研究者データベース

藤岡 優子(フジオカ ユウコ)
遺伝子病制御研究所 疾患制御研究部門
准教授

基本情報

所属

  • 遺伝子病制御研究所 疾患制御研究部門

職名

  • 准教授

学位

  • 博士(薬学)(北海道大学)

科研費研究者番号

  • 80399964

J-Global ID

研究活動情報

論文

  • Yuji Kubota, Yuko Fujioka, Ashwini Patil, Yusuke Takagi, Daisuke Matsubara, Masatomi Iijima, Isao Momose, Ryosuke Naka, Kenta Nakai, Nobuo N. Noda, Mutsuhiro Takekawa
    Nature Communications 13 1 2022年12月 [査読有り]
     
    Abstract Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes. By making use of such distinct characteristics of the MEK1 mutants, we identified cancer- and RASopathy-signature genes that may serve as diagnostic markers or therapeutic targets for these diseases. In particular, two AKT-inhibitor molecules, PHLDA1 and 2, are simultaneously upregulated by oncogenic ERK signaling, and mediate cancer-specific ERK-AKT crosstalk. The combined expression of PHLDA1/2 is critical to confer resistance to ERK pathway-targeted therapeutics on cancer cells. Finally, we propose a therapeutic strategy to overcome this drug resistance. Our data provide vital insights into the etiology, diagnosis, and therapeutic strategy of cancers and RASopathies.
  • Jin Cui, Yuta Ogasawara, Ikuko Kurata, Kazuaki Matoba, Yuko Fujioka, Nobuo N Noda, Masakatsu Shibasaki, Takumi Watanabe
    Journal of the American Chemical Society 2022年09月15日 
    Selective modulation of autophagy is a promising therapeutic strategy, especially for cancer treatment. However, the lack of specific autophagy inhibitors limits this strategy. The formation of the ATG12-ATG5-ATG16L1 complex is essential for targeting the ATG12-ATG5 conjugate to proper membranes and to generate LC3-II for the progression of autophagy. Thus, targeting ATG5-ATG16L1 protein-protein interactions (PPIs) might inhibit early stage autophagy with high specificity. In this paper, we report that a stapled peptide derived from ATG16L1 exhibits potent binding affinity to ATG5, striking resistance to proteolysis, and significant autophagy inhibition activities in cells.
  • Noriyuki Kodera, Daisuke Noshiro, Sujit K Dora, Tetsuya Mori, Johnny Habchi, David Blocquel, Antoine Gruet, Marion Dosnon, Edoardo Salladini, Christophe Bignon, Yuko Fujioka, Takashi Oda, Nobuo N Noda, Mamoru Sato, Marina Lotti, Mineyuki Mizuguchi, Sonia Longhi, Toshio Ando
    Nature nanotechnology 16 2 181 - 189 2021年02月 [査読有り]
     
    Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.
  • Yuko Fujioka, Nobuo N Noda
    Current opinion in cell biology 69 23 - 29 2021年01月11日 [査読有り]
     
    Autophagy is an intracellular degradation system that contributes to cellular homeostasis. Autophagosome formation is a landmark event in autophagy, which sequesters and delivers cytoplasmic components to the lysosome for degradation. Based on selectivity, autophagy can be classified into bulk and selective autophagy, which are mechanistically distinct from each other, especially in the requirement of cargos for autophagosome formation. Recent studies revealed that liquid-like biomolecular condensates, which are formed through liquid-liquid phase separation, regulate the autophagosome formation of both bulk and selective autophagy. Here, we focus on recent findings on the involvement of biomolecular condensates in autophagy regulation and discuss their significance.
  • Akinori Yamasaki, Jahangir Md Alam, Daisuke Noshiro, Eri Hirata, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N Noda
    Molecular cell 77 6 1163 - 1175 2020年03月19日 [査読有り][通常論文]
     
    Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.
  • Yuko Fujioka, Jahangir Md Alam, Daisuke Noshiro, Kazunari Mouri, Toshio Ando, Yasushi Okada, Alexander I May, Roland L Knorr, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N Noda
    Nature 578 7794 301 - 305 2020年02月 [査読有り]
     
    Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.
  • Masaya Yamaguchi, Kenji Satoo, Hironori Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki, Nobuo N Noda
    Journal of molecular biology 430 3 249 - 257 2018年02月02日 [査読有り][通常論文]
     
    Atg8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for Atg8, and it activates the C-terminal Gly116 of Atg8 using ATP. Here, we report the crystal structure of Atg8 bound to the C-terminal domain of Atg7 in an unprecedented mode. Atg8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-Atg8 structures. Instead, Atg8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes Atg8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.
  • Hironori Suzuki, Takuo Osawa, Yuko Fujioka, Nobuo N Noda
    Current opinion in structural biology 43 10 - 17 2017年04月 [査読有り][通常論文]
     
