研究者情報

マスター

アカウント（マスター）

• 氏名

  坂口　和靖(サカグチ　カズヤス), サカグチ　カズヤス

所属（マスター）

• 理学研究院　化学部門　有機・生命化学分野

所属（マスター）

• 理学研究院　化学部門　有機・生命化学分野

独自項目

syllabus

• 2021, 化学特別講義, Advanced Chemistry, 修士課程, 総合化学院, 生物化学、構造生物学、創薬科学、タンパク質科学、クライオ電子顕微鏡、膜輸送体
• 2021, 化学特別講義, Advanced Chemistry, 修士課程, 総合化学院, protein evolution, microbial evolution, drug resistance, fitness landscape, protein engineering, evolutionary biology, population genetics, stochastic processes
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, protein evolution, microbial evolution, drug resistance, fitness landscape, protein engineering, evolutionary biology, population genetics, stochastic processes
• 2021, 生物化学先端講義, Intermediate Biological Chemistry, 修士課程, 総合化学院, 生体分子、タンパク質、立体構造、機能制御、フォールディング、分子認識、酵素、バイオインフォマティクス
• 2021, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 修士課程, 総合化学院, 遺伝情報，タンパク質構造，分子論的理解，生合成機構，動物細胞，二次代謝産物，バイオポリマー，環境浄化
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 遺伝情報，タンパク質構造，分子論的理解，生合成機構，動物細胞，二次代謝産物，バイオポリマー，環境浄化
• 2021, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 修士課程, 工学院, 遺伝情報，タンパク質構造，分子論的理解，生合成機構，動物細胞，二次代謝産物，バイオポリマー，環境浄化
• 2021, 先端総合化学特論Ⅱ, Modern Trends in Chemical Sciences and Engineering II, 博士後期課程, 総合化学院, protein evolution, microbial evolution, drug resistance, fitness landscape, protein engineering, evolutionary biology, population genetics, stochastic processes
• 2021, 生命分子化学特論, Modern Trends in Biomolecular Chemistry, 博士後期課程, 工学院, 遺伝情報，タンパク質構造，分子論的理解，生合成機構，動物細胞，二次代謝産物，バイオポリマー，環境浄化
• 2021, 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), Freshman Seminar, 学士課程, 全学教育, 生命と化学反応、タンパク質、遺伝子と遺伝情報、免疫、癌、細胞、シグナルの伝達、構造と機能
• 2021, 化学実験Ⅴ, Laboratory Work in Chemistry V, 学士課程, 理学部, 生化学、分子生物学、タンパク質、遺伝子、酵素、ホスファターゼ、精製、酵素反応速度論、DNA、遺伝子組み換え、大腸菌
• 2021, 化学実験Ｅ, Laboratory Work in Chemistry E, 学士課程, 理学部, 生化学、分子生物学、タンパク質、遺伝子、酵素、ホスファターゼ、精製、酵素反応速度論、DNA、遺伝子組み換え、大腸菌
• 2021, 情報生化学, Biochemical Regulation, 学士課程, 理学部, 複製、転写、翻訳、DNA, RNA、タンパク質、染色体、遺伝子、プロセシング、変異、修復、翻訳後修飾
• 2021, 生物化学Ⅰ, Biochemistry Ⅰ, 学士課程, 理学部, 生体分子、アミノ酸、タンパク質、糖、脂質、核酸、DNA、RNA、構造と機能、酵素
• 2021, 化学特別講義Ⅱ, Special Lectures in Chemistry II, 学士課程, 理学部, protein evolution, microbial evolution, drug resistance, fitness landscape, protein engineering, evolutionary biology, population genetics, stochastic processes

PositionHistory

• 教育研究評議会評議員, 2014年4月1日, 2016年3月31日
• 大学院総合化学院長, 2014年4月1日, 2016年3月31日

researchmap

プロフィール情報

学位

• 理学博士（九州大学）

プロフィール情報

• 姓

  坂口

• 名

  和靖

• ID各種

  200901070729888644

対象リソース

https://wwwchem.sci.hokudai.ac.jp/~biochem/
http://barato.sci.hokudai.ac.jp/~biochem/

業績リスト

研究キーワード

• 阻害剤   酵素   分化   細胞周期   癌   脱リン酸化   翻訳後修飾   多量体構造   ホスファターゼ   p53   ナノバイオマテリアル   タンパク質科学   ペプチド科学   生物化学   posttranslational modification   oligomer   phosphatase   p53   Nanobiomaterial Science   Protein Science   Peptide Science   生化学   

研究分野

• ナノテク・材料 / 生体化学
• ライフサイエンス / 構造生物化学
• ライフサイエンス / 機能生物化学
• ナノテク・材料 / 生物分子化学

委員歴

• 2013年 - 現在   日本プロテインホスファターゼ研究会   世話人会
• 2007年 - 現在   日本ペプチド学会   評議員   日本ペプチド学会
• 2022年 - 2023年   日本生化学会   代議員
• 2018年 - 2020年   日本ペプチド学会   広報担当理事
• 2014年 - 2018年   日本ペプチド学会   渉外担当理事   日本ペプチド学会
• 2013年 - 2018年   日本生化学会   「生化学」誌企画委員
• 2011年 - 2014年   日本プロテオーム学会   理事   日本プロテオーム学会
• 2008年 - 2010年   日本生化学会   代議員   日本生化学会

受賞

• 2010年10月 日本生化学会 2010 年度（第18 回） JB 論文賞
   PPM1D430, a Novel Alternative Splicing Variant of the Human PPM1D, can Dephosphorylate p53 and Exhibits Specific Tissue Expression 

  受賞者: 中馬吉郎;栗橋渉;水上洋平;梨本健紘;八木寛陽;坂口和靖

論文

• Biomineralization through a Symmetry-Controlled Oligomeric Peptide

  Tatsuya Sakaguchi, Natsumi Nakagawa, Kenta Mine, Jose Isagani B. Janairo, Rui Kamada, James G. Omichinski, Kazuyasu Sakaguchi

  Biomimetics 8 8 606 - 606 2023年12月14日 

  Biomineralization peptides are versatile tools for generating nanostructures since they can make specific interactions with various inorganic metals, which can lead to the formation of intricate nanostructures. Previously, we examined the influence that multivalency has on inorganic structures formed by p53 tetramer-based biomineralization peptides and noted a connection between the geometry of the peptide and its ability to regulate nanostructure formation. To investigate the role of multivalency in nanostructure formation by biomineralization peptides more thoroughly, silver biomineralization peptides were engineered by linking them to additional self-assembling molecules based on coiled-coil peptides and multistranded DNA oligomers. Under mild reducing conditions at room temperature, these engineered biomineralization peptides self-assembled and formed silver nanostructures. The trimeric forms of the biomineralization peptides were the most efficient in forming a hexagonal disk nanostructure, with both the coiled-coil peptide and DNA-based multimeric forms. Together, the results suggest that the spatial arrangement of biomineralization peptides plays a more important role in regulating nanostructure formation than their valency.
• Highly Similar Tetramerization Domains from the p53 Protein of Different Mammalian Species Possess Varying Biophysical, Functional and Structural Properties.

  Shuya Sakaguchi, Natsumi Nakagawa, Haytham M Wahba, Junya Wada, Rui Kamada, James G Omichinski, Kazuyasu Sakaguchi

  International journal of molecular sciences 24 23 2023年11月22日 

  The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein.
• Bilayer Hydrogel Composed of Elastin-Mimetic Polypeptides as a Bio-Actuator with Bidirectional and Reversible Bending Behaviors

  Rui Kamada, Hiromitsu Miyazaki, Jose Isagani Janairo, Yoshiro Chuman, Kazuyasu Sakaguchi

  Molecules 2023年07月
• Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells.

  Tatsuki Tsuruoka, Emiri Nakayama, Takuya Endo, Shingo Harashima, Rui Kamada, Kazuyasu Sakaguchi, Toshiaki Imagawa

  Journal of cell science 2023年05月22日 

  The tumor suppressor p53 plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function: it encode input information and are decoded to induce distinct cellular phenotypes. However it remains unclear to what extent the temporal dynamics of p53 reflects the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 by etoposide is highly dependent on the cell cycle but not by UV. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway.
• Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA.

  Itsumi Tani, Yui Oikawa, Seiyo Doi, Jose Isagani B Janairo, Rui Kamada, Kazuyasu Sakaguchi

  Biochemical and biophysical research communications 642 35 - 40 2023年01月29日 

  The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low.
• Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA

  Itsumi Tani, Yui Oikawa, Seiyo Doi, Jose Isagani B. Janairo, Rui Kamada, Kazuyasu Sakaguchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 642 35 - 40 2023年01月 

  The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low. (c) 2022 Published by Elsevier Inc.
• Lipid Droplet Formation Is Regulated by Ser/Thr Phosphatase PPM1D via Dephosphorylation of Perilipin 1

  Rui Kamada, Sae Uno, Nozomi Kimura, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi

  International Journal of Molecular Sciences 23 19 12046 - 12046 2022年10月10日 

  Hypertrophy and hyperplasia of white adipocytes induce obesity, leading to diseases such as type 2 diabetes and hypertension, and even cancer. Hypertrophy of white adipocytes is attributed to the excessive storage of the energy form of triglycerides in lipid droplets (LDs). LDs are fat storage organelles that maintain whole-body energy homeostasis. It is important to understand the mechanism of LD formation for the development of obesity therapy; however, the regulatory mechanisms of LD size and formation are not fully understood. In this study, we demonstrated that the PPM family phosphatase PPM1D regulates LD formation. PPM1D specific inhibitor, SL-176 significantly decreased LD formation via two different pathways: dependent of and independent of adipocyte-differentiation processes. In the mature white adipocytes after differentiation, LD formation was found to be controlled by PPM1D via dephosphorylation of Ser511 of perilipin 1. We found that inhibition of PPM1D in mature white adipocytes significantly reduced the size of the LDs via dephosphorylation of Ser511 of perilipin 1 but did not change the lipolysis sensitivity and the total amount of lipid in cells. Collectively, the results of this study provide evidence that PPM1D plays an important role in LD formation in mature adipocytes.
• Stereoselective Interaction of Helix-Hairpin-Helix Motif Peptides with DNA Structure

  Rui Kamada, Shuya Sakaguchi, Shoma Kura, Kaori Takamatsu, Tetsuya Yamamoto, Toshiaki Imagawa, Kazuyasu Sakaguchi

  Chemistry Letters 51 10 1029 - 1032 2022年10月05日
• Role of active site arginine residues in substrate recognition by PPM1A

  Itsumi Tani, Shogo Ito, Yukiko Shirahata, Yutaka Matsuyama, James G. Omichinski, Yasuyuki Shimohigashi, Rui Kamada, Kazuyasu Sakaguchi

  Biochemical and Biophysical Research Communications 581 1 - 5 2021年12月 [査読有り]
   

  Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.
• PPM1D Is a Therapeutic Target in Childhood Neural Tumors

  Jelena Milosevic, Diana Treis, Susanne Fransson, Gabriel Gallo-Oller, Baldur Sveinbjörnsson, Nina Eissler, Keiji Tanino, Kazuyasu Sakaguchi, Tommy Martinsson, Malin Wickström, Per Kogner, John Inge Johnsen

  Cancers 13 23 6042 - 6042 2021年11月30日 

  Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.
• Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors

  Rui Kamada, Fuki Kudoh, Shogo Ito, Itsumi Tani, Jose Isagani B. Janairo, James G. Omichinski, Kazuyasu Sakaguchi

