEMISTRY, 270, 10, 5519, 5526, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 1995年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A mutant form of SecY, SecY(-d)1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY(-d)1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY(-d)1 form of SecY. Thus, in the presence of both secY(+) and secY(-d)1, Syd increases the effective SecY(+)/SecY(-d)1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

皮膚透過性バリアの形成に重要な脂質セラミドの産生機構　　　　　　　　　　　　　　　
木原章雄, 進化する皮膚研究最前線～皮膚機能の基本から臨床，応用研究まで～, 2021年, ［招待有り］
日本語

小胞体における新たな脂肪酸α酸化経路　　　　　　　　　　　　　　　
木原 章雄, 酵素工学ニュース, 82, 22, 25, 2019年10月, ［査読有り］, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

スフィンゴ脂質クオリティによる生体防御　　　　　　　　　　　　　　　
木原 章雄, The Lipid, 30, 28, 34, 2019年10月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

極長鎖脂質による表皮および涙液における透過性バリア形成　　　　　　　　　　　　　　　
木原 章雄, 医学のあゆみ, 269, 983, 988, 2019年06月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

The role of PNPLA1 in ω-O-acylceramide synthesis and skin barrier function
Hirabayashi T, Murakami M, Kihara A, Biochim Biophys Acta, 1864, 6, 869, 879, 2019年06月, ［査読有り］, ［招待有り］, ［国際誌］
The human genome encodes nine enzymes belonging to the patatin-like phospholipase domain-containing lipase (PNPLA)/Ca2+-independent phospholipase A2 (iPLA2) family. Although most PNPLA/iPLA2 enzymes are widely distributed and act on phospholipids or neutral lipids as (phospho)lipases to play homeostatic roles in lipid metabolism, the function of PNPLA1 remained a mystery until a few years ago. However, the recent finding that mutations in the human PNPLA1 gene are linked to autosomal recessive congenital ichthyosis (ARCI), as well as evidence obtained from biochemical and gene knockout studies, has shed light on the function of this enzyme in skin-specific sphingolipid metabolism rather than glycerophospholipid metabolism. PNPLA1 is specifically expressed in differentiated keratinocytes and plays a crucial role in the biosynthesis of ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide. In this review, we overview the biosynthetic route and biological role of epidermal ω-O-acylceramide, highlight the function of PNPLA1 as a bona fide acylceramide synthase required for proper skin barrier function and keratinocyte differentiation, and summarize the mutations of PNPLA1 currently identified in ARCI patients. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau., 英語

パルミトイル化　　　　　　　　　　　　　　　
木原章雄, 生体の科学, 69, 490, 491, 2018年10月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

脂質抗原提示分子CD1dによる抗原認識機構の解析　　　　　　　　　　　　　　　
日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実, 日本生化学会大会プログラム・講演要旨集, 91回, [3P, 090], 2018年09月
(公社)日本生化学会, 日本語

スフィンゴ脂質長鎖塩基の代謝と脂肪酸α酸化　　　　　　　　　　　　　　　
木原 章雄, Plant Morphol, 30, 5, 14, 2018年06月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

脂質による皮膚バリア形成と疾患制御　　　　　　　　　　　　　　　
村上誠, 木原章雄, 実験医学, 36, 160, 167, 2018年06月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

スフィンゴ脂質代謝と疾患制御　　　　　　　　　　　　　　　
木原 章雄, 実験医学, 36, 46, 52, 2018年06月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

鎖長の違いによる脂肪酸クオリティ — 極長鎖・超長鎖脂肪酸の産生機構と病態　　　　　　　　　　　　　　　
木原 章雄, 医学のあゆみ, 264, 966, 971, 2018年03月, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides.
Kihara A, Progress in lipid research, 63, 50, 69, 2016年07月, ［査読有り］, ［国際誌］
Ceramide (Cer) is a structural backbone of sphingolipids and is composed of a long-chain base and a fatty acid. Existence of a variety of Cer species, which differ in chain-length, hydroxylation status, and/or double bond number of either of their hydrophobic chains, has been reported. Ceramide is produced by Cer synthases. Mammals have six Cer synthases (CERS1-6), each of which exhibits characteristic substrate specificity toward acyl-CoAs with different chain-lengths. Knockout mice for each Cer synthase show corresponding, isozyme-specific phenotypes, revealing the functional differences of Cers with different chain-lengths. Cer diversity is especially prominent in epidermis. Changes in Cer levels, composition, and chain-lengths are associated with atopic dermatitis. Acylceramide (acyl-Cer) specifically exists in epidermis and plays an essential role in skin permeability barrier formation. Accordingly, defects in acyl-Cer synthesis cause the cutaneous disorder ichthyosis with accompanying severe skin barrier defects. Although the molecular mechanism by which acyl-Cer is generated was long unclear, most genes involved in its synthesis have been identified recently. In Cer degradation pathways, the long-chain base moiety of Cer is converted to acyl-CoA, which is then incorporated mainly into glycerophospholipids. This pathway generates the lipid mediator sphingosine 1-phosphate. This review will focus on recent advances in our understanding of the synthesis and degradation pathways, physiological functions, and pathology of Cers/acyl-Cers., 英語

明らかとなったスフィンゴ脂質の代謝経路：フィトスフィンゴシン代謝による奇数鎖脂肪酸の産生
木原 章雄, 化学と生物, 54, 2, 75, 76, 2016年02月, ［査読有り］, ［招待有り］
公益社団法人 日本農芸化学会, 日本語

セラミドによる皮膚バリア形成　　　　　　　　　　　　　　　
木原 章雄, 実験医学, 33, 15, 117, 122, 2015年09月, ［招待有り］
日本語

スフィンゴ脂質の多様性と多機能性　　　　　　　　　　　　　　　
木原 章雄, 実験医学, 33, 15, 50, 56, 2015年09月, ［招待有り］
日本語

Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids.
Kihara A, Biochimica et biophysica acta, 1841, 5, 766, 72, 2014年05月, ［査読有り］, ［国際誌］
The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a well-known lipid mediator. As a lipid mediator, S1P must be present in extracellular space and bind to its cell surface receptors (S1P1-5). However, most S1P, synthesized intracellularly, is metabolized without being released into extracellular space, in other words, without functioning as a lipid mediator in the vast majority of cells except those supplying plasma and lymph S1P such as blood cells and endothelial cells. Instead, intracellular S1P plays an important role as an intermediate of the sole sphingolipid-to-glycerophospholipid metabolic pathway. The degradation of S1P by S1P lyase is the first irreversible reaction (committed step) of this pathway. This metabolic pathway is conserved in eukaryotes from yeast to human, indicating its much older origin than the function of S1P as a lipid mediator, which is found to be present only in vertebrates and chordates. The sphingolipid-to-glycerophospholipid metabolism takes place ubiquitously in mammalian tissues, and its defect causes an aberration of several tissue functions as well as abnormal lipid metabolism. Although this metabolic pathway has been known for over four decades, only recently the precise reactions and enzymes involved in this pathway have been revealed. This review will focus on the recent advances in our understanding of the sphingolipid metabolic pathway via S1P and its physiological and pathological roles. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology., 5, 英語

Metabolism of very long-chain fatty acids: genes and pathophysiology.
Sassa T, Kihara A, Biomolecules & therapeutics, 22, 2, 83, 92, 2014年02月, ［査読有り］, ［国際誌］
Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology., 2, 英語

スフィンゴ脂質の代謝と関連疾患　　　　　　　　　　　　　　　
木原 章雄, 医学のあゆみ, 248, 1112, 1118, 2014年, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

スフィンゴシン1-リン酸の代謝経路の全容と中間代謝体としての重要性
木原章雄, 生化学, 85, 553, 557, 2013年07月, ［査読有り］
7

スフィンゴシン1-リン酸の代謝経路　　　　　　　　　　　　　　　
木原 章雄, 遺伝子医学 MOOK「最新生理活性脂質研究 –実験手法，基礎的知識とその応用–」, 24, 175, 179, 2013年, ［招待有り］
日本語, 記事・総説・解説・論説等（学術雑誌）