    In autophagy, which is an intracellular degradation system that is conserved among eukaryotes, degradation targets are sequestered through the de novo synthesis of a double-membrane organelle, the autophagosome, which delivers them to the lysosomes for degradation. The core autophagy machinery comprising 18 autophagy-related (Atg) proteins in yeast plays an essential role in autophagosome formation; however, the molecular role of each Atg factor and the mechanism of autophagosome formation remain elusive. Recent years have seen remarkable progress in structural biological studies on the core autophagy machinery, opening new avenues for autophagy research. This review summarizes recent advances in structural biological and mechanistic studies on the core autophagy machinery and discusses the molecular mechanisms of autophagosome formation.
  • Hayashi Yamamoto, Yuko Fujioka, Sho W Suzuki, Daisuke Noshiro, Hironori Suzuki, Chika Kondo-Kakuta, Yayoi Kimura, Hisashi Hirano, Toshio Ando, Nobuo N Noda, Yoshinori Ohsumi
    Developmental cell 38 1 86 - 99 2016年07月11日 [査読有り][通常論文]
     
    Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
  • Fan Wu, Yasunori Watanabe, Xiang-Yang Guo, Xin Qi, Peng Wang, Hong-Yu Zhao, Zheng Wang, Yuko Fujioka, Hui Zhang, Jin-Qi Ren, Tian-Cheng Fang, Yu-Xian Shen, Wei Feng, Jun-Jie Hu, Nobuo N Noda, Hong Zhang
    Molecular cell 60 6 914 - 29 2015年12月17日 [査読有り][通常論文]
     
    Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.
  • Nobuo N Noda, Yuko Fujioka
    Cellular and molecular life sciences : CMLS 72 16 3083 - 96 2015年08月 [査読有り][通常論文]
     
    Autophagosome formation, a landmark event in autophagy, is accomplished by the concerted actions of Atg proteins. Among all Atg proteins, Atg1 kinase in yeast and its counterpart in higher eukaryotes, ULK1 kinase, function as the most upstream factor in this process and mediate autophagy initiation. In this review, we summarize current knowledge of the structure, molecular function, and regulation of Atg1 family kinases in the initiation of autophagy.
  • Yuko Fujioka, Sho W Suzuki, Hayashi Yamamoto, Chika Kondo-Kakuta, Yayoi Kimura, Hisashi Hirano, Rinji Akada, Fuyuhiko Inagaki, Yoshinori Ohsumi, Nobuo N Noda
    Nature structural & molecular biology 21 6 513 - 21 2014年06月 [査読有り][通常論文]
     
    Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established. Here we studied the interactions of yeast Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13 binds tandem microtubule interacting and transport domains in Atg1, using an elongated helix-loop-helix region. Atg13 also binds Atg17, using a short region, thereby bridging Atg1 and Atg17 and leading to Atg1-complex formation. Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17. These observations update the autophagy-initiation model as follows: upon starvation, dephosphorylated Atg13 binds both Atg1 and Atg17, and this promotes PAS assembly and autophagy progression.
  • Nobuo N Noda, Yuko Fujioka, Takao Hanada, Yoshinori Ohsumi, Fuyuhiko Inagaki
    EMBO reports 14 2 206 - 11 2013年02月 [査読有り][通常論文]
     
    Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12-Atg5 conjugate functions as an E3-like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12-Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.
  • Masaya Yamaguchi, Kazuaki Matoba, Ryoko Sawada, Yuko Fujioka, Hitoshi Nakatogawa, Hayashi Yamamoto, Yoshihiro Kobashigawa, Hisashi Hoshida, Rinji Akada, Yoshinori Ohsumi, Nobuo N Noda, Fuyuhiko Inagaki
    Nature structural & molecular biology 19 12 1250 - 6 2012年12月 [査読有り][通常論文]
     
    Autophagy requires ubiquitin-like Atg8 and Atg12 conjugation systems, where Atg7 has a critical role as the sole E1 enzyme. Although Atg7 recognizes two distinct E2s, Atg3 and Atg10, it is not understood how Atg7 correctly loads these E2s with their cognate ubiquitin-like proteins, Atg8 and Atg12. Here, we report the crystal structures of the N-terminal domain of Atg7 bound to Atg10 or Atg3 of thermotolerant yeast and plant homologs. The observed Atg7-Atg10 and Atg7-Atg3 interactions, which resemble each other but are quite distinct from the canonical E1-E2 interaction, makes Atg7 suitable for transferring Atg12 to Atg10 and Atg8 to Atg3 by a trans mechanism. Notably, in vitro experiments showed that Atg7 loads Atg3 and Atg10 with Atg8 and Atg12 in a nonspecific manner, which suggests that cognate conjugate formation in vivo is not an intrinsic quality of Atg7.
  • Nobuo N Noda, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 287 20 16256 - 66 2012年05月11日 [査読有り][通常論文]
     
    Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.
  • Nobuo N Noda, Kenji Satoo, Yuko Fujioka, Hiroyuki Kumeta, Kenji Ogura, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Molecular cell 44 3 462 - 75 2011年11月04日 [査読有り][通常論文]
     
    E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.
  • Hiroyuki Kumeta, Masahiro Watanabe, Hitoshi Nakatogawa, Masaya Yamaguchi, Kenji Ogura, Wakana Adachi, Yuko Fujioka, Nobuo N Noda, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Journal of biomolecular NMR 47 3 237 - 41 2010年07月 [査読有り][通常論文]
  • Yuko Fujioka, Nobuo N Noda, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 285 2 1508 - 15 2010年01月08日 [査読有り][通常論文]
     
    Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5.Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 A resolution and its CCD at 2.5 A resolution. The CCD and full-length Atg16 each exhibit an extended alpha-helix, 90 and 130 A, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.
  • Yukiko Kabeya, Nobuo N Noda, Yuko Fujioka, Kuninori Suzuki, Fuyuhiko Inagaki, Yoshinori Ohsumi
    Biochemical and biophysical research communications 389 4 612 - 5 2009年11月27日 [査読有り][通常論文]
     
    Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.
  • Kenji Satoo, Nobuo N Noda, Hiroyuki Kumeta, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The EMBO journal 28 9 1341 - 50 2009年05月06日 [査読有り][通常論文]
     
    Atg8 is conjugated to phosphatidylethanolamine (PE) by ubiquitin-like conjugation reactions. Atg8 has at least two functions in autophagy: membrane biogenesis and target recognition. Regulation of PE conjugation and deconjugation of Atg8 is crucial for these functions in which Atg4 has a critical function by both processing Atg8 precursors and deconjugating Atg8-PE. Here, we report the crystal structures of catalytically inert human Atg4B (HsAtg4B) in complex with processed and unprocessed forms of LC3, a mammalian orthologue of yeast Atg8. On LC3 binding, the regulatory loop and the N-terminal tail of HsAtg4B undergo large conformational changes. The regulatory loop masking the entrance of the active site of free HsAtg4B is lifted by LC3 Phe119, so that a groove is formed along which the LC3 tail enters the active site. At the same time, the N-terminal tail masking the exit of the active site of HsAtg4B in the free form is detached from the enzyme core and a large flat surface is exposed, which might enable the enzyme to access the membrane-bound LC3-PE.
  • Nobuo N Noda, Hiroyuki Kumeta, Hitoshi Nakatogawa, Kenji Satoo, Wakana Adachi, Junko Ishii, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Genes to cells : devoted to molecular & cellular mechanisms 13 12 1211 - 8 2008年12月 [査読有り][通常論文]
     
    Autophagy is a non-selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double-membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin-like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease-related protein aggregates, respectively. Using X-ray crystallography and NMR, we herein report the structural basis for Atg8-Atg19 and LC3-p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side-chains of Trp and Leu in a four-amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL-containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.
  • Yuko Fujioka, Nobuo N Noda, Minako Matsushita, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 64 Pt 11 1046 - 8 2008年11月01日 [査読有り][通常論文]
     
    Atg16 is a scaffold protein that interacts with Atg12-Atg5 protein conjugates via its N-terminal domain and self-assembles via its coiled-coil domain, thus forming a multimeric Atg12-Atg5-Atg16 complex that is essential for autophagy. The coiled-coil domain of Atg16 was expressed, purified and crystallized. The crystal belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 127.7, c = 77.8 A. Self-rotation functions and volume-to-weight ratio values suggested that the crystal may contain six molecules per asymmetric unit. Since the domain does not contain a methionine residue, selenomethionine-labelled crystals were prepared with a leucine-to-methionine substitution in the coiled-coil domain and these crystals were used for the collection of single-wavelength anomalous dispersion data to 2.5 A resolution.
  • Nobuo N Noda, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Journal of synchrotron radiation 15 Pt 3 266 - 8 2008年05月 [査読有り][通常論文]
     
    Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12-Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12-Atg5-Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12-Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12-Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12-Atg5-Atg16N ternary complex was formed by mixing purified Atg12-Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 A resolution.
  • Hiroyuki Kumeta, Kenji Ogura, Souichirou Adachi, Yuko Fujioka, Nobuhiro Tanuma, Kunimi Kikuchi, Fuyuhiko Inagaki
    Journal of biomolecular NMR 40 3 219 - 24 2008年03月 [査読有り][通常論文]
  • Yuko Fujioka, Nobuo N Noda, Kiyonaga Fujii, Kohki Yoshimoto, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 283 4 1921 - 8 2008年01月25日 [査読有り][通常論文]
     
    Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.
  • Takao Hanada, Nobuo N Noda, Yoshinori Satomi, Yoshinobu Ichimura, Yuko Fujioka, Toshifumi Takao, Fuyuhiko Inagaki, Yoshinori Ohsumi
    The Journal of biological chemistry 282 52 37298 - 302 2007年12月28日 [査読有り][通常論文]
     
    Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.
  • Yumiko Kimura, Arisa Nagao, Yuko Fujioka, Akiko Satou, Takahiro Taira, Sanae M M Iguchi-Ariga, Hiroyoshi Ariga
    International journal of oncology 31 4 829 - 36 2007年10月 [査読有り][通常論文]
     
    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1beta/KAP1, a transcriptional corepressor. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome.
  • Masaya Yamaguti, Nobuo N Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 63 Pt 5 443 - 5 2007年05月01日 [査読有り][通常論文]
     
    Atg10 is an E2-like enzyme that catalyzes the conjugation reaction between Atg12 and Atg5. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Microcrystals of Saccharomyces cerevisiae Atg10 were obtained by the free-interface diffusion method using polyethylene glycol and sodium acetate as precipitants. Using these precipitants, large crystals suitable for data collection were obtained using the sitting-drop vapour-diffusion method. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 51.61, c = 256.16 A, and are estimated to contain two protein molecules per asymmetric unit. A native data set was collected to 2.3 A resolution from a single crystal.
  • Yuya Yamada, Nobuo N Suzuki, Takao Hanada, Yoshinobu Ichimura, Hiroyuki Kumeta, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 282 11 8036 - 43 2007年03月16日 [査読有り][通常論文]
     
    Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.
  • Minako Matsushita, Nobuo N Suzuki, Keisuke Obara, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 282 9 6763 - 72 2007年03月02日 [査読有り][通常論文]
     
    Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5.Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97A resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.
  • Wakana Adachi, Nobuo N Suzuki, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 63 Pt 3 200 - 3 2007年03月01日 [査読有り][通常論文]
     
    The vacuole hydrolase aminopeptidase 1 (Ape1) is a cargo protein transported to the vacuole by the cytosol-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ape1 is processed into mature Ape1 (mApe1). mApe1 has been expressed, purified and crystallized in two crystal forms. Form I belongs to space group P2(1), with unit-cell parameters a = 120.6, b = 219.5, c = 133.1 A, beta = 116.5 degrees. Form II belongs to space group R3, with unit-cell parameters a = 141.2, c = 349.4 A. Diffraction data were collected from these crystals to a resolution of 2.5 A for form I and 1.83 A for form II. Self-rotation functions and the volume-to-weight ratio values suggest that forms I and II contain 12 and four mApe1 molecules per asymmetric unit, respectively, and that mApe1 exists as a tetrahedral dodecamer in both crystal forms.
  • Kenji Satoo, Nobuo N Suzuki, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 63 Pt 2 99 - 102 2007年02月01日 [査読有り][通常論文]
     
    The reversible modification of Atg8 with phosphatidylethanolamine (PE) is crucial for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Atg4 is a cysteine protease that is responsible for the processing and deconjugation of Atg8. Human Atg4B (HsAtg4B; a mammalian orthologue of yeast Atg4) and LC3 (a mammalian orthologue of yeast Atg8) were expressed and purified and two complexes, one consisting of HsAtg4B(1-354) and LC3(1-120) (complex I; the product complex) and the other consisting of HsAtg4B(1-354) and LC3(1-124) (complex II; the substrate complex), were crystallized using polyethylene glycol 3350 as a precipitant. In both complexes His280 of HsAtg4B was mutated to alanine. The crystals belong to the same space group P2(1)2(1)2(1), with unit-cell parameters a = 47.5, b = 91.8, c = 102.6 A for complex I and a = 46.9, b = 90.9, c = 102.5 A for complex II. Diffraction data were collected to a resolution of 1.9 A from both crystals.
  • Takashi Ushimaru, Tomofumi Nakagawa, Yuko Fujioka, Katsue Daicho, Makiko Naito, Yuzo Yamauchi, Hideko Nonaka, Katsumi Amako, Kazuki Yamawaki, Norio Murata
    Journal of Plant Physiology 163 11 1179 - 1184 2006年11月01日 [査読有り][通常論文]
     
    Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance. © 2005 Elsevier GmbH. All rights reserved.
  • Sato Kenji, Suzuki Nobuo N, Sugawara Kenji, Fujioka Yuko, Mizushima Noboru, Ohsumi Yoshinori, Inagaki Fuyuhiko
    AUTOPHAGY 2 4 354  2006年10月 [査読有り][通常論文]
  • Yuya Yamada, Nobuo N Suzuki, Yuko Fujioka, Yoshinobu Ichimura, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 62 Pt 10 1016 - 7 2006年10月01日 [査読有り][通常論文]
     
    Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Crystals of Saccharomyces cerevisiae Atg3 have been obtained by the sitting-drop vapour-diffusion method using ammonium sulfate and lithium sulfate as precipitants. A native data set was collected from a single crystal to 2.5 A resolution. The crystals belong to space group P4(1) or P4(3), with unit-cell parameters a = 59.33, c = 115.22 A, and are expected to contain one protein molecule per asymmetric unit.
  • Kazuya Honbou, Satoru Yuzawa, Nobuo N Suzuki, Yuko Fujioka, Hideki Sumimoto, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 62 Pt 10 1018 - 20 2006年10月01日 [査読有り][通常論文]
     
    p40(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for production of the superoxide that kills invasive microorganisms. Full-length p40(phox) was expressed in Escherichia coli, purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 20,000 as a precipitant. Diffraction data were collected to 3.0 A resolution at 100 K using synchrotron radiation. The crystal belongs to space group C222(1), with unit-cell parameters a = 146.27, b = 189.81, c = 79.88 A. This crystal was estimated to contain two or three protein molecules per asymmetric unit from the acceptable range of volume-to-weight ratio values.
  • Minako Matsushita, Nobuo N Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section F, Structural biology and crystallization communications 62 Pt 10 1021 - 3 2006年10月01日 [査読有り][通常論文]
     
    Atg5 is a novel 34 kDa protein that is covalently modified by Atg12, a ubiquitin-like modifier, and forms a complex with Atg16. The Atg12-Atg5-Atg16 complex localizes to autophagosome precursors and plays an essential role in autophagosome formation. Saccharomyces cerevisiae Atg5 in complex with the N-terminal regions of Atg16 was expressed, purified and crystallized in four crystal forms. Forms I, II and III belong to space group P2(1), with unit-cell parameters a = 66.3, b = 104.4, c = 112.1 A, beta = 92.1 degrees (form I), a = 79.5, b = 101.4, c = 95.1 A, beta = 98.6 degrees (form II) or a = 56.9, b = 101.2, c = 66.5 A, beta = 100.6 degrees (form III). Form IV belongs to space group P4(2)2(1)2, with unit-cell parameters a = 73.3, c = 148.1 A. Diffraction data were collected from all crystal forms and high-resolution data to beyond 2.0 A resolution were obtained from a form IV crystal.
  • Yuko Hagio, Yumiko Kimura, Takahiro Taira, Yuko Fujioka, Sanae M M Iguchi-Ariga, Hiroyoshi Ariga
    Journal of cellular biochemistry 97 1 145 - 55 2006年01月01日 [査読有り][通常論文]
     
    MM-1 was identified as a c-Myc-binding protein and has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting HDAC1 complex via TIF1 beta/KAP1. In this study, originally isolated MM-1 was found to be a fusion protein comprised of the N-terminal 13 amino acids from the sequence of chromosome 14 and of the rest of the amino acids from that of chromosome 12 and was found to be expressed ubiquitously in all human tissues. Four splicing isoforms of MM-1, MM-1alpha, MM-1beta, MM-1gamma, and MM-1delta, which are derived from the sequence of chromosome 12, were then identified. Of these isoforms, MM-1alpha, MM-1gamma, and MM-1delta were found to be expressed in tissue-specific manners and MM-1beta was found to be expressed ubiquitously. Although all of the isoforms potentially possessed c-Myc- and TIF1beta-binding activities, MM-1beta and MM-1delta were found to be mainly localized in the cytoplasm and MM-1alpha and MM-1gamma were found to be localized in the nucleus together with both c-Myc and TIF1beta. Furthermore, when repression activities of MM-1 isoforms toward c-Myc transcription activity were examined by reporter gene assays in HeLa cells, MM-1alpha, MM-1gamma, and MM-1gamma, but not MM-1beta, were found to repress transcription activity of c-Myc, and the degrees of repression by MM-1gamma and MM-1delta were smaller than those by MM-1 and MM-1alpha. These results suggest that each MM-1 isoform distinctly regulates c-Myc transcription activity in respective tissues.
  • Kenji Sugawara, Nobuo N Suzuki, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
    The Journal of biological chemistry 280 48 40058 - 65 2005年12月02日 [査読有り][通常論文]
     
    Reversible modification of Atg8 with phosphatidylethanolamine is crucial for autophagy, the bulk degradation system conserved in eukaryotic cells. Atg4 is a novel cysteine protease that processes and deconjugates Atg8. Herein, we report the crystal structure of human Atg4B (HsAtg4B) at 1.9-A resolution. Despite no obvious sequence homology with known proteases, the structure of HsAtg4B shows a classical papain-like fold. In addition to the papain fold region, HsAtg4B has a small alpha/beta-fold domain. This domain is thought to be the binding site for Atg8 homologs. The active site cleft of HsAtg4B is masked by a loop (residues 259-262), implying a conformational change upon substrate binding. The structure and in vitro mutational analyses provide the basis for the specificity and catalysis of HsAtg4B. This will enable the design of Atg4-specific inhibitors that block autophagy.
  • Yoshiaki Kamada, Yuko Fujioka, Nobuo N Suzuki, Fuyuhiko Inagaki, Stephan Wullschleger, Robbie Loewith, Michael N Hall, Yoshinori Ohsumi
    Molecular and cellular biology 25 16 7239 - 48 2005年08月 [査読有り][通常論文]
     