  Pharmacology & Therapeutics 215 107622 - 107622 2020年11月 [査読有り][招待有り]
   

  Protein phosphatases and kinases control multiple cellular events including proliferation, differentiation, and stress responses through regulating reversible protein phosphorylation, the most important post-translational modification. Members of metal-dependent protein phosphatase (PPM) family, also known as PP2C phosphatases, are Ser/Thr phosphatases that bind manganese/magnesium ions (Mn2+/Mg2+) in their active center and function as single subunit enzymes. In mammals, there are 20 isoforms of PPM phosphatases: PPM1A, PPM1B, PPM1D, PPM1E, PPM1F, PPM1G, PPM1H, PPM1J, PPM1K, PPM1L, PPM1M, PPM1N, ILKAP, PDP1, PDP2, PHLPP1, PHLPP2, PP2D1, PPTC7, and TAB1, whereas there are only 8 in yeast. Phylogenetic analysis of the DNA sequences of vertebrate PPM isoforms revealed that they can be divided into 12 different classes: PPM1A/PPM1B/PPM1N, PPM1D, PPM1E/PPM1F, PPM1G, PPM1H/PPM1J/PPM1M, PPM1K, PPM1L, ILKAP, PDP1/PDP2, PP2D1/PHLPP1/PHLPP2, TAB1, and PPTC7. PPM-family members have a conserved catalytic core region, which contains the metal-chelating residues. The different isoforms also have isoform specific regions within their catalytic core domain and terminal domains, and these regions may be involved in substrate recognition and/or functional regulation of the phosphatases. The twenty mammalian PPM phosphatases are involved in regulating diverse cellular functions, such as cell cycle control, cell differentiation, immune responses, and cell metabolism. Mutation, overexpression, or deletion of the PPM phosphatase gene results in abnormal cellular responses, which lead to various human diseases. This review focuses on the structures and biological functions of the PPM-phosphatase family and their associated diseases. The development of specific inhibitors against the PPM phosphatase family as a therapeutic strategy will also be discussed.
• Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.

  Mathieu Lussier-Price, Xavier H Mascle, Laurent Cappadocia, Rui Kamada, Kazuyasu Sakaguchi, Haytham M Wahba, James G Omichinski

  Structure (London, England : 1993) 28 5 573 - 585 2020年05月05日 

  The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.
• Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner

  Xavier H. Mascle, Christina Gagnon, Haytham M. Wahba, Mathieu Lussier-Price, Laurent Cappadocia, Kazuyasu Sakaguchi, James G. Omichinski

  Structure 28 2 157 - 168.e5 2020年02月
• The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein.

  Nakagawa N, Sakaguchi S, Nomura T, Kamada R, Omichinski JG, Sakaguchi K

  Biochemical and biophysical research communications 521 3 681 - 686 2019年11月 [査読有り][通常論文]
   

  The p53 protein plays a number of roles in protecting organisms from different genotoxic stresses and this includes DNA damage induced by acetaldehyde, a metabolite of alcohol. Since the common tree shrew ingests high levels of alcohol as part of its normal diet, this suggests that its p53 protein may possess unique properties. Using a combination of biophysical and modeling studies, we demonstrate that the tetramerization domain of the tree shrew p53 protein is considerably more stable than the corresponding domain from humans despite sharing almost 90% sequence identity. Based on modeling and mutagenesis studies, we determine that a glutamine to methionine substitution at position 354 plays a key role in this difference. Given the link between stability of the p53 tetramerization domain and its transcriptional activity, the results suggest that this enhanced stability could lead to important consequences at p53-regulated genes in the tree shrew.
• Inhibition of lipid droplet formation by Ser/Thr protein phosphatase PPM1D inhibitor, SL-176

  Rui Kamada, Nozomi Kimura, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi

  PLOS ONE 14 2 e0212682  2019年02月 [査読有り][通常論文]
   

  Obesity is a worldwide public health problem, which is associated with various severe diseases including diabetes, hypertension, atherosclerosis, and cancer. Recent studies have revealed that combination treatment of several different compounds using low doses is effective to reduce side effects. Thus, there is a need to develop an efficient inhibitor for reducing lipid droplets with a divergent target/pathway. Ser/Thr protein phosphatase PPM1D is involved in cellular metabolic processes and is a promising target for anti-obesity treatment. We have previously developed a potent and specific PPM1D inhibitor, SL-176. In this study, we demonstrated that significant reduction of lipid droplet formation in adipocytes by the PPM1D specific inhibitor, SL-176. Using Oil-red O staining and fluorescent imaging of lipid droplet, we found that treatment of SL-176 significantly suppressed lipid droplet formation of 3T3-L1 cells both in amount and in size. SL-176 also repressed mRNA and protein expression of PPARγ and C/EBPα, adipogenic markers, at nontoxic conditions. Thus, SL-176 is a unique and potent inhibitor of lipid droplet formation that acts via PPM1D, a novel target toward inhibiting adipocyte differentiation.
• PPM1D enhances retinoic acid-induced differentiation in human embryonic carcinoma cell line

  Ogasawara, S, Chuman, Y, Michiba, T, Kamada, R, Imagawa, T, Sakaguchi, K

  J. Biochem. 165 6 471 - 477 2019年 [査読有り][通常論文]
   

  The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation.
• Interferon Stimulation Creates Chromatin Marks And Establishes Transcriptional Memory

  Rui Kamada, Wenjing Yang, Yubo Zhang, Mira C. Patel, Yanqin Yang, Ryota Ouda, Anup Dey, Yoshiyuki Wakabayashi, Kazuyasu Sakaguchi, Takashi Fujita, Tomohiko Tamura, Jun Zhu, Keiko Ozato

  Proceedings of the National Academy of Sciences of the USA 115 39 E9162 - E9171 2018年09月 [査読有り][通常論文]
  
• Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells.

  Hirotaka Watanabe, Kojiro Ishibashi, Hiroki Mano, Sho Kitamoto, Nanami Sato, Kazuya Hoshiba, Mugihiko Kato, Fumihiko Matsuzawa, Yasuto Takeuchi, Takanobu Shirai, Susumu Ishikawa, Yuka Morioka, Toshiaki Imagawa, Kazuyasu Sakaguchi, Suguru Yonezawa, Shunsuke Kon, Yasuyuki Fujita

  Cell reports 23 13 3721 - 3729 2018年06月26日 [査読有り][通常論文]
   

  p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.
• Synthesis of bimetallic PdAg nanoparticles through an oligomerization- controlled biomineralization peptide

  Jose Isagani B. Janairo, Kazuyasu Sakaguchi

  Materials Science Forum 928 77 - 82 2018年 

  Peptide – mediated biomineralization is an emerging and promising biomimetic approach for the synthesis of nanomaterials. This nature – inspired technique of producing inorganic nanostructures depends on the biomineralization peptide to control the shape and morphology of the prevailing inorganic nanostructure. One of the challenges in peptide – mediated biomineralization is controlling the 3D arrangement and orientation of the peptide. Recently, we have developed a peptide platform that can specify and direct the geometric arrangement and spatial orientation of the biomineralization peptide. The peptide platform is composed of two segments: a metal binding sequence, and the tetramerization domain of the tumor suppressor p53 protein, which acts as the oligomerization control element. The resulting fusion peptide exhibits a spatially – fixed and well – controlled assembly of the palladium binding sequence. This present study demonstrates the utility and efficacy of this peptide platform to bimetallic materials. Monodispersed 5 nm bimetallic PdAg nanoparticles were synthesized using the oligomerization – controlled biomineralization peptide. The synthesis was carried out in an aqueous environment, void of harsh reagents. When other fusion biomineralization peptides were used to synthesize bimetallic PdAg nanoparticles, less ordered nanoparticles were yielded. The results highlight the importance of controlled assembly on bimetallic nanoparticle formation through biomineralization. The presented method offers a straightforward manner of creating monodispersed and extremely small nanoparticles, which are useful in a wide array of applications.
• Quantitative single cell analysis for transcriptional activity of p53 hetero-tetramers between wild-type protein and oligomerization domain

  Yu Toguchi, Rui Kamada, Madoka Kanno, Toshiaki Imagawa, Kazuyasu Sakaguchi

  Chemistry Letters 47 2 217 - 220 2018年 [査読有り][通常論文]
   

  p53 acts as a transcriptional factor for tumor suppression via tetramerization. Although the dominant-negative effect occurs by hetero-oligomerization between wild-type and mutant p53, the precise mechanism remains unclear. Here, we report an analysis of the transcriptional ability of each hetero-tetramer p53 at the physiological protein expression level in single cells. Quantitative fitting analysis showed that hetero-tetramers which contain more than two wild-type p53 have substantial transcriptional ability. These results suggest that the two DNA binding domains are important for transcription.
• Synergic strategies for the enhanced self-assembly of biomineralization peptides for the synthesis of functional nanomaterials

  Jose Isagani B. Janairo, Tatsuya Sakaguchi, Kenta Mine, Rui Kamada, Kazuyasu Sakaguchi

  Protein and Peptide Letters 25 1 4 - 14 2018年01月01日 [査読有り][招待有り]
   

  Introduction: Peptide-mediated biomineralization is a promising bioinspired technique of nanostructure formation. The biomineralization peptide is responsible for the regulation of the growth and morphology of the inorganic nanostructure. The 3D properties of the biomineralization peptide is a crucial factor in which the success of creating functional nanomaterials depends on. However, given the relatively short sequence of most biomineralization peptides, controlling the multivalency and spatial orientation of the peptide can be a challenging endeavor. Objective: This mini-review details recent advances in enhancing the self-assembly and 3D properties of the biomineralization peptide. The design and creation of fusion peptides is highlighted, which involves the conjugation of the biomineralization peptide with a control element. The control element is responsible for directing the self-assembly of the biomineralization peptide. Conclusion: A variety of control elements that can direct the self-assembly of biomineralization peptides are available. The control element can be a small organic molecule such as a biphenyl, or a peptide segment such as the p53 tetramerization domain or the amyloid peptide. The high diversity of existing control elements further increases the ability of peptide-mediated biomineralization to create functional nanomaterials.
• Inhibition of Ser/Thr phosphatase PPM1D induces neutrophil differentiation in HL-60 cells

  Rui Kamada, Fuki Kudoh, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi

  JOURNAL OF BIOCHEMISTRY 162 4 303 - 308 2017年10月 [査読有り][通常論文]
   

  Protein phosphatase Magnesium-dependent 1, Delta (PPM1D) is a wild-type p53-inducible Ser/Thr phosphatase that acts as a negative regulator of the p53 tumor suppressor. Gene amplification and overexpression of PPM1D have been reported in various cancers including leukemia and neuroblastoma. Therefore, PPM1D is a promising target in cancer therapy. It has been reported that PPM1D knockout mice exhibit neutrophilia in blood and show a defective immune response. Here, we found that inhibition of PPM1D induced neutrophil differentiation of human promyelocytic leukemia cell line HL-60. The combination of a PPM1D inhibitor and all-trans retinoic acid significantly increased their differentiation efficiency. The PPM1D inhibitor also induced G1 arrest in HL-60 cells. Our results suggest that PPM1D may be a potential therapeutic target for blood cell diseases including leukemia.
• Heterochiral Jun and Fos bZIP peptides form a coiled-coil heterodimer that is competent for DNA binding

  Rui Kamada, Natsumi Nakagawa, Taiji Oyama, Kazuyasu Sakaguchi

  JOURNAL OF PEPTIDE SCIENCE 23 7-8 644 - 649 2017年07月 [査読有り][通常論文]
   