Very long-chain fatty acids: elongation, physiology and related disorders.
Kihara A, Journal of Biochemistry, 152, 5, 387, 95, 2012年11月, ［査読有り］, ［国際誌］
Very long-chain fatty acids (VLCFAs) are fatty acids (FAs) with a chain-length of ≥22 carbons. Mammals have a variety of VLCFAs differing in chain-length and the number of double bonds. Each VLCFA exhibits certain functions, for example in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function and anti-inflammation. These functions are elicited not by free VLCFAs themselves, but through their influences as components of membrane lipids (sphingolipids and glycerophospholipids) or precursors of inflammation-resolving lipid mediators. VLCFAs are synthesized by endoplasmic reticulum membrane-embedded enzymes through a four-step cycle. The most important enzymes determining the tissue distribution of VLCFAs are FA elongases, which catalyze the first, rate-limiting step of the FA elongation cycle. Mammals have seven elongases (ELOVL1-7), each exhibiting a characteristic substrate specificity. Several inherited disorders are caused by mutations in genes involved in VLCFA synthesis or degradation. In this review, I describe the molecular mechanism of FA elongation and the responsible enzymes in mammals and yeast, as well as VLCFA-related disorders in human., 5, 英語

生理活性脂質とは
木原章雄, 五十嵐靖之, 臨床検査, 56, 107, 116, 2012年, ［招待有り］

オートファジーと小胞輸送におけるスフィンゴ脂質の役割
木原 章雄, 生体の科学「細胞の分子構造と機能 –核以外の細胞小器官–」, 63, 482, 485, 2012年, ［招待有り］

脂肪酸の多彩な代謝
木原章雄, 生化学, 82, 7, 591, 605, 2010年, ［招待有り］
7, 日本語

Ceramide biosynthesis in keratinocyte and its role in skin function.
Mizutani Y, Mitsutake S, Tsuji K, Kihara A, Igarashi Y, Biochimie, 91, 6, 784, 90, 2009年06月, ［査読有り］, ［国際誌］
The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1-CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health., 6, 英語

Production and release of sphingosine 1-phosphate and the phosphorylated form of the immunomodulator FTY720.
Kihara A, Igarashi Y, Biochimica et Biophysica Acta, 1781, 9, 496, 502, 2008年09月, ［査読有り］, ［国際誌］
The bioactive lipid molecule sphingosine 1-phosphate (S1P) binds to specific cell surface receptors and regulates several cellular processes. S1P is abundant in plasma, and physiologically its most important target cells are lymphocytes and vascular endothelial cells. S1P plays a pivotal role in the immune system by regulating lymphocyte egress from the thymus and secondary lymphoid organs. The immunomodulator FTY720 impairs this egress, causing lymphopenia. Platelets had long been considered to be the major source of plasma S1P, however recent studies revealed the importance of erythrocytes as a major supply. The sphingosine analog FTY720 is a prodrug, and FTY720 phosphate (FTY720-P) its functional form. Although both erythrocytes and platelets can produce S1P, only platelets synthesize and release FTY720-P. This review will focus on the recent advances in our understanding of the metabolism and release of S1P and FTY720-P, especially in platelets and erythrocytes., 9, 英語

Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.
Kihara A, Mitsutake S, Mizutani Y, Igarashi Y, Progress in Lipid Research, 46, 2, 126, 44, 2007年03月, ［査読有り］, ［国際誌］
Sphingolipids are major lipid constituents of the eukaryotic plasma membrane. Without certain sphingolipids, cells and/or embryos cannot survive, indicating that sphingolipids possess important physiological functions that are not substituted for by other lipids. One such role may be signaling. Recent studies have revealed that some sphingolipid metabolites, such as long-chain bases (LCBs; sphingosine (Sph) in mammals), long-chain base 1-phosphates (LCBPs; sphingosine 1-phosphate (S1P) in mammals), ceramide (Cer), and ceramide 1-phosphate (C1P), act as signaling molecules. The addition of phosphate groups to LCB/Sph and Cer generates LCBP/S1P and C1P, respectively. These phospholipids exhibit completely different functions than those of their precursors. In this review, we describe recent advances in understanding the functions of LCBP/S1P and C1P in mammals and in the yeast Saccharomyces cerevisiae. Since LCB/Sph, LCBP/S1P, Cer, and C1P are mutually convertible, regulation of not only the total amount of the each lipid but also of the overall balance in cellular levels is important. Therefore, we describe in detail their metabolic pathways, as well as the genes involved in each reaction., 2, 英語

多彩な機能を持つスフィンゴ脂質，スフィンゴシン(長鎖塩基)１-リン酸の生理的役割，生成/代謝，細胞内動態
木原章雄, 生化学, 78, 8, 725, 737, 2006年08月, ［招待有り］
8, 日本語, 書評論文,書評,文献紹介等

Lipid asymmetry of the eukaryotic plasma membrane: functions and related enzymes.
Ikeda M, Kihara A, Igarashi Y, Biological & Pharmaceutical Bulletin, 29, 8, 1542, 6, 2006年08月, ［査読有り］, ［国内誌］
Biological membranes are composed of lipid bilayers. Major lipid components of the eukaryotic plasma membrane include glycerophospholipids, sphingolipids, and cholesterol. Lipids are irregularly distributed between the two leaflets, thus causing lipid asymmetry, or within the same leaflet, forming a lipid microdomain. Glycerophospholipids and sphingolipids both contribute to the lipid asymmetry, whereas cholesterol and sphingolipids form lipid microdomains. Maintenance of proper lipid asymmetry is required for the mechanical stability of the membrane and for vesicular transport. On the other hand, local or global changes in lipid asymmetry are important for cell cycle progression, apoptosis, and platelet coagulation. Three classes of lipid translocases, P-type ATPases, ABC transporters, and scramblases, are known to be involved in the regulation of lipid asymmetry. In this review, we describe the physiological and pathological functions of lipid asymmetry and the current knowledge of lipid translocases., 8, 英語

生理活性脂質スフィンゴシン1−リン酸の機能と代謝
木原章雄, 岩城壮一郎, 五十嵐靖之, 蛋白質核酸酵素, 50, 11, 1443, 1448, 2005年09月, ［招待有り］
11, 日本語

ヒトSphingolipid C4-Hydroxylase,hDES2,のクローニングとケラチノサイトの分化におけるファイトセラミド生成とhDES2発現の相関について
水谷有紀子, 木原章雄, 五十嵐靖之, 脂質生化学研究, 46, 2004年

酵母におけるスフィンゴイド塩基１−リン酸の役割と細胞内動態
木原章雄, 五十嵐靖之, 蛋白質核酸酵素, 47, 431, 437, 2002年03月, ［招待有り］
4 Suppl

マウス,セラミド合成酵素のクローニングおよび酵素学的解析について
水谷有紀子, 木原章雄, 五十嵐靖之, 生化学, 74, 8, 2002年

酵母セラミド合成酵素の同定とその遺伝子欠損株の解析　　　　　　　　　　　　　　　
木原 章雄, ファルマシア, 38, 549, 2002年, ［招待有り］

Erratum: Intracellular stability of α fragments of β-galactosidase: Effects of amino-terminally fused polypeptides (Biochemical and Biophysical Research Communications (1995) 215 No 2 (452-458))
T. Homma, T. Yoshihisa, A. Kihara, Y. Akiyama, K. Ito, Biochemical and Biophysical Research Communications, 227, 642, 1996年01月01日

セラミド研究の新展開 〜基礎から応用へ〜　　　　　　　　　　　　　　　
木原 章雄, スフィンゴ脂質恒常性維持のためのセラミド分解経路
セラミド研究会編，食品化学新聞社, 2019年06月, ［分担執筆］

Bioactive lipid mediators: current reviews and protocols　　　　　　　　　　　　　　　
木原 章雄, Sphingolipid metabolism via sphingosine 1-phosphate
Springer Japan, 2015年, ［分担執筆］

ここまできたセラミド研究最前線 セラミド –基礎と応用–　　　　　　　　　　　　　　　
大野祐介, 木原章雄, 極長鎖脂肪酸伸長とセラミド合成
セラミド研究会編，食品化学新聞社, 2011年, ［分担執筆］

バイオとナノの融合 II 新生命科学の応用　　　　　　　　　　　　　　　
五十嵐靖之, 木原章雄, スフィンゴ脂質の生理活性と代謝調節
北海道大学 COE 研究成果編集委員編，北海道大学出版会, 2007年, ［分担執筆］

Sphingolipid Biology　　　　　　　　　　　　　　　
Kihara A, Igarashi Y, Synthesis, metabolism, and trans-bilayer movement of long-chain base.
Springer-Verlag, 2006年, ［分担執筆］

MEMBRANE PROTEINS : Structure, Function and Expression Control　　　　　　　　　　　　　　　
Ito K, Akiyama Y, Kihara A, Matsuo E, Homma T, Shirai Y, Yoshihisa T, Taura T, Shimoike T, Sone M, Matsumoto G, The SecY Complex and the FtsH complex in the E. coli Plasma Membrane.
Kyushu University Press/ Karger Medical and Scientific Publishers, 1997年, ［分担執筆］