    The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex 1 and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2(D239A)) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORC1, function. YPK2(D239A) also suppressed the lethality of tor2Delta cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2Delta cells. In contrast, Ypk2(D239A) has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.
  • Nobuo N Suzuki, Kohki Yoshimoto, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Autophagy 1 2 119 - 26 2005年07月 [査読有り][通常論文]
     
    Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.
  • Satoru Yuzawa, Kenji Ogura, Masataka Horiuchi, Nobuo N Suzuki, Yuko Fujioka, Mikio Kataoka, Hideki Sumimoto, Fuyuhiko Inagaki
    The Journal of biological chemistry 279 28 29752 - 60 2004年07月09日 [査読有り][通常論文]
     
    The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O(2).) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).
  • Satoru Yuzawa, Masashi Yokochi, Yuko Fujioka, Kenji Ogura, Hideki Sumimoto, Fuyuhiko Inagaki
    Journal of biomolecular NMR 29 3 451 - 2 2004年07月 [査読有り][通常論文]
  • Kenji Sugawara, Nobuo N Suzuki, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Genes to cells : devoted to molecular & cellular mechanisms 9 7 611 - 8 2004年07月 [査読有り][通常論文]
     
    Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 A resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABAA receptor-associated protein (GABARAP), contains a ubiquitin core with two alpha helices, alpha1 and alpha2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among alpha1, alpha2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins.
  • Shukichi Hashida, Satoru Yuzawa, Nobuo N Suzuki, Yuko Fujioka, Takayuki Takikawa, Hideki Sumimoto, Fuyuhiko Inagaki, Hirotada Fujii
    The Journal of biological chemistry 279 25 26378 - 86 2004年06月18日 [査読有り][通常論文]
     
    The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.
  • Satoru Yuzawa, Nobuo N Suzuki, Yuko Fujioka, Kenji Ogura, Hideki Sumimoto, Fuyuhiko Inagaki
    Genes to cells : devoted to molecular & cellular mechanisms 9 5 443 - 56 2004年05月 [査読有り][通常論文]
     
    The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the production of reactive oxygen species. p47(phox) is a cytosolic component of the NADPH oxidase and plays an important role in the assembly of the activated complex. The structural determination of the tandem SH3 domains of p47(phox) is crucial for elucidation of the molecular mechanism of the activation of p47(phox). We determined the X-ray crystal structure of the tandem SH3 domains with the polybasic/autoinhibitory region (PBR/AIR) of p47(phox). The GAPPR sequence involved in PBR/AIR forms a left-handed polyproline type-II helix (PPII) and interacts with the conserved SH3 binding surfaces of the SH3 domains simultaneously. These SH3 domains are related by a 2-fold pseudosymmetry axis at the centre of the binding groove and interact with the single PPII helix formed by the GAPPR sequence with opposite orientation. In addition, a number of intra-molecular interactions among the SH3 domains, PBR/AIR and the linker tightly hold the architecture of the tandem SH3 domains into the compact structure and stabilize the autoinhibited form synergistically. Phosphorylation of the serine residues in PBR/AIR could destabilize and successively release the intra-molecular interactions. Thus, the overall structure could be rearranged from the autoinhibitory conformation to the active conformation and the PPII ligand binding surfaces on the SH3 domains are now unmasked, which enables their interaction with the target sequence in p22(phox).
  • Kenji Sugawara, Nobuo N Suzuki, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
    Acta crystallographica. Section D, Biological crystallography 59 Pt 8 1464 - 5 2003年08月 [査読有り][通常論文]
     