  Coiled coils, consisting of at least two -helices, have important roles in the regulation of transcription, cell differentiation, and cell growth. Peptides composed of d-amino acids (d-peptides) have received great attention for their potential in biomedical applications, because they give large diversity for the design of peptidyl drug and are more resistant to proteolytic digestion than l-peptides. However, the interactions between l-peptides/l-protein and d-peptides in the formation of complex are poorly understood. In this study, stereoisomer-specific peptides were constructed corresponding to regions of the basic-leucine-zipper domains of Jun and Fos proteins. basic-leucine-zipper domains consist of an N-terminal basic domain, which is responsible for DNA binding, and a C-terminal domain that enables homodimerization or heterodimerization via formation of a coiled-coil. By combining peptides with different stereochemistries, the d-l heterochiral Jun-Fos heterodimer formation induced DNA binding by the basic domains of Jun-Fos. Our study provides new insight into the interaction between l-peptide and d-peptide enantiomers for developing d-peptide materials and drugs. Copyright (c) 2017 European Peptide Society and John Wiley & Sons, Ltd.
• Oligomerization enhances the binding affinity of a silver biomineralization peptide and catalyzes nanostructure formation

  Tatsuya Sakaguchi, Jose Isagani B. Janairo, Mathieu Lussier-Price, Junya Wada, James G. Omichinski, Kazuyasu Sakaguchi

  SCIENTIFIC REPORTS 7 1 1400  2017年05月 [査読有り][通常論文]
   

  Binding affinity and specificity are crucial factors that influence nanostructure control by biomineralization peptides. In this paper, we analysed the role that the oligomeric state of a silver biomineralization peptide plays in regulating the morphology of silver nanostructure formation. Oligomerization was achieved by conjugating the silver specific TBP biomineralization peptide to the p53 tetramerization domain peptide (p53Tet). Interestingly, the TBP-p53Tet tetrameric peptide acted as a growth catalyst, controlling silver crystal growth, which resulted in the formation of hexagonal silver nanoplates without consuming the peptide. The TBP-p53Tet peptide caps the surface of the silver crystals, which enhances crystal growth on specific faces and thereby regulates silver nanostructure formation in a catalytic fashion. The present findings not only provide an efficient strategy for controlling silver nanostructure formation by biomineralization peptides, but they also demonstrate that in this case the oligomeric peptides play a unique catalytic role.
• Tetramer Formation of Tumor Suppressor Protein p53: Structure, Function, and Applications

  Kamada, R, Toguchi, Y, Nomura, T, Sakaguchi, K

  Biopolymers: Peptide Science 106 4 598 - 612 2016年11月 [査読有り][招待有り]
  
• PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin

  Yuuki Kozakai, Rui Kamada, Junya Furuta, Yuhei Kiyota, Yoshiro Chuman, Kazuyasu Sakaguchi

  SCIENTIFIC REPORTS 6 33272  2016年09月 [査読有り][通常論文]
   

  An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1.
• Patterning nanofibrils through the templated growth of multiple modified amyloid peptides

  Hiroki Sakai, Ken Watanabe, Fuki Kudoh, Rui Kamada, Yoshiro Chuman, Kazuyasu Sakaguchi

  SCIENTIFIC REPORTS 6 31993  2016年08月 [査読有り][通常論文]
   

  There has been considerable interest in the patterning of functionalized nanowires because of the potential applications of these materials to the construction of nanodevices. A variety of biomolecular building blocks containing amyloid peptides have been used to functionalize nanowires. However, the patterning of self-assembled nanowires can be challenging because of the difficulties associated with controlling the self-assembly of these functionalized building blocks. Herein, we present a versatile approach for the patterning of nanowires based on the combination of templated fibril growth with a versatile functionalization method using our structure-controllable amyloid peptides (SCAPs). Using this approach, we have succeeded in the formation of multi-type nanowires with tandem domain structures in high yields. Given that the mixing-SCAP method can lead to the formation of tandem fibrils, it is noteworthy that our method allowed us to control the initiation of fibril formation from the gold nanoparticles, which were attached to a short fibril as initiation points. This approach could be used to prepare a wide variety of fibril patterns, and therefore holds great potential for the development of novel self-assembled nanodevices.
• Effective Cellular Morphology Analysis for Differentiation Processes by a Fluorescent 1,3a,6a-Triazapentalene Derivative Probe in Live Cells

  Rui Kamada, Fumi Tano, Fuki Kudoh, Nozomi Kimura, Yoshiro Chuman, Ayumi Osawa, Kosuke Namba, Keiji Tanino, Kazuyasu Sakaguchi

  PLOS ONE 11 8 e0160625  2016年08月 [査読有り][通常論文]
   

  Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a, 6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells.
• Quantitative Correlation between the Protein Expression Level in Escherichia Coli and Thermodynamic Stability of Protein In Vitro

  Junya Wada, Hiromitsu Miyazaki, Rui Kamada, Kazuyasu Sakaguchi

  CHEMISTRY LETTERS 45 2 185 - 187 2016年02月 [査読有り][通常論文]
   

  Protein expression using Escherichia coli is a common and important method for recombinant protein production. Herein, we quantitatively analyzed the correlation between protein expression in vivo and thermodynamic structure stability in vitro using the tetramerization domain of tumor suppressor protein p53. We found a strong positive relationship between the expression level and the thermodynamic stability. Our study suggests that a minimum thermodynamic stability of a protein is required for substantial protein expression in bacterial cells.
• Effect of C-terminal Region of Ser/Threonine Phosphatase PPM1D on its Location.

  Kudoh, F, Kamada, R, Ito, S, Kiyota, Y, Sakaguchi, K

  Peptide Sci. 2015 221 - 222 2016年 [査読有り][通常論文]
  
• Novel inhibitors targeting PPM1D phosphatase potently suppress cancer cell proliferation

  Sari Ogasawara, Yuhei Kiyota, Yoshiro Chuman, Ayano Kowata, Fumihiko Yoshimura, Keiji Tanino, Rui Kamada, Kazuyasu Sakaguchi

  BIOORGANIC & MEDICINAL CHEMISTRY 23 19 6246 - 6249 2015年10月 [査読有り][通常論文]
   

  Protein phosphatase magnesium-dependent 1 delta (PPM1D, Wip1) is a p53 inducible serine/threonine phosphatase. PPM1D is a promising target protein in cancer therapy since overexpression, missense mutations, truncating mutations, and gene amplification of PPM1D are reported in many tumors, including breast cancer and neuroblastoma. Herein, we report that a specific inhibitor, SL-176 that can be readily synthesized in 10 steps, significantly inhibits proliferation of a breast cancer cell line overexpressing PPM1D and induces G2/M arrest and apoptosis. SL-176 decreases PPM1D enzyme activity potently and specifically in vitro. These results demonstrate that SL-176 could be a useful lead compound in the development of effective anti-cancer agents. (C) 2015 Elsevier Ltd. All rights reserved.
• Antiproliferative Activity of Silver Nanoplates on Human Promyelocytic Leukemia Cell Lines

  Tatsuya Sakaguchi, Kenta Mine, Fuki Kudoh, Rui Kamada, Kazuyasu Sakaguchi

  CHEMISTRY LETTERS 44 3 327 - 329 2015年03月 [査読有り][通常論文]
   

  The physical and chemical properties of silver nanomaterials are highly dependent on their shape and size. Recently, Ag nanoparticles have been reported to be useful for medicinal and pharmaceutical applications including anticancer activity. In this study, we showed that Ag nanoplates possess significantly higher antiproliferative activity on human promyelocytic leukemia cells, HL-60, than spherical nanoparticles. The triangular Ag nanoplates induced apoptosis in the cells and were located in the same subcellular compartment as the spherical Ag nanoparticles.
• Synthesis of yellow and red fluorescent 1,3a, 6a-triazapentalenes and the theoretical investigation of their optical properties

  Kosuke Namba, Ayumi Osawa, Akira Nakayama, Akane Mera, Fumi Tano, Yoshiro Chuman, Eri Sakuda, Tetsuya Taketsugu, Kazuyasu Sakaguchi, Noboru Kitamura, Keiji Tanino

  CHEMICAL SCIENCE 6 2 1083 - 1093 2015年 [査読有り][通常論文]
   

  To expand the originally developed fluorescent 1,3a, 6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a, 6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a, 6a-triazapentalene and 2-(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a, 6a-triazapentalene were synthesized. The former exhibited yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large Stokes shifts, and the 1,3a, 6a-triazapentalene system enabled the same fluorescent chromophore to cover the entire region of visible wavelengths. The potential applications of the 1,3a, 6a-triazapentalenes as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a, 6a-triazapentalene system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical calculations were performed to investigate the optical properties of the 1,3a, 6a-triazapentalenes. These calculations revealed that the excitation involves a significant charge-transfer from the 1,3a, 6a-triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths showed a good correlation with the experimental ones, and thus the system could enable the theoretical design of substituents with the desired optical properties.
• Structural and Functional Characterization of the Phosphorylation-Dependent Interaction between PML and SUMO1

  Laurent Cappadocia, Xavier H. Mascle, Veronique Bourdeau, Samuel Tremblay-Belzile, Malik Chaker-Margot, Mathieu Lussier-Price, Junya Wada, Kazuyasu Sakaguchi, Muriel Aubry, Gerardo Ferbeyre, James G. Omichinski

  STRUCTURE 23 1 126 - 138 2015年01月 [査読有り][通常論文]
   

  PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to un-phosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.
• Function of Proto-oncogene Product PPM1D and Development of PPM1D Inhibitors for Cancer Chemotherapy

  Kamada R, Chuman Y, Kozakai Y, Sakaguchi K

  Seikagaku. The Journal of Japanese Biochemical Society 87 5 531 - 538 日本生化学会 2015年 [査読有り][通常論文]
  
• Inhibition of C-terminal truncated PPM1D enhances the effect of doxorubicin on cell viability in human colorectal carcinoma cell line

  Yuuki Kozakai, Rui Kamada, Yuhei Kiyota, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 24 5593 - 5596 2014年12月 [査読有り][通常論文]
   

  PPM1D is a p53-inducible Ser/Thr phosphatase. One of the main functions of PPM1D in normal cells is to act as a negative regulator of the p53 tumor suppressor by dephosphorylating p53 and several kinases. PPM1D is considered an oncoprotein owing to both its functions and the fact that gene amplification and overexpression of PPM1D are reported in several tumors. Recently, PPM1D mutations resulting in C-terminal truncated alterations were found in brainstem gliomas and colorectal cancers, and these mutations enhanced the activity of PPM1D. Therefore, C-terminal truncated PPM1D should be also considered as a potential candidate target of anticancer drugs. Here we showed that combination treatment with PPM1D-specific inhibitor SPI-001 and doxorubicin suppressed cell viability of HCT-116 cells overexpressing C-terminal truncated PPM1D through p53 activation compared with doxorubicin alone. Our results suggest that combination treatment with PPM1D inhibitor and doxorubicin may be a potential anti-cancer treatment in PPM1D-mutated cancer cells. (C) 2014 Elsevier Ltd. All rights reserved.
• Effects of Buffer on the Structure and Catalytic Activity of Palladium Nanomaterials Formed by Biomineralization

  Jose Isagani B. Janairo, Kazuyasu Sakaguchi

  CHEMISTRY LETTERS 43 8 1315 - 1317 2014年08月 [査読有り][通常論文]
   