多様なセラミドによる皮膚透過性バリア形成　　　　　　　　　　　　　　　
木原章雄
ADVENT Forum in Japan 2022（サノフィー株式会社医学セミナー）, 2022年09月30日, 公開講演，セミナー，チュートリアル，講習，講義等
2022年09月30日 - 2022年09月30日, ［招待講演］

脂質による体表面バリア形成の分子機構の解明　　　　　　　　　　　　　　　
木原章雄
革新的先端研究開発支援事業（AMED-CREST/PRIME）脂質領域公開シンポジウム, 2022年09月08日, 日本語, 口頭発表（一般）
2022年09月08日 - 2022年09月08日, ［招待講演］

セラミドの多様性と皮膚バリア機能　　　　　　　　　　　　　　　
木原章雄
第94回日本生化学会大会, 2021年11月03日, 日本語, シンポジウム・ワークショップパネル（指名）
2021年11月03日 - 2021年11月05日, ［招待講演］

マイバム脂質の産生機構とドライアイ防止における役割　　　　　　　　　　　　　　　
木原章雄
第14回箱根ドライアイクラブ, 2021年05月28日, 日本語, 口頭発表（招待・特別）
2021年05月28日 - 2021年05月29日, ［招待講演］

セラミドの多様性と皮膚透過性バリア機能　　　　　　　　　　　　　　　
木原章雄
第13回セラミド研究会学術集会, 2020年11月27日, 日本語, 口頭発表（招待・特別）
2020年11月27日 - 2020年11月27日, ［招待講演］

哺乳類セラミドの多様性を生み出す分子機構　　　　　　　　　　　　　　　
木原章雄
第14回スフィンゴテラピィ研究会, 2020年01月25日, 日本語, 口頭発表（一般）
2020年01月23日 - 2020年01月25日

Molecular mechanism producing the skin barrier lipid acylceramide　　　　　　　　　　　　　　　
Kihara, A
60th International Conference on the Bioscience of Lipids, 2019年06月21日, 英語, 口頭発表（一般）
2019年06月17日 - 2019年06月21日

Acylceramide production and skin barrier formation by the fatty acid omega-hydroxylase CYP4F22/Cyp4f39　　　　　　　　　　　　　　　
Itoh N, Ohno Y, Sassa T, Kihara A
FEBS Special Meeting on Sphingolipid Biology (13th Sphingolipid Club & 10th International Ceramide Conference), 2019年05月08日, 英語, ポスター発表
［国際会議］

極長鎖脂肪酸含有脂質による体表面バリア形成　　　　　　　　　　　　　　　
木原章雄
第6回JFAS (Japan/Joy of Fatty Acids Secrets/Society), 2019年03月24日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

皮膚透過性バリアを形成するアシルセラミドの産生機構と皮膚角化症魚鱗癬の分子機構　　　　　　　　　　　　　　　
木原 章雄
第57回日本薬学会東北支部大会, 2018年10月20日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

極長鎖脂質による体表面バリア形成の分子機構　　　　　　　　　　　　　　　
木原 章雄
第40回生体膜と薬物の相互作用シンポジウム, 2018年10月18日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

皮膚透過性バリア脂質アシルセラミドの産生の分子機構と産生不全による病態　　　　　　　　　　　　　　　
木原 章雄
第91回日本生化学会大会, 2018年09月25日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

A decrease in 2-hydroxygalactosylceramide in the brain of the Sjögren-Larsson syndrome gene knockout mice　　　　　　　　　　　　　　　
金武司, 佐々貴之, 野尻光希, 木原章雄
59th International Conference on the Bioscience of Lipids, 2018年09月05日, 英語, ポスター発表
［国際会議］

皮膚バリア脂質アシルセラミドの合成経路と魚鱗癬病態の分子機構の解明　　　　　　　　　　　　　　　
木原 章雄
第42回蛋白質と酵素の構造に関する九州シンポジウム, 2018年08月31日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

脂質による体表面バリア形成　　　　　　　　　　　　　　　
木原 章雄
第174回東京脂質談話会, 2018年04月18日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Long-chain base metabolism and α-oxidation in the ER　　　　　　　　　　　　　　　
木原 章雄
Gordon Research Conference on Glycolipid & Sphingolipid Biology, 2018年02月12日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

スフィンゴ脂質代謝酵素の同定と脂質修飾による調節　　　　　　　　　　　　　　　
木原 章雄
2017年度生命科学系学会合同年次大会, 2017年12月07日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

スフィンゴ脂質長鎖塩基の代謝経路の解明　　　　　　　　　　　　　　　
木原 章雄
日本植物学会第81回大会, 2017年09月08日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

フィトスフィンゴシン代謝と小胞体α酸化の分子機構の解明　　　　　　　　　　　　　　　
木原章雄, 北村拓也, 関直哉, 小原隆
第12回スフィンゴテラピィ研究会, 2017年07月16日, 日本語, 口頭発表（一般）
［国内会議］

Skin barrier formation by acylceramide　　　　　　　　　　　　　　　
木原 章雄
Hokkaido University–Korean Society for Molecular and Cellular Biology Joint Symposium, 2017年07月04日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

脂質による体表面バリア形成機構と病態　　　　　　　　　　　　　　　
木原 章雄
九州大学公開講演会 最新化学談話シリーズ 平成29年度第1回談話会, 2017年05月26日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

スフィンゴ脂質の代謝と生理機能　　　　　　　　　　　　　　　
木原 章雄
2016年度遺伝研研究会「単細胞システム細胞装置のダイナミズム」, 2017年03月28日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

セラミドによる皮膚バリア形成の分子機構と病態　　　　　　　　　　　　　　　
木原 章雄
第41回日本薬学会関東支部学術講演会, 2016年11月19日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

セラミド多様性の分子機構　　　　　　　　　　　　　　　
木原 章雄
第9回セラミド研究会学術集会, 2016年10月28日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Human CYP4F22 in production of acylceramide for skin permeability barrier formation　　　　　　　　　　　　　　　
木原 章雄
21st International Symposium on Microsomes and Drug Oxidations, 2016年10月03日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

シェーグレン・ラルソン症候群モデルマウスを用いた病態のメカニズム解析　　　　　　　　　　　　　　　
木原章雄, 永沼達郎, 金武司, 佐々貴之
第11回スフィンゴテラピィ研究会, 2016年07月16日, 日本語, 口頭発表（一般）
［国内会議］

Kihara A. Identification of genes involved in epidermal acylceramide production　　　　　　　　　　　　　　　
木原 章雄
Gordon Research Conference on Glycolipid & Sphingolipid Biology, 2016年03月07日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Metabolic pathways and genes involved in the synthesis and degradation of ceramides　　　　　　　　　　　　　　　
木原 章雄
6th International Singapore Lipid Symposium, 2015年11月30日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

表皮におけるアシルセラミド産生の分子機構の解明　　　　　　　　　　　　　　　
木原 章雄
第8回セラミド研究会学術集会, 2015年10月30日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

スフィンゴ脂質代謝と皮膚バリア形成　　　　　　　　　　　　　　　
木原 章雄
第120回バイオセミナー（長浜バイオ大学）, 2015年09月29日, 日本語, 公開講演，セミナー，チュートリアル，講習，講義等
［招待講演］, ［国内会議］

Ceramide metabolism and skin barrier formation　　　　　　　　　　　　　　　
木原 章雄
International Conference of Chung-Ang University on Life Science and Medical Reseach, 2015年09月23日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Metabolism, function in skin barrier formation, and pathology of ceramide　　　　　　　　　　　　　　　
木原 章雄
International Conference of the Korean Society for Molecular and Cellular Biology 2015, 2015年09月23日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Molecular mechanism generating acylceramide essential for skin barrier　　　　　　　　　　　　　　　
木原 章雄
第10回スフィンゴテラピィ研究会, 2015年06月18日, 英語, 口頭発表（一般）
［国内会議］

セラミドによる皮膚バリア形成　　　　　　　　　　　　　　　
木原 章雄
東京都医学総合研究所セミナー, 2015年05月28日, 中国語, 公開講演，セミナー，チュートリアル，講習，講義等
［招待講演］, ［国内会議］

Molecular mechanism of the production of acylceramide, the key lipid for skin barrier formation　　　　　　　　　　　　　　　
木原 章雄
8th International Ceramide Conference (iCC8)/ 11th Sphingolipid Club (SLC) joint meeting, 2015年05月07日, 英語, 口頭発表（一般）
［国際会議］

Phytosphingosine is a source of odd-numbered fatty acids via a novel pathway involving the MPO1 gene　　　　　　　　　　　　　　　
木原 章雄
6th International conference on phospholipase A2 and lipid mediators, 2015年02月11日, 英語, ポスター発表
［国際会議］