    Aut7/Apg8 is located in the intermediate structures of the autophagosome and plays an essential role in autophagosome formation. The processed form, cleaved at a C-terminus of Gly120 and called LC3-I, was expressed, purified and crystallized in two crystal forms. One form belongs to space group I4(1), with unit-cell parameters a = 84.39, c = 36.89 A. The other form belongs to space group P4(1) or P4(3), with unit-cell parameters a = 60.48, c = 35.28 A. From the latter form, a complete diffraction data set was collected to 2.1 A resolution.
  • Satoru Yuzawa, Nobuo N Suzuki, Yuko Fujioka, Kenji Ogura, Hideki Sumimoto, Fuyuhiko Inagaki
    Acta crystallographica. Section D, Biological crystallography 59 Pt 8 1479 - 80 2003年08月 [査読有り][通常論文]
     
    p47(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for the production of the superoxide which kills invasive microorgamisms. A recombinant form of a histidine-tagged tandem SH3 domain of the p47(phox)-containing polybasic autoinhibited region was expressed in Escherichia coli and purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 6000 as a precipitant. Diffraction data were collected to 2.15 A resolution at 100 K using synchrotron radiation. The crystal belongs to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 100.02, c = 44.94 A. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.6 A(3) Da(-1) and a solvent content of 52% by volume.
  • Yuko Fujioka, Takahiro Taira, Yuichi Maeda, Shinya Tanaka, Hiroshi Nishihara, Sanae M. M. Iguchi-Ariga, Kazuo Nagashima, Hiroyoshi Ariga
    Journal of Biological Chemistry 276 48 45137 - 45144 2001年11月30日 [査読有り][通常論文]
     
    The c-myc oncogene product (c-Myc) is a transcription factor that dimerizes with Max and recognizes the E-box sequence, and it plays key functions in cell proliferation, differentiation, and apoptosis. We previously showed that MM-1 bound to myc box II within the transactivation domain of c-Myc and repressed the E-box-dependent transcriptional activity of c-Myc. Here we report that MM-1 showed features of a tumor suppressor. In an EST data base search for cDNAs homologous to MM-1, we found a frequent substitution of amino acid 157 of MM-1, from alanine to arginine (A157R), and the substitution was observed more in tumor cells than in normal cells. A survey of the A157R mutation of MM-1 in 57 cultured cancer cells and 90 tissues from cancer patients showed that the A157R was present in about 50-60% of leukemia/lymphoma cells and in more than 75% of squamous cell carcinoma of tongue cancer. Although both the A157R and the wild-type MM-1 bound to c-Myc, only A157R lost the activities to repress both the E-box-dependent transcriptional activity of c-Myc and the myc/ras cooperative transforming activity in rat 3Y1 cells. Furthermore, the wild-type MM-1, but not A157R, arrested the growth of 3Y1 cells. The human MM-1 gene was mapped at chromosome 12q12-12q13, where many chromosome abnormalities in cancer cells have been reported. The results suggest that MM-1 is a novel candidate for a tumor suppressor that controls the transcriptional activity of c-Myc.

その他活動・業績

共同研究・競争的資金等の研究課題

  • 相分離によるオートファジーの駆動原理
    日本医療研究開発機構(AMED):基盤研究事業部(研究企画課) 革新的先端研究開発支援事業 PRIME
    研究期間 : 2020年10月 -2024年03月 
    代表者 : 藤岡 優子
  • 液-液相分離によるメンブレンコンタクトの構築基盤の解明
    日本学術振興会:科学研究費助成事業 学術変革領域研究(A)
    研究期間 : 2021年09月 -2023年03月 
    代表者 : 藤岡 優子
  • 液-液相分離によるオートファジー関連結合反応の制御機構
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 藤岡 優子
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 藤岡 優子
     
    われわれはオートファジーの開始点PASの構成因子であるVac8に着目し構造機能研究を行った。その結果、PASは液体のような性質を持つ複合体(液滴)であり、Vac8はAtg13を介してPASと液胞膜を繋ぎとめる働きをしていることを明らかにした。また飢餓で誘導されるPAS(飢餓PAS)とCvt経路に働くPAS(選択的PAS)では、その構成因子が異なることが知られていたが、われわれはVac8とAtg13の役割は共通であることを示唆した。
  • オートファジー始動複合体の作動状態の活写
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2017年04月 -2019年03月 
    代表者 : 藤岡 優子
     
    オートファジーの進行に必要な多数のAtgタンパク質群は,飢餓条件下PASに局在し,分解対象を包む膜である隔離膜の形成に働く.Atg1,Atg13, Atg17, Atg29, Atg31からなるタンパク質複合体であるAtg1複合体は,数十コピーからなる高次の多量体を形成することでPASの足場を形成し,オートファジーの始動に働くと考えられているが,その構築基盤は未だ不明である.本研究では,実際にPASの中核として機能する状態のAtg1複合体の動的構造を明らかにすることで,PASの実体および形成メカニズムを明らかにする. 近年,液-液相分離という原理で形成される膜のないオルガネラ(membraneless organelles)が注目を集めている.これら膜のないオルガネラには,核酸や天然変性領域を持つタンパク質が多く含まれる.我々はPASの性質や構成因子の分子間相互作用様式が,液-液相分離を介した膜のないオルガネラに酷似していることを見出した.そこで本年度は光褪色後蛍光回復法や蛍光相関分光法などの定量的な解析により、PASが細胞質との間でAtg因子を活発にやり取りしていること,PAS内のAtg因子は細胞質内と同等の高い運動性を保持していることを示した.さらにPASが形成される過程においてAtg1複合体液滴同士の融合やオストヴァルト熟成が見られることを示し,PASが液-液相分離により形成された液滴であることを証明した.また,PASの中核を担うAtg1複合体について,試験管内で液-液相分離することを示し,Atg13が相分離に必須の役割を持つことを示した.高速AFM像と蛍光顕微鏡像を同時観察可能な装置を用いて、液-液相分離状態のAtg13-Atg17-Atg29-Atg31複合体の微細構造観察を行ない,その規則性のない動的内部構造を明らかにした.
  • オートファジー始動複合体の動的構造解析
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2015年04月 -2017年03月 
    代表者 : 藤岡 優子
     