  Effects of a buffer on the structure and catalytic activity of Pd nanostructures obtained from biomineralization were evaluated. Significant structural differences attributed to the buffer were observed. In addition, the Pd nanostructures prepared in buffered solutions had enhanced catalytic activity in the reduction of aminonitrophenol isomers. Our findings emphasize that the buffer selection is critical for biomineralization. Moreover, these findings indicate that the buffer can be possibly used to control the structure and activity of Pd nanomaterials, which is a very simple and cost-effective way compared with other methods.
• Effects of biomineralization peptide topology on the structure and catalytic activity of Pd nanomaterials

  Jose Isagani B. Janairo, Tatsuya Sakaguchi, Kenji Hara, Atsushi Fukuoka, Kazuyasu Sakaguchi

  CHEMICAL COMMUNICATIONS 50 66 9259 - 9262 2014年08月 [査読有り][通常論文]
   

  Highly branched, coral-like Pd nanostructures were formed using a biomineralization peptide conjugated to an oligomeric peptide that simultaneously controls the spatial orientation, arrangement and valency. The Pd nanocoral showed very high catalytic activity in the reduction of nitrophenol. The results highlight the importance of topological arrangement in nanostructure formation and catalytic activity.
• Formation of functionalized nanowires by control of self-assembly using multiple modified amyloid peptides

  Hiroki Sakai, Ken Watanabe, Yuya Asanomi, Yumiko Kobayashi, Yoshiro Chuman, Lihong Shi, Takuya Masuda, Thomas Wyttenbach, Michael T. Bowers, Kohei Uosaki, Kazuyasu Sakaguchi

  Advanced Functional Materials 23 39 4881 - 4887 2013年10月18日 [査読有り][通常論文]
   

  Amyloid peptides have great potential as building blocks in the creation of functional nanowires due to their natural ability to self-assemble into nanofibrillar structures and because they can be easily modified with various functional groups. However, significant modifications of an amyloid peptide generally alter its self-assembly property, making it difficult to construct functionalized fibrils with a desired structure and function. In this study, a very effective method to overcome this problem is demonstrated by using our structure-controllable amyloid peptides (SCAPs) terminated with a three-amino-acid-residue cap. The method consists on mixing two or more structurally related amyloid peptides with a fraction of modified SCAPs which co-assemble into a fibril. This SCAP-mixing method provides remarkable control over the self-assembly process both on the small oligomers level and the macroscopic fibrils level. Furthermore, it is shown that the modified peptides imbedded in the resulting fibril can subsequently be functionalized to generate nanowires with the desired properties, highlighting the importance of this SCAP method for nanotechnology applications. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Effects of E/Z Configuration of Fluoroalkene-containing HDAC Inhibitors on Selectivity for HDAC Isoforms

  Yoshiro Chuman, Mariko Ueyama, Satoshi Sano, Fei Wu, Yuhei Kiyota, Takayoshi Higashi, Satoshi Osada, Kazuyasu Sakaguchi

  CHEMISTRY LETTERS 42 8 833 - 835 2013年08月 [査読有り][通常論文]
   

  Histone deacetylase (HDAC) inhibitors belong to a new class of potential anticancer agents. It may be possible to reduce some of the toxicity by specifically targeting only the HDAC isoform. Here, stereoisomeric HDAC inhibitors containing fluoroalkene were analyzed for their specificity toward HDAC isoforms. Z-Form 1(Z) showed high affinity to HDACs whereas E-isoform 1(E) had lower affinity to HDAC1 and HDAC4. These data suggested that introduction of alkene with E/Z configuration to HDAC inhibitor can be a new strategy to develop the isoform-selective HDAC inhibitors.
• Inhibition of tumor suppressor protein p53-dependent transcription by a tetramerization domain peptide via hetero-oligomerization

  Junya Wada, Rui Kamada, Toshiaki Imagawa, Yoshiro Chuman, Kazuyasu Sakaguchi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 22 8 2780 - 2783 2012年04月 [査読有り][通常論文]
   

  Tumor suppressor protein p53 induces cell cycle arrest, apoptosis, and senescence in response to cellular stresses. The p53 tetramer formation is essential for its functions. Despite of these crucial functions of p53 for integrity of genome, activation of the p53 signal pathway causes low induced pluripotent stem (iPS) cell generation efficiency. In this study, we report transient inhibition of p53-dependent transcription using a p53 tetramerization domain peptide that contains cell penetrating and nuclear localization signals. The peptide was efficiently introduced into cells and inhibited p21 expression via hetero-tetramerization with endogenous p53 protein. This method can be applied towards safe and efficient iPS cell generation. (C) 2012 Elsevier Ltd. All rights reserved.
• 癌原遺伝子産物PPM1Dの相互作用因子の同定と核小体形成における機能解析

  小境 夕紀, 清田 雄平, 八木 寛陽, 中馬 吉郎, 坂口 和靖

  日本プロテオーム学会大会要旨集 2012 138 - 138 日本プロテオーム学会（日本ヒトプロテオーム機構） 2012年
• A small molecule inhibitor of p53-inducible protein phosphatase PPM1D

  Yagi Hiroaki, Chuman Yoshiro, Kozakai Yuuki, Imagawa Toshiaki, Takahashi Yu, Yoshimura Fumihiko, Tanino Keiji, Sakaguchi Kazuyasu

  Bioorganic & Medicinal Chemistry Letters 22 1 729 - 732 2012年 [査読有り][通常論文]
  
• Phosphatase assay for multi-phosphorylated substrates using phosphatase specific-motif antibody

  Yoshiro Chuman, Kanako Iizuka, Takeshi Honda, Hitoshi Onoue, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi

  JOURNAL OF BIOCHEMISTRY 150 3 319 - 325 2011年09月 [査読有り][通常論文]
   

  Protein phosphorylation plays central roles in a wide variety of signal transduction pathways and most phosphorylated proteins contain multi-phosphorylated sites. PPM1 type Ser/Thr protein phosphatase family is known to show rigid substrate specificity unlike other Ser/Thr phosphatase PPP family including PP1, PP2A and PP2B. PPM1 type phosphatases are reported to play important roles in growth regulation and in cellular stress signalling. In this study, we developed a phosphatase assay of PPM1D using phosphatase motif-specific antibody. PPM1D is a member of PPM1 type Ser/Thr phosphatase and known to dephosphorylate Ser(P)-Gln sequence. The gene amplification and overexpression of PPM1D were reported in many human cancers. We generated the monoclonal antibody specific for the Ser(P)-Gln sequence, named 3G9-H11. The specificity of this method using ELISA enables the convenient measurement of the dephosphorylation level of only PPM1D target residues of substrate peptides with multiple phosphorylated sites in the presence of multiple phosphatases. In addition, the antibody was applicable to immunoblotting assay for PPM1D function analysis. These results suggested that this method should be very useful for the PPM1D phosphatase assay, including high-throughput analysis and screening of specific inhibitors as anti-cancer drugs. The method using phosphatase motif-specific antibody can be applied to other PPM1 phosphatase family.
• Formation process and structure of polyproline self-assembled monolayer on gold surface

  Han Ying, Noguchi Hidenori, Kazuyasu Sakaguchi, Uosaki Kohei

  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 241 2011年03月27日 [査読有り][通常論文]
  
• Effective method for formation of functionalized nanowires using amyloid peptides

  Sakai Hiroki, Watanabe Ken, Chuman Yoshiro, Uosaki Kohei, Sakaguchi Kazuyasu

  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 241 2011年03月27日 [査読有り][通常論文]
  
• Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation.

  Kamada, R, Nomura, T, Anderson, C. W, Sakaguchi, K

  J. Biol. Chem. 286 252 - 258 2011年 [査読有り][通常論文]
  
• Probing phenylalanine environments in oligomeric structures with pentafluorophenylalanine and cyclohexylalanine.

  Nomura, T, Kamada, R, Ito, I, Sakamoto, K, Chuman, Y, Ishimori, K, Shimohigashi, Y, Sakaguchi, K

  Biopolymers 95 6 410 - 419 2011年 [査読有り][通常論文]
  
• Novel chemical inhibitors specific for p53-inducible protein phosphatase PPM1D

  Yagi Hiroaki, Chuman Yoshiro, Yoshimura Fumihiko, Tanino Keiji, Sakaguchi Kazuyasu

  Abstracts of Papers of the American Chemical Society 241 2011年 [査読有り][通常論文]
  
• Design of Potent Inhibitors of Human RAD51 Recombinase Based on BRC Motifs of BRCA2 Protein: Modeling and Experimental Validation of a Chimera Peptide

  Julian Nomme, Axelle Renodon-Corniere, Yuya Asanomi, Kazuyasu Sakaguchi, Alicja Z. Stasiak, Andrzej Stasiak, Bengt Norden, Vinh Tran, Masayuki Takahashi

  JOURNAL OF MEDICINAL CHEMISTRY 53 15 5782 - 5791 2010年08月 

  We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.
• Enhancement of transcriptional activity of mutant p53 tumor suppressor protein through stabilization of tetramer formation by calix[6]arene derivatives

  Rui Kamada, Wataru Yoshino, Takao Nomura, Yoshiro Chuman, Toshiaki Imagawa, Takanori Suzuki, Kazuyasu Sakaguchi

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 15 4412 - 4415 2010年08月 [査読有り][通常論文]
   

  Li-Fraumeni syndrome, a hereditary disorder characterized by familial clusters of early-onset multiple tumors, is caused by mutation of the TP53 gene, which encodes the p53 tumor suppressor protein. Mutation of Arg337 to histidine in the tetramerization domain of p53 is most frequently observed in Li-Fraumeni syndrome. This mutation is reported to destabilize the tetrameric structure of p53. We designed and synthesized calix[6] arene derivatives, which have six imidazole or pyrazole groups at the upper rim. In this study, we report, for the first time, the enhancement of the in vivo transcriptional activity of the most common Li-Fraumeni p53 mutant by imidazole-calix[6] arene through stabilization of the oligomer formation. (C) 2010 Elsevier Ltd. All rights reserved.
• Drastic effects on fibril formation of amyloid-$β$ peptides by the addition of amino acid residue units to the termini

  Asanomi Yuya, Kobayashi Yumiko, Sakai Hiroki, Masuda Takuya, Chen Xinjiang, Chuman Yoshiro, Uosaki Kohei, Sakaguchi Kazuyasu

  Protein and peptide letters 17 4 458 - 463 Bentham Science Publishers 2010年04月 [査読有り][通常論文]
   

  We report that the addition of amino acids to the amyloid peptide dramatically affected the structure and the rate of formation of amyloid fibrils. The attachment of three lysines to A beta(10-35) gave the formation of remarkably long fibrils, while three glutamates resulted in a faster formation rate of the fibrils.
• Fluoroalkene Modification of Mercaptoacetamide-based Histone Deacetylase Inhibitors

  OSADA S, SANO S, KODAMA H, UEYAMA M, CHUMAN Y, SAKAGUCHI K

  Bioorg. Med. Chem. 18 2 605 - 611 2010年 [査読有り][通常論文]
   

  Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.
• Tetramerization of tumor suppressor protein p53

  Rui Kamada, Kazuyasu Sakaguchi

  Seikagaku 82 6 484 - 493 2010年 [査読有り][通常論文]
  
• Characterization of a new cancer-associated mutant of p53 with a missense mutation (K351N) in the tetramerization domain

  Michela Muscolini, Elisa Montagni, Silvana Caristi, Takao Nomura, Rui Kamada, Silvia Di Agostino, Marco Corazzari, Mauro Piacentini, Giovanni Blandino, Antonio Costanzo, Kazuyasu Sakaguchi, Loretta Tuosto

  CELL CYCLE 8 20 3396 - 3405 2009年10月 [査読有り][通常論文]
   

  Inactivation of the tumor suppressor p53 is central to carcinogenesis and acquisition of resistance to drug-induced apoptosis. The majority of alterations are missense mutations and occur within the DNA-binding domain. However, little is known about the point mutations in the tetramerization domain (TD). Here we investigated the properties of a new p53 mutant (Lys 351 to Asn) in the TD identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution significantly reduces the thermodynamic stability of p53 tetramers without affecting the overall half-life of the protein. Moreover, p53 K351N has a reduced ability to bind DNA and to trans-activate its specific target gene promoters, such as bax. Data obtained from the analysis of p53 subcellular localization revealed that K351N mutation inhibits the nuclear export of p53 and accumulation in the cytoplasm induced by cisplatin treatment. These results identify p53 K351N as a new cancer associated mutant with reduced tumor suppressor activity and altered functions in response to apoptotic stimuli.
• Transcriptional Activity of p53 in Individual Living Mammalian Cells.