Regulation of fatty aldehyde dehydrogenases by lipid modification　　　　　　　　　　　　　　　
木原章雄
2nd International symposium on protein modification in pathogenic dysregulation of signaling, 2015年01月24日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

長鎖塩基の吸収と代謝の分子メカニズム　　　　　　　　　　　　　　　
木原 章雄
第7回セラミド研究会学術集会, 2014年10月31日, 日本語, 口頭発表（一般）
［国内会議］

長鎖塩基のグリセロ脂質への代謝経路と代謝酵素の同定　　　　　　　　　　　　　　　
木原 章雄
日本植物学会第78回大会, 2014年09月12日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

セラミドの合成と分解の経路・遺伝子の同定　　　　　　　　　　　　　　　
木原 章雄
第9回スフィンゴテラピィ研究会, 2014年07月18日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

表皮セラミドと長鎖塩基の代謝と生理機能　　　　　　　　　　　　　　　
木原 章雄
ifia JAPAN 2014（第19回国際食品素材／添加物展・会議）, 2014年05月22日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

The fatty acid elongase ELOVL1 and the P450 family member CYP4F22 are involved in the production of epidermal acylceramide　　　　　　　　　　　　　　　
木原 章雄
Gordon Research Conference (Glycolipid & Sphingolipid Biology), 2014年01月16日, 英語, 口頭発表（一般）
［国際会議］

皮膚アシルセラミド産生における脂肪酸伸長酵素と水酸化酵素の解析　　　　　　　　　　　　　　　
木原 章雄
第6回セラミド研究会学術集会, 2013年11月07日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

アシルセラミドの産生と皮膚バリア形成における役割　　　　　　　　　　　　　　　
木原 章雄
第21回未来創薬・医療イノベーションセミナー, 2013年09月17日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

極長鎖脂肪酸の生理機能の解明　　　　　　　　　　　　　　　
木原章雄
第85回日本生化学会大会, 2012年12月14日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

スフィンゴシン1-リン酸代謝経路の全容解明とシェーグレン・ラルソン症候群原因遺伝子の関与　　　　　　　　　　　　　　　
木原章雄, 中原かなえ, 大國礼, 北村拓也, 安部健介, 永沼達郎, 大野祐介
第5回セラミド研究会 学術集会, 2012年10月12日, 日本語, 口頭発表（一般）
［国内会議］

極長鎖脂肪酸の皮膚バリア，小胞輸送における役割　　　　　　　　　　　　　　　
木原章雄
第54回日本脂質生化学会, 2012年06月08日, 日本語
［招待講演］, ［国内会議］

The complete sphingosine 1-phosphate metabolic pathway involves the Sjögren-Larsson syndrome gene.　　　　　　　　　　　　　　　
Kihara A
Gordon Research Conference (Glycolipid & Sphingolipid Biology), 2012年04月25日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

スフィンゴシン1-リン酸の代謝経路の全容解明　　　　　　　　　　　　　　　
木原章雄
日本薬学会第132年会, 2012年03月29日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

The fatty aldehyde dehydrogenase ALDH3A2 is involved in the sphingosine 1-phosphate metabolic pathway.　　　　　　　　　　　　　　　
Kihara A
52nd International Conference on the Bioscience of Lipids, 2011年09月01日, 英語, ポスター発表
［国際会議］

The Sjögren-Larsson syndrome-causative gene ALDH3A2 is involved in the sphingosine 1-phosphate-metabolic pathway.　　　　　　　　　　　　　　　
Kihara A
The 30th Naito Conference on Membrane Dynamics and Lipid Biology [II], 2011年06月30日, 英語, 口頭発表（一般）
［国際会議］

スフィンゴシン１−リン酸代謝経路の解明：シェーグレン・ラルソン症候群原因遺伝子ALDH3A2の関与　　　　　　　　　　　　　　　
木原章雄, 中原かなえ, 安部 健介
第53回日本脂質生化学会, 2011年05月13日, 日本語, 口頭発表（一般）
［国内会議］

脂質非対称を感知するセンサー分子とシグナル伝達経路の解析　　　　　　　　　　　　　　　
木原章雄, 小原圭介
日本薬学会第131年会, 2011年03月31日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

C24アシルCoAの産生とC24セラミド合成による制御機構　　　　　　　　　　　　　　　
木原章雄
第3回セラミド研究会 学術集会, 2010年11月12日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

極長鎖脂肪酸の代謝経路と調節機構　　　　　　　　　　　　　　　
木原章雄
神戸大学医学部グローバルＣＯＥ学術講演会, 2010年10月19日, 日本語, 口頭発表（基調）
［招待講演］, ［国内会議］

Specificity of elongation of saturated, very long-chain fatty acids provides a link to C24 sphingolipid synthesis.　　　　　　　　　　　　　　　
Kihara A
The 27th Naito Conference on Membrane Dynamics and Lipid Biology [I], 2010年06月30日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Elucidation of the pathway of very long-chain fatty acid synthesis and its relationship to sphingolipid metabolism　　　　　　　　　　　　　　　
Kihara A
Gordon Research Conferences (Glycolipid & Sphingolipid Biology), 2010年02月09日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Metabolism and functions of sphingosine 1-phosphate and FTY720.　　　　　　　　　　　　　　　
木原章雄
第31回生体膜と薬物の相互作用シンポジウム, 2009年11月30日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Rim101経路による脂質非対称感知システムの分子機構　　　　　　　　　　　　　　　
木原章雄, 小原圭介
第82回日本生化学会大会, 2009年10月24日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Synthesis of very long-chain fatty acid and its relationship to sphingolipid metabolism　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
50th International Conference on the Bioscience of Lipids, 2009年09月04日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

脂肪酸の多彩な代謝と機能　　　　　　　　　　　　　　　
木原章雄
日本生化学会北海道支部第46回例会, 2009年07月17日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Synthesis of very long-chain fatty acid and its relationship to sphingolipid metabolism and transport.　　　　　　　　　　　　　　　
Kihara A
4th International conference on phospholipase A2 and lipid mediators, 2009年05月27日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

脂質非対称変化による酵母スフィンゴイド塩基トランスロカーゼ発現誘導の分子機構　　　　　　　　　　　　　　　
木原章雄, 池田未佳, 小原圭介, 五十嵐靖之
第31回日本分子生物学会年会／第81回日本生化学会大会 合同大会, 2008年12月11日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

細胞膜脂質非対称感知システムによるスフィンゴイド塩基トランスロカーゼRsb1の発現誘導機構　　　　　　　　　　　　　　　
木原章雄
第176回酵母細胞研究会例会, 2008年12月08日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Phs1ファミリーによるホスファチジルイノシトール輸送とスフィンゴ脂質代謝に及ぼす影響　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会, 2007年12月12日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Phs1ファミリーによるスフィンゴ脂質輸送・代謝調節　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第49回日本脂質生化学会, 2007年06月06日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Palmitoylation of sphingosine kinase and sphingosine 1-phosphate receptor 1 by DHHC Cys-rich domain-containing palmitoyltransferase　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
4th NIBB-EMBL Symposium (Biology of Protein Conjugation Structure and Function), 2006年12月05日, 英語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国際会議］

Palmitoylation of the sphingosine 1-phosphate receptor S1P1 and sphingosine kinase　　　　　　　　　　　　　　　
Kihara A, Ohno Y, Igarashi Y
47th International Conference on the Bioscience of Lipids, 2006年09月, 英語, ポスター発表
［国際会議］

Palmitoylation of the key enzymes in sphingosine 1-phosphate (S1P) signaling, sphingosine kinase and S1P receptor　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
FASEB Summer Research conferences (Protein Lipidation, Signaling & Membrane Domain), 2006年07月26日, 英語, 口頭発表（一般）
［国際会議］

DHHC 蛋白質によるスフィンゴシン１−リン酸関連因子のパルミトイル化　　　　　　　　　　　　　　　
木原章雄, 平賀由起, 大野祐介, 五十嵐靖之
第28回日本分子生物学会年会, 2005年12月10日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

マウススフィンゴシンキナーゼ１の２つの転写バリアントに見られる酵素学的性質の違い　　　　　　　　　　　　　　　
木原章雄, 穴田佳大, 五十嵐靖之
第78回日本生化学大会, 2005年10月19日, 英語, 口頭発表（一般）
［国内会議］

スフィンゴ脂質シグナリングと膜動態の解明　　　　　　　　　　　　　　　
木原章雄
第78回日本生化学大会, 2005年10月19日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

生理活性脂質スフィンゴシン1−リン酸の細胞内代謝機構　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第42回日本生化学会北海道支部例会, 2005年07月22日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