    オートファジーの進行には多数のAtgタンパク質群が必要だが,それらの殆どは飢餓条件下,PAS(pre-autophagosomal structure) に局在し,分解対象を包む膜である隔離膜の形成に働く.飢餓時には,脱リン酸化状態のAtg13がキナーゼであるAtg1と結合し,さらにAtg17-Atg29-Atg31複合体が結合することにより5者複合体(Atg1複合体)が形成される.細胞内ではAtg1複合体の各構成因子が50分子程度ずつ存在することから,Atg1複合体はより高次の多量体を形成することでPASの足場を形成すると考えられているが,その構造基盤は未だ不明である.本研究では,Atg1複合体の動的構造を明らかにすることで,PASの実体および形成メカニズムを明らかにする. 本年度は,主に,高速AFMを用いてAtg17-Atg29-Atg31複合体およびAtg1を詳しく解析した.Atg17-Atg29-Atg31複合体については,Atg17のS字構造のみならず,Atg31-Atg29複合体がおおきく移動して新たに膜小胞と相互作用しうる凹面を形成する様子や,Atg29の天然変性領域がAtg17と相互作用する様子など,これまで捉えられていなかった極めて重要な観察結果を得ることに成功した.一方、Atg1については,自己リン酸化を受けたものと受けないものを比較し,リン酸化を受けたもののみ,天然変性領域内に微細な二次構造を形成することが判明した.引き続き円二色性測定やNMR測定などのin vitro解析を行って高速AFMのデータの裏づけをとっている.
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 藤岡 優子
     
    オートファジーの膜動態に重要なタンパク質Atg8は,リン脂質によって修飾され膜に局在することによってオートファジーの進行に寄与することが知られている.Atg4は脂質化されたAtg8を脱脂質化する酵素でありAtg8のリサイクルにも寄与しているが,その制御機構は明らかになっていなかった.本研究ではAtg4の活性の制御について試験管内で検討した.その結果,Atg4の活性は他のAtgタンパク質との相互作用やAtg1によるリン酸化では影響を受けなかった一方、Atg4自身の領域で制御を受けることを示唆するデータを得た.Atg4は細胞内局在等の変化に応じて自身の活性を制御する能力を有しているのかもしれない.
  • オートファジーの始動を制御するATG1キナーゼ複合体の分子機構の解明
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 藤岡 優子
     
    Atg1はオートファジー関連タンパク質の中で唯一のタンパク質キナーゼである。Atg1のキナーゼ活性はAtg13との結合によって制御されているという報告があるものの、その詳細な分子メカニズムは依然不明である。本研究は、Atg1とAtg13の複合体と、その複合体に結合することによってAtg1の活性化に寄与するAtg17-Atg29-Atg31の3者複合体、さらにはAtg1-Atg13-Atg17-Atg29-Atg31の5者複合体の立体構造解析を行うことによって、その相互作用様式を明らかにし、Atg1のキナーゼ活性の調節メカニズムを解明することを目的としている。 昨年度に報告した通り、これまでにAtg1-Atg13複合体と、Atg13-Atg17-Atg29-Atg31の4者複合体について結晶構造を決定することに成功した。また立体構造をもとに行った生化学的解析などから、飢餓によるAtg13の脱リン酸化が、オートファゴソーム形成の足場であるPAS(pre-autophagosomal structure)の形成、さらにはオートファジーの始動を引き起こす分子機構の一端が明らかとなり、本年度論文として報告した。 その発展として、脱リン酸化したAtg13がAtg17の二か所に結合することによって、Atg17同士が架橋され凝集するという現象を発見した。この発見は5者複合体の結晶化が本質的に困難であることを示唆しているが、PASの形成機構そのものを表している可能性があるため、本年度は予定を変更して高速原子間力顕微鏡を用いて複合体の形成過程の可視化に取り組んだ。その結果、個別のタンパク質の可視化に成功したので、今後は引き続きタンパク質を区別するタグの開発や、凝集するタンパク質を観察する手法の開発などを行いながら、5者複合体の凝集過程の観察を行う予定である。


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