  Imagawa, T, Terai, T, Yamada, Y, Kamada, R, Sakaguchi, K

  Anal. Biochem. 387 249 - 256 2009年 [査読有り][通常論文]
  
• Effects of tumor-associated mutations in the p53 tetramerization domain on oligomerization state and transcriptional activity.

  Kamada, R, Terai, T, Nomura, T, Chuman, Y, Imagawa, T, Sakaguchi, K

  Adv. Exp. Med. Biol. 611 567 - 568 2009年 [査読有り][通常論文]
  
• Oxidation of methionine residue at hydrophobic core destabilizes p53 tetrameric structure

  NOMURA Takao, KAMADA Rui, ITO Issaku, CHUMAN Yoshiro, SHIMOHIGASHI Yasuyuki, SAKAGUCHI Kazuyasu

  Biopolymers 91 1 78 - 84 2009年 [査読有り][通常論文]
  
• PPM1D430, a Novel Alternative Splicing Variant of the Human PPM1D, can Dephosphorylate p53 and Exhibits Specific Tissue Expression

  Yoshiro Chuman, Wataru Kurihashi, Yohei Mizukami, Takehiro Nashimoto, Hiroaki Yagi, Kazuyasu Sakaguchi

  JOURNAL OF BIOCHEMISTRY 145 1 1 - 12 2009年01月 [査読有り][通常論文]
   

  PPM1D is a PPM1 type protein phosphatase and is induced in response to DNA damage. PPM1D-deficient mice show defects in spermatogenesis and lymphoid cell functions but the mechanisms underlying these phenotypes remain unknown. In our current study, we identify and characterize an alternative splicing variant (denoted PPM1D430) of human PPM1D at both the mRNA and protein level. PPM1D430 comprises the common 420 residues of the known PPM1D protein (PPM1D605) and contains a stretch of PPM1D430-specific 10 amino acids. Semi-quantitative reverse transcriptionpolymerase chain reaction (RTPCR) analysis revealed that PPM1D430 mRNA is also induced in response to the genotoxic stress in a p53-dependent manner. In vitro phosphatase analysis and PPM1D430-specific RNA interference analysis further indicated that PPM1D430 can dephosphorylate Ser15 of human p53 both in vitro and in vivo. On the other hand, expression profiling of this gene by RTPCR analysis of a human tissue cDNA panel revealed that PPM1D430 is expressed exclusively in testes and in leucocytes whereas PPM1D605 is ubiquitous. In addition, PPM1D430 shows a different subcellular localization pattern and protein stability when compared with PPM1D605 under some conditions. Our current findings thus suggest that PPM1D430 may exert specific functions in immune response and/or spermatogenesis.
• Differential Recognition of Phosphorylated Transactivation Domains of p53 by Different p300 Domains

  POLLEY Smarajit, POLLEY Smarajit, GUHA Soumi, ROY Neeladri Sekhar, KAR Sanchari, SAKAGUCHI Kazuyasu, CHUMAN Yoshiro, SWAMINATHAN V, KUNDU Tapas, ROY Siddhartha

  J. Mol. Biol. 376 1 8 - 12 2008年 [査読有り][通常論文]
  
• Characterization of the Active Site and a Unique Uncompetitive Inhibitor of the PPM1-type Protein Phosphatase PPM1D

  Chuman, Y, Yagi, H, Fukuda, T, Nomura, T, Matsukizono, M, Shimohigashi, Y, Sakaguchi K

  Protein Pept. Lett. 15 938 - 948 2008年 [査読有り][通常論文]
  
• PILLARS OF IMMUNOLOGY

  Stern Lawrence J, Hunt Donald F, Henderson Robert A, Shabanowitz Jeffrey, Sakaguchi Kazuyasu, Michel Hanspeter, Sevilir Noelle, Cox Andrea L, Appella Ettore, Engelhard Victor H

  J Immunol 179 2665  2007年 [査読有り][通常論文]
  
• Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies

  Takeshi Honda, Ayami Matsushima, Kazunori Sumida, Yoshiro Chuman, Kazuyasu Sakaguchi, Hitoshi Onoue, Ian A. Meinertzhagen, Yasuyuki Shimohigashi, Miki Shimohigashi

  JOURNAL OF COMPARATIVE NEUROLOGY 499 3 404 - 421 2006年11月 [査読有り][通常論文]
   

  Pigment-dispersing factor (PDF) is an 18-mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti-Uca beta-PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF-like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF-like peptide, we prepared 10 anti-Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti-Uca beta-PDH pAb and anti-Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF-like peptide possessing both N- and C-terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform.
• Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.

  Higashimoto Y, Asanomi Y, Takakusagi S, Lewis MS, Uosaki K, Durell SR, Anderson CW, Appella E, Sakaguchi K

  Biochemistry 45 6 1608 - 1619 2006年02月 [査読有り][通常論文]
  
• Evidence for a relationship between activity and the tetraprotomeric assembly of solubilized pig gastric H/K-ATPase

  K Abe, S Kaya, K Taniguchi, Y Hayashi, T Imagawa, M Kikumoto, K Oiwa, K Sakaguchi

  JOURNAL OF BIOCHEMISTRY 138 3 293 - 301 2005年09月 [査読有り][通常論文]
   

  Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C(12)E(s)) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5 '-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K(0.5) = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K(0.5)= 28 mM) and H/K-ATPase (K(0.5) = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/KATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K(+) in C(12)E(8)-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C(12)E(8)-solubilized gastric H/K-ATPase is a tetraprotomer.
• Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (m8) is essential for Na,K-ATPaseActivity

  T Imagawa, T Yamamoto, S Kaya, K Sakaguchi, K Taniguchi

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 19 18736 - 18744 2005年05月 [査読有り][通常論文]
   

  The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K-0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K-0.5(Na,EP)) or Na, K-ATPase activity (K-0.5(Na,ATPase)), were increased by around 2 similar to 8- fold in the case of T774A, V920E, and E954A. The apparent K-0.5 values for K+, as estimated by the Na,K-ATPase (K-0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K-0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K-0.5(K,ATPase). The apparent K-0.5 values for ATP (K-0.5(EP)), as estimated by phosphorylation ( a high affinity ATP effect), were increased by 1.6 similar to 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na, K-ATPase activity (K-0.5(ATPase)) and ATP-induced inhibition (K-i,0.5(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.
• Mechanism of Amyloid-Like Fibrillar Aggregation of Mutant Peptide of p53 Tetramerization Domain

  ASANOMI Yuya, TAKAKUSAGI Satoru, CHUMAN Yoshiro, KAYA Shunji, IMAGAWA Toshiaki, UOSAKI Kohei, SAKAGUCHI Kazuyasu

  Peptide science : proceedings of the ... Japanese Peptide Symposium 2004 0 313 - 316 2005年03月 [査読無し][通常論文]
  
• Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues

  K Isozaki, H Fukahori, T Honda, N Shirasu, K Okada, T Nose, K Sakaguchi, Y Shimohigashi

  LETTERS IN PEPTIDE SCIENCE 10 5-6 511 - 522 2003年 [査読有り][通常論文]
   

  The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [D-Ala(2), Leu(5)]-enkephalyl-Cys(Npys)(6) and [D-Ala(2),Leu(CH(2)SNpys)(5)]enkephalin, and dynorphin A analogues of [D-Ala(2),Cys(Npys)(12)]dynorphin A-(1-13 ) amide and [D-Ala(2),Cys(Npys)(8)]dynorphin A-(1-9) amide have been found to affinity-label all of the delta, mu (rat brain), and kappa (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the delta opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the delta opioid receptor. Then, using a mutant delta receptor with the Cys60-->Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [D-Ala(2),Cys(Npys)(12)]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type delta receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant delta receptors. These results indicate that the delta-Cys60 residue has a free thiol group, which is labeled by [D-Ala(2),Cys(Npys)(12)]dynorphin A-(1-13) amide.
• Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element

  T Kuwata, C Gongora, Y Kanno, K Sakaguchi, T Tamura, T Kanno, Basrur, V, R Martinez, E Appella, T Golub, K Ozato

  MOLECULAR AND CELLULAR BIOLOGY 22 21 7439 - 7448 2002年11月 [査読有り][通常論文]
   

  ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-gamma) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in WN-y-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-y-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-y-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-y. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-y-dependent gene regulation in an immune system-specific manner.
• Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor binding

  M Kawano, K Okada, T Honda, T Nose, K Sakaguchi, T Costa, Y Shimohigashi

  JOURNAL OF PEPTIDE SCIENCE 8 10 561 - 569 2002年10月 [査読有り][通常論文]
   

  Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(=Arg-TYr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position I of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the p opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds. Copyright (C) 2002 European Peptide Society and John Wiley Sons, Ltd.
• Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents.

  Higashimoto Y, Saito S, Tong XH, Hong A, Sakaguchi K, Appella E, Anderson CW

  The Journal of biological chemistry 275 30 23199 - 23203 2000年07月 [査読有り][通常論文]
  
• Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase - Effect on Mdm2 binding

  K Sakaguchi, S Saito, Y Higashimoto, S Roy, CW Anderson, E Appella

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 13 9278 - 9283 2000年03月 [査読有り][通常論文]
   

  The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr's but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser's Thr's was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser's. Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm(2) binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).
• Identification of gel-separated tumor marker proteins by mass spectrometry

  AC Bergman, T Benjamin, A Alaiya, M Waltham, K Sakaguchi, B Franzen, S Linder, T Bergman, G Auer, E Appella, PJ Wirth, H Jornvall

  ELECTROPHORESIS 21 3 679 - 686 2000年02月 [査読有り][通常論文]
   

  Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these overexpressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.
• Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation

  DV Bulavin, S Saito, MC Hollander, K Sakaguchi, CW Anderson, E Appella, AJ Fornace

  EMBO JOURNAL 18 23 6845 - 6854 1999年12月 [査読有り][通常論文]
   

  Components of the ras signaling pathway contribute to activation of cellular p53, In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site, Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46, Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.
• Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas

  Y Chuman, AC Bergman, T Ueno, S Saito, K Sakaguchi, AA Alaiya, B Franzen, T Bergman, D Arnott, G Auer, E Appella, H Jornvall, S Linder

  FEBS LETTERS 462 1-2 129 - 134 1999年11月 [査読有り][通常論文]
   

  A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located, Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors. (C) 1999 Federation of European Biochemical Societies.
• Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth

  SE Pike, L Yao, J Setsuda, KD Jones, B Cherney, E Appella, K Sakaguchi, H Nakhasi, CD Atreya, J Teruya-Feldstein, P Wirth, G Gupta, G Tosato

  BLOOD 94 7 2461 - 2468 1999年10月 [査読有り][通常論文]
   

  Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180, Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases. This is a US government work. There are no restrictions on its use.
• Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53.