生理活性脂質スフィンゴシン１−リン酸の機能と代謝調節機構　　　　　　　　　　　　　　　
木原章雄
バイオウイーク in Sapporo 2005, 2005年07月05日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Post-translational modification of the yeast and mammalian sphingosine kinase　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
FASEB Summer Research conferences (Lysophospholipid Mediators in Health and Disease), 2005年06月12日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytoplasmic side of the endoplasmic reticulum membrane　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第77回日本生化学大会, 2004年10月15日, 日本語, 口頭発表（一般）
［国内会議］

Posttranslational modification of the yeast sphingosine kinase, Lcb4p, by palmitoylation and phosphorylation　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
Gordon Research Conferences (Glycolipid & Sphingolipid Biology), 2004年07月27日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

Post-translational modification of yeast long-chain base kinase, Lcb4p　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
1st, Sapporo Summer Conference, Sapporo Sphingolipid Symposium, 2004年07月21日, 英語, 口頭発表（招待・特別）
［国際会議］

酵母スフィンゴシンキナーゼ Lcb4p の翻訳後修飾　　　　　　　　　　　　　　　
木原章雄, 岩城壮一郎, 黒津芙美子, 五十嵐靖之
第46回日本脂質生化学会, 2004年06月19日, 日本語, 口頭発表（一般）
［国内会議］

パルミトイルトランスフェラーゼ Akr1p による酵母スフィンゴシンキナーゼ Lcb4p の翻訳後修飾　　　　　　　　　　　　　　　
木原章雄, 黒津芙美子, 岩城壮一郎, 五十嵐靖之
第26回日本分子生物学会年会, 2003年12月10日, 日本語, 口頭発表（一般）
［国内会議］

Characterization of phytosphingosine resistant yeast mutants isolated by transposon insertion　　　　　　　　　　　　　　　
木原章雄, 黒津芙美子, 岩城壮一郎, 佐野孝光, 五十嵐靖之
第76回日本生化学大会, 2003年10月17日, 日本語, 口頭発表（一般）
［国内会議］

Sphingosine-1-phosphate lyase, SPL, is involved in the differentiation of F9 embryonal carcinoma cells to primitive endoderm　　　　　　　　　　　　　　　
Kihara A, Ikeda M, Kariya Y, Igarashi Y
FASEB Summer Research Conferences (Lysophospholipid and Related Lipids in Biology and Diseases, 2003年06月29日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

スフィンゴシン１−リン酸リアーゼ遺伝子欠損による分化応答の解析　　　　　　　　　　　　　　　
木原章雄
日本生化学大会，分子生物学会，日本生物物理学会の合同シンポジウム, 2002年11月22日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

スフィンゴシン１−リン酸リアーゼ遺伝子欠損による分化応答の解析　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第75回日本生化学大会, 2002年10月14日, 日本語, 口頭発表（一般）
［国内会議］

スフィンゴシン１−リン酸 (S1P) リアーゼ欠損細胞を用いた細胞分化応答の解析　　　　　　　　　　　　　　　
木原章雄
群馬大学生体調節研究所シンポジウム「脂質メディエーターの動態制御と生体機能」, 2002年08月30日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Topological analysis of sphingoid base 1-phosphate phosphatases and identification of transporter protein responsible for resistance to sphingoid bases in yeast　　　　　　　　　　　　　　　
Kihara A, Igarashi Y
Gordon Research Conferences (Glycolipid & Sphingolipid Biology), 2002年01月28日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

脂質ホスファターゼによるスフィンゴイド塩基１−リン酸の ER 膜への取り込み　　　　　　　　　　　　　　　
木原章雄, 五十嵐靖之
第24回日本分子生物学会年会, 2001年12月10日, 日本語, 口頭発表（一般）
［国内会議］

PI 3-K との相互作用を介した Apg6p/Beclin の Golgi - エンドソーム間輸送及びオートファジーにおける役割　　　　　　　　　　　　　　　
木原章雄, 野田健司, 吉森保, 大隅良典
第23回日本分子生物学会年会, 2000年12月16日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Apg6p と相互作用する因子の同定及びその複合体の解析　　　　　　　　　　　　　　　
木原章雄, 野田健司, 大隅良典
第73回日本生化学大会, 2000年10月12日, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Apg6p 複合体の同定と解析　　　　　　　　　　　　　　　
木原章雄, 野田健司, 大隅良典
第33回酵母遺伝学フォーラム, 2000年08月08日, 日本語, ポスター発表
［国内会議］

FtsH による膜蛋白質の processive degradation　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 千葉志信, 伊藤 維昭
第72回日本生化学大会, 1999年10月08日, 日本語, 口頭発表（一般）
［国内会議］

λ CII 蛋白質の分解に関わる新たな大腸菌因子 HflD の同定及び解析　　　　　　　　　　　　　　　
木原章雄, 伊藤維昭
第21回日本分子生物学会年会, 1998年12月17日, 日本語
［国内会議］

膜結合型 ATP 依存性プロテアーゼ FtsH による膜タンパク質YccAの分解　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第71回日本生化学会大会, 1998年10月16日, 日本語
［国内会議］

膜結合型プロテアーゼ FtsH によるタンパク質のクオリティーコントロール　　　　　　　　　　　　　　　
木原章雄, 伊藤維昭
蛋白質研究所セミナー「蛋白質社会の不可逆的リモデリング」, 1998年06月, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

新たな膜タンパク質基質、YccA を用いた FtsH プロテアーゼの制御機構の解析　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第20回日本分子生物学会年会, 1997年12月17日, 日本語
［国内会議］

HflKCはペリプラズム側から FtsH プロテアーゼを制御する　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第70回日本生化学会大会, 1997年09月25日, 日本語
［国内会議］

FtsH-interacting membrane proteins and their roles in the FtsH functions　　　　　　　　　　　　　　　
Kihara A, Akiyama Y, Ito K
EMBO Workshop on Cellular Functions of AAA Proteins, 1997年05月, 英語
［国際会議］

FtsH の HflKC との複合体形成および活性調節　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 吉久徹, 伊藤維昭
第69回日本生化学会大会、第 19 回日本分子生物学会年会 合同年会, 1996年08月28日, 日本語
［国内会議］

FtsH と膜結合プロテアーゼ HflK/C との複合体形成　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第18回日本分子生物学会年会, 1995年12月09日, 日本語
［国内会議］

多機能膜 ATPase FtsH の機能と構造の解析　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
ワークショップ AAA ファミリー蛋白の機能, 1995年03月, 日本語, 口頭発表（一般）
［国内会議］

蛋白質膜透過因子 SecY の分解系の遺伝解析　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第17回日本分子生物学会年会, 1994年12月, 日本語
［国内会議］

単独 SecY の分解に FtsH が関与する　　　　　　　　　　　　　　　
木原章雄, 秋山芳展, 伊藤維昭
第67回日本生化学会大会, 1994年09月, 日本語
［国内会議］

ホスホエノールピルビン酸カルボキシラーゼの保存性 Arg438 の変異による四次構造の変化とアロステリック調節能の変化　　　　　　　　　　　　　　　
木原章雄, 寺田和豊, 泉井桂
第66回日本生化学会大会, 1993年10月, 日本語
［国内会議］

生化学II　　　　　　　　　　　　　　　
北海道大学

生化学I　　　　　　　　　　　　　　　
北海道大学

薬理学III　　　　　　　　　　　　　　　
北海道大学

多様なセラミドによる生体バリア形成
科学研究費助成事業 基盤研究(S)
2022年04月27日 - 2027年03月31日
木原 章雄
日本学術振興会, 基盤研究(S), 北海道大学, 22H04986

セラミド多様性を創出・維持する代謝経路とその破綻に起因する病態の分子機構の解明　　　　　　　　　　　　　　　
科学研究費補助金(基盤研究(A))
2018年04月 - 2022年03月
木原 章雄
文部科学省, 研究代表者, 競争的資金

脂質による体表面バリア形成の分子機構の解明　　　　　　　　　　　　　　　
AMED-CREST
2015年12月 - 2021年03月
木原 章雄
国立研究開発法人 日本医療研究開発機構, 研究代表者, 競争的資金

極長鎖脂質による生体機能調節とその破綻による病態の分子機構の解明　　　　　　　　　　　　　　　
科学研究費補助金(新学術領域研究)
2018年04月 - 2020年03月
木原 章雄
文部科学省, 研究代表者, 競争的資金