  Delphin C, Ronjat M, Deloulme JC, Garin G, Debussche L, Higashimoto Y, Sakaguchi K, Baudier J

  The Journal of biological chemistry 274 15 10539 - 10544 1999年04月 [査読有り][通常論文]
  
• Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

  AC Bergman, AA Alaiya, W Wendler, B Binetruy, M Shoshan, K Sakaguchi, T Bergman, U Kronenwett, G Auer, E Appella, H Jornvall, S Linder

  CELLULAR AND MOLECULAR LIFE SCIENCES 55 3 467 - 471 1999年03月 [査読有り][通常論文]
   

  Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.
• Effects of mutations on tetramer formation of tumor suppressor protein p53

  Y Higashimoto, MS Lewis, P Kennedy, AM Gronenborn, GM Clore, E Appella, K Sakaguchi

  PEPTIDE SCIENCE - PRESENT AND FUTURE 303 - 305 1999年 [査読有り][通常論文]
  
• Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth

  SE Pike, L Yao, KD Jones, B Cherney, E Appella, K Sakaguchi, H Nakhasi, J Teruya-Feldstein, P Wirth, G Gupta, G Tosato

  JOURNAL OF EXPERIMENTAL MEDICINE 188 12 2349 - 2356 1998年12月 [査読有り][通常論文]
   

  All endothelial cell inhibitor sas purified from supernatant of an Epstein-Barr virus-immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not calls of other lineages, and suppressed angiogenesis in vivo. We have named this NH2 terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.
• Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles

  Y Kawakami, PF Robbins, Wang, X, JP Tupesis, MR Parkhurst, XQ Kang, K Sakaguchi, E Appella, SA Rosenberg

  JOURNAL OF IMMUNOLOGY 161 12 6985 - 6992 1998年12月 [査読有り][通常論文]
   

  To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides, An HLA-Al-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK), CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV), HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC), Replacement of either cysteine residue with alpha-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.
• Posttranslational modifications involved in the DNA damage response.

  CW Anderson, E Appella, K Sakaguchi

  JOURNAL OF PROTEIN CHEMISTRY 17 6 527 - 527 1998年08月 [査読有り][通常論文]
  
• Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells

  CA Pise-Masison, M Radonovich, K Sakaguchi, E Appella, JN Brady

  JOURNAL OF VIROLOGY 72 8 6348 - 6355 1998年08月 [査読有り][通常論文]
   

  Inhibition of p53 function, through either mutation or interaction with viral or cellular transforming proteins, correlates strongly with the oncogenic potential. Only a small percentage of human T cell lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations, and mutated p53 genes have been found in only one-fourth of adult T-cell leukemia cases. In previous studies, we demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-1-transformed cells. Further, the viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53 through a mechanism involving the first 52 amino acids of p53. Here we show for the first time that phosphorylation of p53 inactivates p53 by blocking its interaction with basal transcription factors. Using two-dimensional peptide mapping, we demonstrate that peptides corresponding to amino acids 1 to 19 and 387 to 393 are hyperphosphorylated in HTLV-1-transformed cells. Moreover, using antibodies specific for phosphorylated Ser15 and Ser392, we demonstrate increased phosphorylation of these amino acids. Since HTLV-1 p53 binds DNA in a sequence-specific manner but fails to interact with TFIID, we tested whether phosphorylation of the N terminus of p53 affected p53-TFIID interaction. Using biotinylated peptides, we show that phosphorylation of Ser15 alone inhibits p53-TFIID interaction. In contrast, phosphorylation at Ser15 and -37 restores TFIID binding and blocks MDM2 binding. Our studies provide evidence that HTLV-1 utilizes the posttranslational modification of p53 in vivo to inactivate function of the tumor suppressor protein.
• Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41

  Munoz-Barroso, I, S Durell, K Sakaguchi, E Appella, R Blumenthal

  JOURNAL OF CELL BIOLOGY 140 2 315 - 323 1998年01月 [査読有り][通常論文]
   

  We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus-1 (HIV-1) envelope glycoprotein-expressing cell and a CD4(+) target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol, We developed a new three-color assay to keep track of the cell into which fluorescent lipids and/or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipid-permissive fusion pore and a solute-permissive fusion pore (FPS), respectively, A synthetic peptide (DP178) corresponding to residues 643-678 of the HIV-1(LAI) gp120-gp41 sequence (Wild, C.T., D.C. Shugars, T.K. Greenwell, C.B. McDanal, and T.J. Matthews, 1994. Proc. Natl. Acad. Sci. USA. 91:12676-12680) completely inhibited FPS at 50 ng/ml, whereas at that concentration there was 20-30% fusion activity measured by the lipid redistribution, The differences detected in lipid mixing versus contents mixing are maintained up to 6 h of coculture of gp120-41-expressing cells with target cells, indicating that DP178 can "clamp" the fusion complex in the lipid mixing intermediate for very long time periods, A peptide from the NH2-terminal of gp41, DP107, inhibited HIV-1(LAI) gp120-gp41-mediated cell fusion at higher concentrations, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations, The inhibition of solute redistribution by DP178 was complete when the peptide was added to the fusion reaction mixture during the first 15 min of coculture, We have analyzed the inhibition data in terms of a fusion pore dilation model that incorporates the recently determined high resolution structure of the gp41 core.
• An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli

  ML Cai, Y Huang, K Sakaguchi, GM Clore, AM Gronenborn, R Craigie

  JOURNAL OF BIOMOLECULAR NMR 11 1 97 - 102 1998年01月 [査読有り][通常論文]
   

  A cost-effective protocol for uniform N-15 and/or C-13 isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.
• DNA damage induces phosphorylation of the amino terminus of p53

  JD Siliciano, CE Canman, Y Taya, K Sakaguchi, E Appella, MB Kastan

  GENES & DEVELOPMENT 11 24 3471 - 3481 1997年12月 [査読有り][通常論文]
   

  Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.
• Increased expression of $α$-enolase in c-jun transformed rat fibroblasts without increased activation of plasminogen

  Bergman Ann-Charlotte, Linder Christina, Sakaguchi Kazuyasu, Sten-Linder Margareta, Alaiya Ayodele A, Franz{\'e}n Bo, Shoshan Maria C, Bergman Tomas, Wiman Bj{\"o}rn, Auer Gert, other

  FEBS letters 417 1 17 - 20 Wiley Online Library 1997年11月 [査読有り][通常論文]
   

  Two-dimensional gel electrophoresis was used to identify polypeptides differentially expressed between normal and c-jun transformed rat fibroblasts, The level of a 49 kDa polypeptide was 3-fold elevated in c-jun transformed cells, Sequence analysis by ion trap mass spectrometry identified the polypeptide as rat alpha-enolase, Enolase functions as a cell surface receptor for plasminogen, suggesting that upregulation may increase plasminogen activation and cell surface proteolysis important for tumor growth, However, no difference was observed between normal and transformed cells in formation of plasmin, suggesting that upregulation of alpha-enolase may contribute to an increased metabolic capacity, but not to increased plasminogen activation, (C) 1997 Federation of European Biochemical Societies.
• Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53

  K Sakaguchi, H Sakamoto, MS Lewis, CW Anderson, JW Erickson, E Appella, D Xie

  BIOCHEMISTRY 36 33 10117 - 10124 1997年08月 [査読有り][通常論文]
   

  Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation, Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The K-d for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be similar to 1-10 mu M. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.
• Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner

  M Fiscella, HL Zhang, SJ Fan, K Sakaguchi, SF Shen, WE Mercer, GF VandeWoude, PM OConnor, E Appella

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 12 6048 - 6053 1997年06月 [査読有り][通常論文]
   

  Exposure of mammalian cells to ionizing radiation (IR) induces a complex array of cellular responses including cell cycle arrest and/or apoptosis. IR-induced GI arrest has been shown to depend on the presence of the tumor suppressor p53, which acts as a transcriptional activator of several genes, p53 also plays a role in the induction of apoptosis in response to DNA damage, and this pathway can be activated by bath transcription-dependent and -independent mechanisms, Here we report the identification of a novel transcript whose expression is induced in response to IR in a p53-dependent manner, and that shows homology to the type 2C protein phosphatases. We have named this novel gent, wip1. In vitro, recombinant Wip1 displayed characteristics of a type 2C phosphatase, including Mg2+ dependence and relative insensitivity to okadaic acid. Studies performed in several cell lines revealed that wip1 accumulation following IR correlates with the presence of wild-type p53, The accumulation of wip1 mRNA following IR was rapid and transient, and the protein was localized to the nucleus, Similar to waf1, ectopic expression of wip1 in human cells suppressed colony formation, These results suggest that Wip1 might contribute to growth inhibitory pathways activated in response to DNA damage in a p53-dependent manner.
• Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T Helper Epitopes (PADRE) for antibody responses in vivo

  MF delGuercio, J Alexander, RT Kubo, T Arrhenius, A Maewal, E Appella, SL Hoffman, T Jones, D Valmori, K Sakaguchi, HM Grey, A Sette

  VACCINE 15 4 441 - 448 1997年03月 [査読有り][通常論文]
   

  Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity;of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development. (C) 1997 Elsevier Science Ltd.
• Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells

  Tsai, V, S Southwood, J Sidney, K Sakaguchi, Y Kawakami, E Appella, A Sette, E Celis

  JOURNAL OF IMMUNOLOGY 158 4 1796 - 1802 1997年02月 [査読有り][通常論文]
   

  The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100(+) melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do not appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol, Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission, Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical ''subdominant'' epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.
• Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

  H Sakamoto, H Kodama, Y Higashimoto, M Kondo, MS Lewis, CW Anderson, E Appella, K Sakaguchi

  INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH 48 5 429 - 442 1996年11月 [査読有り][通常論文]
   

  A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the nonphosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)(2))-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifugation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the alpha-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. (C) Munksgaard 1996.
• The proteolytic environment involved in MHC class II-restricted antigen presentation can be modulated by the p41 form of invariant chain

  B Fineschi, K Sakaguchi, E Appella, J Miller

  JOURNAL OF IMMUNOLOGY 157 8 3211 - 3215 1996年10月 [査読有り][通常論文]
   

  During biosynthesis, MHC class II associates with invariant chain which exists in two forms, p31 and p41. Both forms of invariant chain prevent peptide binding to class II, facilitate transport, and enhance class II localization to Ag-processing compartments, In spite of these shared functions, presentation of some Ags can be selectively enhanced by expression of p41, Here we show that p41 can function as a protease inhibitor: 1) the functional and biochemical consequences of p41 expression can be mimicked by inhibiting cysteine proteases in vivo; 2) the amount of intracellular active cysteine proteases is dramatically decreased in p41-positive cells; and 3) a polypeptide corresponding to the p41-unique region is a potent inhibitor of cathepsin L in vitro, These data suggest that p41 can enhance Ag presentation by reducing the proteolytic activity of the Ag-processing compartment, thus protecting a subset of antigenic epitopes from excessive degradation.
• Chemical synthesis and applications of phosphopeptides

  Sakaguchi Kazuyasu, Roller Peter P, Appella Ettore

  Genetic engineering 249  Springer, Boston, MA 1996年 [査読有り][通常論文]
  
• IDENTIFICATION OF A TYROSINASE EPITOPE RECOGNIZED BY HLA-A24-RESTRICTED, TUMOR-INFILTRATING LYMPHOCYTES

  XQ KANG, Y KAWAKAMI, M ELGAMIL, RF WANG, K SAKAGUCHI, YANNELLI, JR, E APPELLA, SA ROSENBERG, PF ROBBINS

  JOURNAL OF IMMUNOLOGY 155 3 1343 - 1348 1995年08月 [査読有り][通常論文]
   

  A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HFA-A24-restricted TIL, TIL 888, as well as HLA-A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti-peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24(+) B cell lines pulsed with the peptide and HLA-A24(+) tyrosinase(+) melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24(+) melanoma patients.
• CLONING OF A NEW GENE ENCODING AN ANTIGEN RECOGNIZED BY MELANOMA-SPECIFIC HLA-A24-RESTRICTED TUMOR-INFILTRATING LYMPHOCYTES

  PF ROBBINS, M ELGAMIL, YF LI, SL TOPALIAN, L RIVOLTINI, K SAKAGUCHI, E APPELLA, Y KAWAKAMI, SA ROSENBERG

  JOURNAL OF IMMUNOLOGY 154 11 5944 - 5950 1995年06月 [査読有り][通常論文]
   

  The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor-infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24-restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and an Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290.
• BACKBONE DYNAMICS OF THE OLIGOMERIZATION DOMAIN OF P53 DETERMINED FROM N-15 NMR RELAXATION MEASUREMENTS

  RT CLUBB, JG OMICHINSKI, K SAKAGUCHI, E APPELLA, AM GRONENBORN, GM CLORE

  PROTEIN SCIENCE 4 5 855 - 862 1995年05月 [査読有り][通常論文]
   

  The backbone dynamics of the tetrameric p53 oligomerization domain (residues 319-360) have been investigated by two-dimensional inverse detected heteronuclear H-1-N-15 NMR spectroscopy at 500 and 600 MHz. (NT1)-N-15, T-2, and heteronuclear NOEs were measured for 39 of 40 non-proline backbone NH vectors at both field strengths. The overall correlation time for the tetramer, calculated from the T-1/T-2 ratios, was found to be 14.8 ns at 35 degrees C. The correlation times and amplitudes of the internal motions were extracted from the relaxation data using the model-free formalism (Lipari G, Szabo A, 1982, J Am Chem Soc 104:4546-4559). The internal dynamics of the structural core of the p53 oligomerization domain are uniform and fairly rigid, with residues 327-354 exhibiting an average generalized order parameter (S-2) Of 0.88 +/- 0.08. The N- and C-termini exhibit substantial mobility and are unstructured in the solution structure of p53. Residues located at the N- and C-termini, in the beta-sheet, in the turn between the alpha-helix and beta-sheet, and at the C-terminal end of the alpha-helix display two distinct internal motions that are faster than the overall correlation time. Fast internal motions (less than or equal to 20 ps) are within the extreme narrowing limit and are of uniform amplitude. The slower motions (0.6-2.2 ns) are outside the extreme narrowing limit and vary in amplitude. Four residues at the tetramer interface exhibit a small degree of conformational averaging as evidenced by N-15 line broadening, possibly due to sliding or rolling of the helices at the interface of the two dimers that form the tetramer.
• REFINED SOLUTION STRUCTURE OF THE OLIGOMERIZATION DOMAIN OF THE TUMOR-SUPPRESSOR P53

  GM CLORE, J ERNST, R CLUBB, JG OMICHINSKI, WMP KENNEDY, K SAKAGUCHI, E APPELLA, AM GRONENBORN

  NATURE STRUCTURAL BIOLOGY 2 4 321 - 333 1995年04月 [査読有り][通常論文]
   

  The NMR solution structure of the oligomerization domain of the tumour suppressor p53 (residues 319-360) has been refined. The structure comprises a dimer of dimers, oriented in an approximately orthogonal manner. The present structure determination is based on 4,472 experimental NMR restraints which represents a three and half fold increase over our previous work in the number of NOE restraints at the tetramerization interface. A comparison with the recently solved 1.7 Angstrom resolution X-ray structure shows that the structures are very similar and that the average angular root-mean-square difference in the interhelical angles is about 1 degrees. The results of recent extensive mutagenesis data and the possible effects of mutations which have been identified in human cancers are discussed in the light of the present structure.
• RECOGNITION OF MULTIPLE EPITOPES IN THE HUMAN-MELANOMA ANTIGEN GP100 BY TUMOR-INFILTRATING T-LYMPHOCYTES ASSOCIATED WITH IN-VIVO TUMOR-REGRESSION

  Y KAWAKAMI, S ELIYAHU, C JENNINGS, K SAKAGUCHI, XQ KANG, S SOUTHWOOD, PF ROBBINS, A SETTE, E APPELLA, SA ROSENBERG

  JOURNAL OF IMMUNOLOGY 154 8 3961 - 3968 1995年04月 [査読有り][通常論文]
   

  Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp 100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.
• INDUCTION OF TUMOR-REACTIVE CTL FROM PERIPHERAL-BLOOD AND TUMOR-INFILTRATING LYMPHOCYTES OF MELANOMA PATIENTS BY IN-VITRO STIMULATION WITH AN IMMUNODOMINANT PEPTIDE OF THE HUMAN-MELANOMA ANTIGEN MART-1

  L RIVOLTINI, Y KAWAKAMI, K SAKAGUCHI, S SOUTHWOOD, A SETTE, PF ROBBINS, FM MARINCOLA, ML SALGALLER, YR YANNELLI, E APPELLA, SA ROSENBERG

  JOURNAL OF IMMUNOLOGY 154 5 2257 - 2265 1995年03月 [査読有り][通常論文]
   

  MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2(+) melanoma patients, as well as from 2 of 4 PBL from HLA-A2(+) healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2(+) MART-1(+) melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2(+) melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.
• THE HUMAN DNA-ACTIVATED PROTEIN-KINASE, DNA-PK, IS ACTIVATED BY DNA BREAKS AND PHOSPHORYLATES NUCLEAR DNA-BINDING PROTEIN SUBSTRATES ON SERINES AND THREONINES FOLLOWING BY GLUTAMINE

  CW ANDERSON, MA CONNELLY, H ZHANG, JD SIPLEY, SP LEESMILLER, K SAKAGUCHI, SJ ULLRICH, SP JACKSON, E APPELLA

  JOURNAL OF PROTEIN CHEMISTRY 13 5 500 - 501 1994年07月 [査読有り][通常論文]
  
• STRUCTURE AND POSTTRANSLATIONAL MODIFICATION OF THE HUMAN P53 PROTEIN

  E APPELLA, M FISCELLA, N ZAMBRANO, SJ ULLRICH, K SAKAGUCHI, H SAKAMOTO, MS LEWIS, D LIN, WE MERCER, CW ANDERSON

  JOURNAL OF PROTEIN CHEMISTRY 13 5 499 - 500 1994年07月 [査読有り][通常論文]
  
• IDENTIFICATION OF THE IMMUNODOMINANT PEPTIDES OF THE MART-1 HUMAN-MELANOMA ANTIGEN RECOGNIZED BY THE MAJORITY OF HLA-A2-RESTRICTED TUMOR-INFILTRATING LYMPHOCYTES

  Y KAWAKAMI, S ELIYAHU, K SAKAGUCHI, PF ROBBINS, L RIVOLTINI, YANNELLI, JR, E APPELLA, SA ROSENBERG

  JOURNAL OF EXPERIMENTAL MEDICINE 180 1 347 - 352 1994年07月 [査読有り][通常論文]
   

  Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART-1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL, when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.
• IDENTIFICATION OF A HUMAN-MELANOMA ANTIGEN RECOGNIZED BY TUMOR-INFILTRATING LYMPHOCYTES ASSOCIATED WITH IN-VIVO TUMOR REJECTION

  Y KAWAKAMI, S ELIYAHU, CH DELGADO, PF ROBBINS, K SAKAGUCHI, E APPELLA, YANNELLI, JR, GJ ADEMA, T MIKI, SA ROSENBERG

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 14 6458 - 6462 1994年07月 [査読有り][通常論文]
   

  The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2(+) melanomas and on all HLA-A2(+) cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2(+) breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2(+) melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types bf tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.
• HIGH-RESOLUTION STRUCTURE OF THE OLIGOMERIZATION DOMAIN OF P53 BY MULTIDIMENSIONAL NMR

  GM CLORE, JG OMICHINSKI, K SAKAGUCHI, N ZAMBRANO, H SAKAMOTO, E APPELLA, AM GRONENBORN

  SCIENCE 265 5170 386 - 391 1994年07月 [査読有り][通常論文]
   

  The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.
• STRUCTURAL ESSENTIALS OF SER-1 IN TETHERED PEPTIDE LIGAND OF HUMAN THROMBIN RECEPTOR FOR PHOSPHOINOSITIDE HYDROLYSIS

  K SAKAGUCHI, H KODAMA, Y OGINO, T COSTA, T NOSE, Y SHIMOHIGASHI

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 67 6 1659 - 1663 1994年06月 [査読有り][通常論文]
   

  In order to inspect the structural elements of Ser-1 in receptor activation by SFLLRNP (one-letter amino acid code), a ligand peptide tethered to the thrombin receptor, a series of analogs with such replacements as D-Ser, Ala, Thr, and Ac-Ser have been synthesized. These analogs were evaluated for their ability to hydrolyze the phosphoinositide in human neuroblastoma SH-EP cells. It was found that the alpha-amino group and L-configuration of Ser-1 are very important in the activation of receptors. N-Acetylation or deletion of Ser1 completely eliminated the activity of SFLLRNP (a half-maximal effective concentration, EC50 = 0.89 muM (1 M = 1 mol dm-3)), and these modifications induced no antagonist activity. Incorporation of D-Ser also drastically diminished the activity, but retained about 50% activity of the maximal response by 100 muM SFLLRNP. The Ser/Ala substitution sustained 30% of the activity of SFLLRNP to elicit a full stimulation. The Ser/Thr substitution, however, enhanced the activity (20%) in spite of its decreased activity (60%) reported for platelet aggregation. These results indicated that the beta-hydroxyl group of Ser-1 is important to receptor activation, but not essential. The effect of chemical modifications on the receptor activities of the tethered ligand is discussed with regard to the efficacy between phosphoinositide hydrolysis and biological activities.
• MOLECULAR-INTERACTIONS BETWEEN INTERFERON CONSENSUS SEQUENCE BINDING-PROTEIN AND MEMBERS OF THE INTERFERON REGULATORY FACTOR FAMILY

  C BOVOLENTA, PH DRIGGERS, MS MARKS, JA MEDIN, AD POLITIS, SN VOGEL, DE LEVY, K SAKAGUCHI, E APPELLA, JE COLIGAN, K OZATO

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 11 5046 - 5050 1994年05月 [査読有り][通常論文]
   

  Interferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor expressed mostly in the cells of the immune system. ICSBP belongs to the IFN regulatory factor (IRF) family, which also includes IRF-1, IRF-2, and the IFN-alpha-stinulated gene factor 3 gamma(ISGF3 gamma). We show here that ICSBP forms a complex with IRF-1 or IRF-2 both in vivo and in vitro and, in the presence or absence of the target DNA, with the IFN-stimulated response element (ISRE). Further, electrophoretic mobility shift assays show that this interaction greatly enhances the otherwise very low binding affinity of ICSBP to the ISRE. We show, on the other hand, that ICSBP inhibits binding of the IFN-alpha-stinulated gene factor 3 gamma to the ISRE. Through these interactions ICSBP is likely to exert complex modulatory functions in the regulation of IFN-stimulated genes.
• CD45 REGULATION OF TYROSINE PHOSPHORYLATION AND ENZYME-ACTIVITY OF SRC FAMILY KINASES