極長鎖脂肪酸の産生及び代謝機構と代謝異常による病態の解明
科学研究費補助金(基盤研究(A))
2014年 - 2017年
木原 章雄
極長鎖脂肪酸の産生に関わる脂肪酸伸長サイクルでは，ELOVL1-7，KAR，HACD1-4，TERによって４つの反応が触媒される。本研究ではHACD遺伝子のノックアウトマウスあるいはノックアウト細胞を用いてミオパチーの分子機構，HACD1-4の基質特異性の違いを解明した。ELOVLの変異マウスや生化学的な解析によって，ELOVLの基質特異性，KARによる活性調節機構を解明した。皮膚バリア形成に重要な極長鎖脂質アシルセラミドの産生に関与する脂肪酸オメガ水酸化酵素CYP4F22を同定し，魚鱗癬発症の分子機構を解明した。TERの脂肪酸伸長以外の新たな機能を明らかにした。
文部科学省, 基盤研究(A), 北海道大学, 研究代表者, 競争的資金, 26251010

スフィンゴシン1-リン酸代謝経路に関わる遺伝子の同定と関連疾患の解析
科学研究費補助金(新学術領域研究(研究領域提案型))
2013年 - 2014年
木原 章雄
スフィンゴシン1-リン酸（S1P）は脂質メディエーターとしての機能だけでなく，スフィンゴ脂質をグリセロリン脂質へ代謝させる経路の分岐点に位置する代謝中間体としての側面も持つ。この代謝経路の反応の詳細や反応に関わる酵素の遺伝子は長い間不明なままであったが，最近我々はその殆どを明らかにすることに成功した。本年度我々は最後まで未同定であったS1P代謝経路の最後の反応を触媒するトランス-2-エノイルCoAレダクターゼをコードする遺伝子としてTERを同定することに成功した。TERはこれまで極長鎖脂肪酸の産生に働くことが報告されており，我々の結果はTERがスフィンゴ脂質代謝と極長鎖脂肪酸産生の２つの機能を持つことを示している。ALDH3A2はS1P代謝に関わる脂肪族アルデヒドデヒドロゲナーゼとして我々が近年同定した遺伝子である。ヒトにおいてALDH3A2遺伝子変異は皮膚神経疾患であるシェーグレン・ラルソン症候群を引き起こすことが知られている。我々はAldh3A2ノックアウトマウスの解析を行い，ミエリン機能に重要な2-ヒドロキシガラクトシルセラミドが減少していることを見いだし，神経症状に対する発症メカニズムの知見を得た。一方，このノックアウトマウスでは明らかな皮膚症状は見られなかったが，この原因としてマウスの表皮ではAldh3A2と同様の活性を示すAldh3B2の発現が高いためであることを明らかにした。我々はこれまでにフィトスフィンゴシンが奇数鎖脂肪酸に代謝されることを酵母を用いた解析から明らかにしてきたが，本年度は動物細胞でも同様にフィトスフィンゴシンが奇数鎖脂肪酸へと代謝されることを明らかにした。この代謝にはS1Pと同様にAldh3A2が関与していた。生体内の奇数鎖脂肪酸の供給源には不明な点が多かったが，我々は酵母の奇数鎖脂肪酸の約６割がフィトスフィンゴシンに由来することを明らかにした。
文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, 研究代表者, 競争的資金, 25116701

パルミトイル化によるタンパク質機能調節と病態の分子機構
科学研究費補助金(新学術領域研究(研究領域提案型))
2013年 - 2014年
木原 章雄
パルイトイル化はタンパク質のシステイン残基にパルミチン酸が付加する翻訳後修飾の１つである。パルミトイル化を受けるシステイン残基には決まったモチーフがないことから，パルミトイル化修飾の有無は予測が難しいことが多い。パルミトイル化を触媒する酵素はプロテインアシルトランスフェラーゼ（PAT）と呼ばれ，ヒトにはDHHC1-22及びDHHC24の23種が存在する。Gタンパク質共役型受容体（GPCR）の多くは7回目の膜貫通領域直下のシステイン残基でパルミトイル化を受けることが知られているが，その反応を触媒するPATについては不明である。我々はGPCRの中でも，スフィンゴシン1-リン酸受容体S1P1に着目し，最近我々が開発した酵母を用いたアッセイ系を利用して，S1P1のPATの同定を行った。その結果，23種のヒトDHHCタンパク質のうち，DHHC2, 3, 6, 7, 10, 15, 20が活性を示すことが明らかとなった。また，これらのうち，DHHC2, 3, 7, 10, 15, 20は別のスフィンゴシン1-リン酸受容体であるS1P5に対しても活性を示した。これらのDHHCタンパク質はGPCRに共通したPATである可能性が高い。癌遺伝子にコードされるRasのパルミトイル化はRasの細胞膜局在と活性に必要である。我々はRasパルミトイル化の阻害剤の探索を行い，9600種の化合物の中から一次スクリーニングで16個の候補化合物を見いだした。さらに，これらを用いて二次スクリーニングを行った結果，現在3個の候補を得ている。スフィンゴシン1-リン酸の代謝に関わるアルデヒドデヒドロゲナーゼALDH3ファミリーの解析により，ALDH3B1がC末端側でパルミトイル化とプレニル化の脂質修飾を受けることを明らかにした。これらの脂質修飾はALDH3B1の細胞膜局在と長鎖アルデヒドに対する活性に重要であった。
文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, 研究代表者, 競争的資金, 25117701

脂質非対称を感知して伝達する分子機構の解明
科学研究費補助金(挑戦的萌芽研究)
2013年 - 2014年
木原 章雄
細胞膜二重層では外層と内層の脂質組成が異なる（脂質非対称）。我々はこれまでに脂質非対称を感知してシグナルを伝える経路としてRim101経路を同定してきた。Rim101経路は外界のアルカリ化に応答する経路としても知られる。従来のモデルでは外界のアルカリ化が起こるとまずセンサータンパク質Rim21がエンドサイトーシスし，その後エンドソーム上で下流因子を集積させて転写因子Rim101を切断・活性化するというものであった。しかし我々は今回，従来のモデルとは異なり，これらのRim101経路の因子群（Rim8，Rim13，Rim20，Snf7）が細胞膜上にパッチ状に集積してシグナルを伝えることを明らかにした。また，ユビキチンリガーゼRSP5の変異体を用いた解析から，この過程にはユビキチン化が関与することも見いだした。マイクロアレイを用いた解析から，脂質非対称が乱れた際にRim101経路依存的に誘導される遺伝子群を抽出し，機能未知のOpt2を同定した。ホスファチジルエタノールアミン（PE）のフリップに関わるLEM3欠損株ではPEが外層に露出しており，外層のPEに結合して毒性を示す薬剤Duramycinに対して感受性を示す。一方，OPT2とLEM３の二重欠損株は耐性を示した。また，PEに結合するRoペプチドにより外層に露出したPEの可視化を行ったところ，LEM3欠損株よりもOPT2とLEM３の二重欠損株ではPEの露出が低下していた。さらに，Opt2を過剰発現させると蛍光標識したPEの取り込み量が減少した。これらのことから，Opt2がこれまでに報告されているフロッパーゼのファミリーには属さない新しいタイプのフロッパーゼであることが示唆された。
文部科学省, 挑戦的萌芽研究, 北海道大学, 研究代表者, 競争的資金, 25650059

極長鎖脂肪酸産生の分子メカニズムと病態,生理機能の解明
科学研究費補助金(基盤研究(B))
2011年 - 2013年
木原 章雄, 佐々 貴之, 小原 圭介, 大野 祐介
炭素数20を超える脂肪酸である極長鎖脂肪酸の細胞内量は長鎖脂肪酸（C11-C20）よりも少ないものの，長鎖脂肪酸では代替できない特長的な生理機能を有し，様々な疾患と関連する。本研究課題において我々はノックアウトマウスや培養細胞を用いた生理学的・細胞生物学的な解析，精製した極長鎖脂肪酸合成酵素を用いた生化学的な解析，酵母を用いた遺伝学的な解析により，極長鎖脂肪酸の皮膚バリアにおける役割，病態との関連（副腎白質ジストロフィー，ミオパチー，精神遅滞），セラミド合成酵素による調節機構，エンドソームを介した小胞輸送での働きを明らかにした。
文部科学省, 基盤研究(B), 北海道大学, 研究代表者, 競争的資金, 23370057

スフィンゴシン1-リン酸代謝経路の解明とシェーグレン・ラルソン症候群の原因究明
科学研究費補助金(新学術領域研究(研究領域提案型))
2011年 - 2012年
木原 章雄
1.スフィンゴシン1-リン酸(SIP)の代謝経路の解明SIPはSIPリアーゼによりヘキサデセナール(アルデヒド)へ分解された後,パルミトイルCoAを経てグリセロリン脂質へ代謝されることが知られている。この過程には少なくともアルデヒドから脂肪酸への酸化,アシルCoAへの変換,二重結合の飽和(還元)の3反応が含まれているはずであるが,触媒酵素の同定とこれらの反応の順序は不明であった。我々は本研究課題が始まる以前に,ヘキサデセナールが脂肪族アルデヒドデヒドロゲナーゼALDH3A2によってヘキサデセン酸に変換されることを見出していた。本申請では酵母Faa1,Faa4,ヒトACSL1,3,5,6がSIP代謝過程に関与するアシルCoA合成酵素であることを明らかにした。また,酵母FAA1,FAA4遺伝子の二重欠損株を用いて放射標識されたスフィンゴシンのトレーサー実験を行なった結果,ヘキサデセン酸が代謝中間体であることも見出した。このことは,SIPの代謝がヘキサデセナール,ヘキサデセン酸,ヘキサデセノイルCoA,パルミトイルCoAと進行していることを表していた。ヘキサデセノイルCoAからパルミトイルCoAの変換にはトランスエノイルCoA還元酵素が関わると予測し,酵母TSC13の変異株を用いた解析を行なった。その結果,この変異株中でSIPのグリセロリン脂質への代謝が低下していることを見出した。2.Aldh3A2ノックアウトマウスの解析EMMAから入手したAldh3A2ノックアウトマウスはジーントラップによる遺伝子欠損であったため,遺伝子が完全に欠損されていない可能性があった。そこで,全身でCreリコンビナーゼを発現するマウスと掛け合わせ,完全欠損を試みた。現在ヘテロノックアウトマウスが完成し,近いうちにホモノックアウトマウスが産まれる予定である。
文部科学省, 新学術領域研究(研究領域提案型), 北海道大学, 研究代表者, 競争的資金, 23116501

脂質非対称を感知するセンサーと細胞内シグナルの分子機構の解明
科学研究費補助金(挑戦的萌芽研究)
2011年 - 2012年
木原 章雄, 小原 圭介
脂質非対称とは細胞膜脂質二重層において外層と内層の脂質分子の組成が異なることを指す。我々はこれまでに脂質非対称変化を感知し,下流に伝達する経路としてRim101経路を同定してきた。本研究において我々はRim101経路の因子の中でもRim21がセンサーとして機能し,Rim9,Dfg16はRim21と複合体を形成してその細胞内局在,安定性を調節していることを明らかにした。また,脂質非対称が変化した際に,細胞機能を保つ役割を持つ新規因子としてOpt2を同定した。
文部科学省, 挑戦的萌芽研究, 北海道大学, 研究代表者, 競争的資金, 23657120

Phs1ファミリーによるホスファチジルイノシトールの細胞内輸送機構の解明
科学研究費補助金(若手研究(A))
2008年 - 2010年
木原 章雄, 佐々 貴之, 小原 圭介
酵母Phs1は極長鎖脂肪酸の伸長に関わる3-ヒドロキシアシルCoA脱水酵素である。Phs1は脂質代謝に関わる因子や小胞輸送因子と相互作用し,スフィンゴ脂質合成やホスファチジルイノシトール(PI)輸送を調節していることを明らかにした。Phs1の哺乳類ホモログは未同定であったが,我々はHACD1-4を同定することに成功した。また,哺乳類の極長鎖脂肪酸伸長経路の律速段階を触媒する酵素の基質特異性を明らかにすることにより,極長鎖脂肪酸伸長経路の全貌を解明した。さらに,極長鎖脂肪酸伸長と疾患との関わりについても知見を得た。
文部科学省, 若手研究(A), 北海道大学, 研究代表者, 競争的資金, 20687008

三量体Gタンパク質共役型受容体の脂質修飾による新たな制御機構の解明
科学研究費補助金(萌芽研究, 挑戦的萌芽研究)
2008年 - 2009年
木原 章雄
三量体Gタンパク質共役型受容体(GPCR)の殆どがパルミトイル化(S-アシル化)修飾を受けることが知られているが,その修飾の役割は受容体毎に異なっている。我々はこれまでにスフィンゴシン1-リン酸受容体S1P_1がパルミトイル化を受け,その修飾が効率的なシグナル伝達とインターナリゼーションに重要であることを明らかにしてきた。本年度の研究において我々はその詳細な分子機構の解析を行なった結果,パルミトイル化がインターナリゼーションに重要なセリン残基のリン酸化に必要であることを見出した。さらに本年度は類縁のGPCRであるスフィンゴシン1-リン酸受容体S1P_2とリゾホスファチジン酸受容体LPA_2のパルミトイル化の役割を解析することにより,GPCRにおけるパルミトイル化の役割を規定する因子の解明を目指した。我々はLPA_2のパルミトイル化を受ける2つのシステイン残基を同定し,そのシステインを含むCXXC配列が,LIMドメイン含有タンパク質TRIP6とアポトーシス関連因子Siva-1との結合に関わることを明らかにした。LPA_2を介したシグナルはアポトーシス刺激と拮抗して細胞の生存に関わるが,この相互作用が失われるとその効果が減弱することも見出した。一方,S1P_2もS1P_1やLPA_2と同様に7回目の膜貫通部位直下の細胞質領域に存在するシステイン残基でパルミトイル化を受けることを見...
文部科学省, 萌芽研究, 挑戦的萌芽研究, 北海道大学, 研究代表者, 競争的資金, 20659007

生理活性スフィンゴ脂質の生理機能と病態の分子機構
科学研究費助成事業
2006年 - 2007年
五十嵐 靖之, 木原 章雄, 光武 進, 水谷 有紀子, 岩城 壮一郎
スフィンゴシン1-リン酸(S1P)は、受容体を介して血管系・免疫系で重要な働きを示す事が明らかになり、創薬のターゲットとして注目を集めている。我々は、この研究課題でS1P産生酵素であるスフィンゴシンキナーゼが、スフィンゴシンキナーゼ1と2で、異なる転写調節を受けている事を明らかにし、S1P産生のメカニズムに新しい知見を加えた。次に血中でのS1Pの供給源としてこれまで血小板が考えられてきたが、赤血球が新たな供給源となる事を明らかにした。S1P受容体の研究では、S1P受容体のC末端がパルミトイル化をうけ、それが、受容体のインターナリゼーションを調節している事を明らかにした。FTY-720はS1P受容体のアゴニストとして、S1P受容体を介した白血球の遊走を制御し、免疫抑制剤としての効果を持つ事が知られている。しかしその詳細な代謝を含めて謎が多い。我々はFTY-720が細胞内でリン酸化された後、LPPファミリーのフォスファターゼによって脱リン酸化を受ける事を明らかにした。
一方、S1Pと並ぶ生理活性スフィンゴ脂質であるセラミド(Cer)に関して、我々はその合成酵素の一つであるLAS3(CerS3)を新たにクローニングした。本酵素はこれまでしられていたLAS5などと比較して、長鎖の脂肪酸を転移する活性を有し、スフィンゴ脂質に特徴的な長鎖構造を作る上で重要であると考えられ興味深い。また、最近新たに見つかったCer代謝酵素の一つであるセラミドキナーゼ(CERK)に関しては、その遺伝子欠損マウスの解析を通して、CERKが小脳、特にプルキンエ細胞に高発現し、マウスの情動性に関わっている可能性を示した。
上述の様に、生理活性スフィンゴ脂質に関してS1P, S1P受容体、Cer, Cer代謝酵素の新たな調節メカニズムを明らかにした。
日本学術振興会, 基盤研究(B), 北海道大学, 18370050

細胞膜脂質非対称の制御機構及び脂質非対称シグナルの解明
科学研究費補助金(若手研究(A))
2005年 - 2007年
木原 章雄
細胞膜脂質二重膜における脂質の外層と内層の組成は異なっており(脂質非対称),ホスファチジルコリンや複合スフィンゴ脂質は外層に存在する。一方,他のグリセロリン脂質は内層に偏っている。近年,この脂質非対称の維持が生体機能に重要であることが明らかとなってきた。我々は以前,酵母においてスフィンゴイド塩基トランスロカーゼRsb1を同定し,この発現がグリセロリン脂質非対称変化により誘導されることを見出した。こめことはグリセロリン脂質の非対称変化を感知するセンサー蛋白質と下流シグナル伝達経路の存在を示唆していた。我々は,本研究において実際にそのシグナル伝達経路としてRim経路を同定することに成功した。この経路は培地中のアルカリや電荷に応答することが報告されており,脂質非対称変化により外層に露出した酸性リン脂質の電荷をRim経路の最上流に位置して細胞膜受容体として考えられているRim21あるいはDfg16が感知していると推定される。本年度はこれらの経路とRsb1発現上昇の関係を詳細に検討し,Rsb1の発現上昇が転写レベルで制御されている事をレポーターアッセイにより定量的に示した。また,脂質非対称変化が実際にRim経路を活性化することを最下流因子Rim101のプロセッシングを指標にして明らかにした。さらに,小胞輸送,細胞壁合成,脂質合成などの異常による間接的な影響でRim経路が活性化しているわ...
文部科学省, 若手研究(A), 北海道大学, 研究代表者, 競争的資金, 17687011

細胞内スフィンゴシン1-リン酸の機能と動態の解明
科学研究費補助金(若手研究(B))
2003年 - 2004年
木原 章雄
スフィンゴシン1-リン酸(S1P)は、細胞外からの受容体を介する作用と細胞内での様々な刺激のセカンドメッセンジャーとしての作用を併せ持つユニークな生理活性脂質である。申請者は、S1PがER脂質二重膜の細胞質側と内腔側で異なった代謝を受けることを見いだした。すなわち、S1Pは細胞質側でS1Pリアーゼ(SPL)により分解されるのに対して、内腔側ではS1Pホスファターゼによって脱リン酸化される。また、SPLを遺伝学的に欠損させたマウスF9細胞を用いた解析から、細胞内S1P自身が細胞増殖作用を持つのでなく、S1Pリアーゼを介して分解される経路の活性化が重要であることを見いだした。我々は動物細胞を用いた解析に加え、酵母の遺伝学を用いて細胞内のスフィンゴイド長鎖塩基1-リン酸(LCBP)(S1P類似体;S1Pと同様の代謝、働きを持つ)の作用経路、動態、生成調節機構の解明を行なっている。これらの解析の中で、LCBP合成酵素であるLcb4pがパルミトイル化、リン酸化、ユビキチン化により複雑に制御されていることを見いだした。パルミトイル化により膜にアンカーしたLcb4pは細胞膜、ER膜に輸送された後、リン酸化、ユビキチン化による修飾を受ける。これらの修飾がシグナルとなり、MVBを介した液胞への輸送後、分解されることを明らかにした。また、LCBPの基質である長鎖塩基(LCB)の脂質二重層での配向...
文部科学省, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 15770078

スフィンゴシン1-リン酸とその受容体の情報伝達機構と生理機能の解析
科学研究費助成事業
2000年 - 2004年
五十嵐 靖之, 木原 章雄, 光武 進, 矢冨 裕, 岡島 史和, 井ノ口 仁一, 和田 淳
スフィンゴ脂質のシグナル機構や受容体の研究で五十嵐班はこの1年間で,48報の原著論文を発表するなど大きな成果を上げた。特に主立った成果として(1)ヒトスフィンゴ脂質C-4水酸化酵素hDES2を始めてクローニングした(FEBS Lett,2004) (2)マスト細胞で抗原刺激での顆粒球放出にセラミドキナーゼが関与していることを明らかにした(JBC,2004) (3)酵母細胞膜の非対称性維持機構の存在,特にスフィンゴ脂質とグリセロリン脂質のクロストーク機構の存在を初めて示した(MBC,2004) (4)哺乳類スフィンゴ脂質代謝のうちで最後まで残されていた3-ケトヒドロキシスフィンゴシン還元酵素(FTV-1)のクローニングに成功した(JBC,2004) (5)酵母細胞スフィンゴシンキナーゼのリン酸化機構とその分解における役割を明らかにした(JBC,2004) (6)日米共同研究でEDG-6とS1Pの結合機構を分子モデルを使って示した(BJ、2005) (7)スフィンゴシンキナーゼ2の特異的阻害剤を合成開発した(BMC,2005)などの成果をあげている。
日本学術振興会, 特定領域研究, 北海道大学, 12140201

血小板由来のリゾ体生理活性脂質の生成、放出機構の研究
科学研究費助成事業
2001年 - 2003年
五十嵐 靖之, 矢冨 裕, 光武 進, 木原 章雄, 和田 淳
1)ヒト血小板からのスフィンゴシン1-リン酸(S1P)とリゾホスファチジン酸(LPA)の放出をそれぞれを2次元TLCで分離する方法をはじめて確立し、両生理活性脂質のトロンビン刺激による放出を初めて確認した。
2)Tigyi G.らの作成したモデルを実験的に証明し,S1Pと相互作用するアミノ酸残基を同定した。この結果により、コンピュテーショナルモデリングの有用性が実験的に示され、S1P受容体におけるリガンドと受容体との相互作用についての情報が得られた。
3)血小板からのスフィンゴシン1-リン酸の放出機構の研究はいくつかの学会で報告したが,まだ最終的なトランスポーターの同定にはいたらなかった。又血小板から分泌されるLPA合成にかかわるホスホリパーゼの精製,同定は精力的に試みたが,最終目的遺伝子の同定はまだできていない。
日本学術振興会, 基盤研究(B), 北海道大学, 13480198

Elucidation of metabolism, function, dynamics, and pathology of lipids　　　　　　　　　　　　　　　
2000年
競争的資金

セラミド合成酵素 Lass6 を用いたフィトセラミドおよびαハイドロキシセラミドの製造方法　　　　　　　　　　　　　　　
特許権
2007-104926

スフィンゴシンキナーゼ活性を有する分泌型ポリペプチドおよびそれをコードするスフィンゴシンキナーゼ遺伝子　　　　　　　　　　　　　　　
特許権
WO2003/031628

UHB大学　　　　　　　　　　　　　　　
2020年11月17日 - 2020年11月17日
講師
講演会
北海道文化放送

道新アカデミー　　　　　　　　　　　　　　　
2019年09月26日 - 2019年09月26日
講師
講演会
北海道新聞

第10回北大セミナーin旭川　　　　　　　　　　　　　　　
2019年07月20日 - 2019年07月20日
講師
出前授業
北海道大学

体を守るバリアとしての脂質　　　　　　　　　　　　　　　
2021年12月
内閣府/総合科学技術・イノベーション会議エビデンス事業
みらいぶっく 学問・大学なび
https://www.sekaiwokaeyo.com/ni019/, ［インターネットメディア］

脂質の長さがもたらすバリア機能を探る　　　　　　　　　　　　　　　
2021年06月
リバネス
研究応援 vol.22
p8-9, ［新聞・雑誌］

ドライアイ防ぐ涙の脂質特定　　　　　　　　　　　　　　　
2020年04月22日
本人以外
北海道新聞
夕刊2面, ［新聞・雑誌］

涙中の特殊な脂質がドライアイを防ぐ 北海道大が解明　　　　　　　　　　　　　　　
2020年04月17日
Science Portal
https://scienceportal.jst.go.jp/newsflash/20200417_01/, ［インターネットメディア］

体守る皮膚の脂質 合成に関わる遺伝子　　　　　　　　　　　　　　　
2020年01月29日
日経新聞
9面, ［新聞・雑誌］

ドライアイ防止 涙の脂質が重要　　　　　　　　　　　　　　　
2018年02月01日
毎日新聞
［新聞・雑誌］

Gene Involved in defective skin barrier and eczema and ichthyosis identified　　　　　　　　　　　　　　　
2017年04月17日
MedicalResearch.com
https://medicalresearch.com/author-interviews/gene-involved-in-defective-skin-barrier-and-eczema-and-ichthyosis-identified/33925/, ［インターネットメディア］

皮膚バリア形成に最も重要な脂質の産生機構解明　　　　　　　　　　　　　　　
2017年03月24日
北海道医療新聞
5面, ［新聞・雑誌］

皮膚守る脂質解明 北大アトピー治療薬に期待　　　　　　　　　　　　　　　
2017年03月09日
日経産業新聞
［新聞・雑誌］

皮膚バリア形成機構 解明 アトピー性皮膚炎など 治療薬開発に期待も　　　　　　　　　　　　　　　
2015年06月19日
本人以外
北海道医療新聞
3面, ［新聞・雑誌］

皮膚の防御機構解明 北大・名大 アトピー治療に糸口　　　　　　　　　　　　　　　
2015年06月11日
本人以外
日刊工業新聞
19面, ［新聞・雑誌］

第61回日本脂質生化学会，実行委員長　　　　　　　　　　　　　　　
2019年07月04日 - 2019年07月05日
企画立案・運営等
学会・研究会等
木原章雄

2nd Japan-Korea Lipid Joint Symposium, Organizer　　　　　　　　　　　　　　　
2018年09月12日 - 2018年09月14日
企画立案・運営等
学会・研究会等
木原章雄

第143回日本薬学会北海道支部，例会長　　　　　　　　　　　　　　　
2016年05月14日 - 2016年05月15日
企画立案・運営等
学会・研究会等
日本薬学会

FASEB Summer Research conferences, Co-organizer　　　　　　　　　　　　　　　
2013年08月04日 - 2013年08月09日
企画立案・運営等
学会・研究会等
五十嵐靖之