  CM BURNS, K SAKAGUCHI, E APPELLA, JD ASHWELL

  JOURNAL OF BIOLOGICAL CHEMISTRY 269 18 13594 - 13600 1994年05月 [査読有り][通常論文]
   

  Previous analyses have suggested that the CD45 tyrosine phosphatase activates src family tyrosine kinases p56(lck) and p59(fyn) by dephosphorylating regulatory COOH-terminal residues. We have examined the status of p56(lck) and p59(fyn) in murine and human CD45(-) T cell lines. Surprisingly, despite the fact that p56(lck) and p59(fyn) were spontaneously hyperphosphorylated, the tyrosine kinase activity of both enzymes was increased in CD45(-) versus CD45(+) cells. In vitro exposure of hyperphosphorylated p56(lck) to CD45 decreased enzyme activity to near-basal levels. Lck from CD45(-) cells was hyperphosphorylated on the cyanogen bromide digestion fragment that contains the negative regulatory residue Tyr-505, and the identity of this site of phosphorylation was confirmed by trypsin digestion followed by high performance liquid chromatography. Loss of CD45 results, therefore, in a paradoxical hyperphosphorylation of the COOH-terminal tyrosine and increased src family kinase enzymatic activity.
• DEFINITION OF SPECIFIC PEPTIDE MOTIFS FOR 4 MAJOR HLA-A ALLELES

  RT KUBO, A SETTE, HM GREY, E APPELLA, K SAKAGUCHI, NZ ZHU, D ARNOTT, N SHERMAN, J SHABANOWITZ, H MICHEL, WM BODNAR, TA DAVIS, DF HUNT

  JOURNAL OF IMMUNOLOGY 152 8 3913 - 3924 1994年04月 [査読有り][通常論文]
   

  Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.
• NATURALLY PROCESSED PEPTIDES LONGER THAN 9 AMINO-ACID-RESIDUES BIND TO THE CLASS-I MHC MOLECULE HLA-A2.1 WITH HIGH-AFFINITY AND IN DIFFERENT CONFORMATIONS

  Y CHEN, J SIDNEY, S SOUTHWOOD, AL COX, K SAKAGUCHI, RA HENDERSON, E APPELLA, DF HUNT, A SETTE, VH ENGELHARD

  JOURNAL OF IMMUNOLOGY 152 6 2874 - 2881 1994年03月 [査読有り][通常論文]
   

  An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide indifferent conformations would appear to have distinct advantages to the immune system.
• THE PERIPHERAL SUBUNIT-BINDING DOMAIN OF THE DIHYDROLIPOYL ACETYLTRANSFERASE COMPONENT OF THE PYRUVATE-DEHYDROGENASE COMPLEX OF BACILLUS-STEAROTHERMOPHILUS - PREPARATION AND CHARACTERIZATION OF ITS BINDING TO THE DIHYDROLIPOYL DEHYDROGENASE COMPONENT

  DS HIPPS, LC PACKMAN, MD ALLEN, C FULLER, K SAKAGUCHI, E APPELLA, RN PERHAM

  BIOCHEMICAL JOURNAL 297 1 137 - 143 1994年01月 [査読有り][通常論文]
   

  The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain of the and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.
• DIRECT IDENTIFICATION OF AN ENDOGENOUS PEPTIDE RECOGNIZED BY MULTIPLE HLA-A2.1-SPECIFIC CYTOTOXIC T-CELLS

  RA HENDERSON, AL COX, K SAKAGUCHI, E APPELLA, J SHABANOWITZ, DF HUNT, VH ENGELHARD

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 21 10275 - 10279 1993年11月 [査読有り][通常論文]
   

  An endogenous peptide recognized by a murine T-cell clone specific for the human class I major histocompatibility complex-encoded molecule HLA-A2.1 was identified through the use of microcapillary high-performance liquid chromatography coupled with electrospray-ionization tandem mass spectrometry. The peptide was associated with HLA-A2.1 on both normal cells and the antigen-processing-mutant cell line T2. This observation demonstrates that a processing mechanism other than that involving the transporter associated with antigen processing (TAP) proteins 1 and 2 can produce peptides that can be recognized by T cells. The peptide was also recognized by four other independently derived murine HLA-A2.1-specific murine T-cell clones. This suggests that xenogeneic responses are directed at a restricted subset of major histocompatibility complex product-associated peptides. Finally, quantitation of this peptide in cell extracts using mass spectrometry showed it to be among the most dominant HLA-A2.1 associated species on human lymphoid cells. The potential relevance of this observation to models of alloreactivity will be discussed. The methodology described should be generally useful for the identification of peptide epitopes recognized by alloreactive, tumor-specific, and autoimmune T cells.
• CHARACTERISTICS OF ENDOGENOUS PEPTIDES ELUTED FROM THE CLASS-I MHC MOLECULE HLA-B7 DETERMINED BY MASS-SPECTROMETRY AND COMPUTER MODELING

  EL HUCZKO, WM BODNAR, D BENJAMIN, K SAKAGUCHI, NZ ZHU, J SHABANOWITZ, RA HENDERSON, E APPELLA, DF HUNT, VH ENGELHARD

  JOURNAL OF IMMUNOLOGY 151 5 2572 - 2587 1993年09月 [査読有り][通常論文]
   

  Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.
• 2-DIMENSIONAL NUCLEAR-MAGNETIC-RESONANCE ANALYSIS OF A LABELED PEPTIDE BOUND TO A CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULE

  PC DRISCOLL, JD ALTMAN, JJ BONIFACE, K SAKAGUCHI, PA REAY, JG OMICHINSKI, E APPELLA, MM DAVIS

  JOURNAL OF MOLECULAR BIOLOGY 232 2 342 - 350 1993年07月 [査読有り][通常論文]
  
• PHOSPHORYLATION AT SER-15 AND SER-392 IN MUTANT P53-MOLECULES FROM HUMAN TUMORS IS ALTERED COMPARED TO WILD-TYPE P53

  SJ ULLRICH, K SAKAGUCHI, SP LEESMILLER, M FISCELLA, WE MERCER, CW ANDERSON, E APPELLA

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 13 5954 - 5958 1993年07月 [査読有り][通常論文]
   

  The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. p53 protein binds DNA, activates transcription, and can be phosphorylated at N- and C-terminal sites. Previously, wild-type p53 was shown to be hyperphosphorylated compared to mutant p53 during p53-mediated growth arrest in vivo. Here we show that Ser-15 and Ser-9 in the N-terminal transactivation domain of wild-type human p53 are phosphorylated in vivo in cells derived from the human glioblastoma line T98G. In [Ile237]p53 and [Ala143]p53, two natural p53 mutants from human tumors that are defective for activation of transcription, phosphorylation at Ser-15 was reduced and phosphorylation at Ser-392 was increased compared to wild-type p53. No change was observed at Ser-9. [His273]p53, a third mutant, had a phosphorylation state similar to that of wild-type p53. We suggest that phosphorylation of Ser-15 may depend on the ability of p53 to adopt a wild-type conformation and may contribute to p53's ability to block cell growth.
• IDENTIFICATION OF A BINDING-SITE FOR THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NUCLEOCAPSID PROTEIN

  K SAKAGUCHI, N ZAMBRANO, ET BALDWIN, BA SHAPIRO, JW ERICKSON, JG OMICHINSKI, GM CLORE, AM GRONENBORN, E APPELLA

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 11 5219 - 5223 1993年06月 [査読有り][通常論文]
   

  The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.
• THE EZRIN-LIKE FAMILY OF TYROSINE KINASE SUBSTRATES - RECEPTOR-SPECIFIC PATTERN OF TYROSINE PHOSPHORYLATION AND RELATIONSHIP TO MALIGNANT TRANSFORMATION

  F FAZIOLI, WT WONG, SJ ULLRICH, K SAKAGUCHI, E APPELLA, PP DIFIORE

  ONCOGENE 8 5 1335 - 1345 1993年05月 [査読有り][通常論文]
   

  A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immunoaffinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.
• SEQUENCE OF A CDNA-ENCODING BOVINE APOLIPOPROTEIN-H

  B GAO, M VIRMANI, E ROMM, E LAZARWESLEY, K SAKAGUCHI, E APPELLA, G KUNOS, L TAKACS

  GENE 126 2 287 - 288 1993年04月 [査読有り][通常論文]
   

  The nucleotide sequence of the ApoH cDNA encoding the bovine apolipoprotein H (ApoH) has been determined. The deduced protein, which contains a 19-amino-acid (aa) signal peptide and the 326-aa mature ApoH, shows 89% and 86% homology with human and rat ApoH, respectively,
• THE HIGH-RESOLUTION STRUCTURE OF THE PERIPHERAL SUBUNIT-BINDING DOMAIN OF DIHYDROLIPOAMIDE ACETYLTRANSFERASE FROM THE PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX OF BACILLUS-STEAROTHERMOPHILUS

  YN KALIA, SM BROCKLEHURST, DS HIPPS, E APPELLA, K SAKAGUCHI, RN PERHAM

  JOURNAL OF MOLECULAR BIOLOGY 230 1 323 - 341 1993年03月 [査読有り][通常論文]
  
• OPTIMUM CROSS-LINKING SPACER LENGTH OF DIMERIC NEUROKININ-B ANALOGS FOR INTERACTION WITH NK-1 TACHYKININ RECEPTORS

  H MATSUMOTO, Y SHIMOHIGASHI, Y TAKANO, K SAKAGUCHI, H KAMIYA, M OHNO

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 66 1 196 - 204 1993年01月 [査読有り][通常論文]
   

  A series of dimeric analogs of neurokinin B (NKB) COOH-terminal heptapeptide were synthesized in order to find an optimum length of cross-linking spacer for bivalent interaction with tachykinin receptors. Dimerization was carried out at the NH2-terminus of heptapeptide with succinyl bis[(GIY)n-OH], in which the number of glycine varies from 0 to 4. Dimers D-(GlY)n-NKB4-10, namely succinyl bis[(GIY)n-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2] (n=0-4), were almost inactive in the assay using rat vas deferens, indicating that they have no interaction with NK-2 receptor subtype. In contrast, these dimers were very active in guinea pig ileum (GPI) containing all of NK-1, 2, and 3 receptors. Relative activity was highest when the cross-linking spacer with oligoglycine of n=2, and decreased sharply for dimers with shorter and longer chain lengths (n=0 and 1; 3 and 4). In order to specify receptor subtype (NK-1 or NK-3) in GPI to which dimers bind, dimers were examined under the conditions that NK-1 receptors are desensitized by substance P methyl ester. All dimers exhibited drastically diminished contractile activity, indicating that dimers exclusively interact with NK-1. This was further confirmed by blocking the response of NK-3 receptors with atropine, which had no effect on the contractile activity of dimers. The results suggested that dimers interact bivalently with NK-1 receptors by bridging adjacent two binding sites.
• INVARIANT CHAIN PEPTIDES IN MOST HLA-DR MOLECULES OF AN ANTIGEN-PROCESSING MUTANT

  A SETTE, S CEMAN, RT KUBO, K SAKAGUCHI, E APPELLA, DF HUNT, TA DAVIS, H MICHEL, J SHABANOWITZ, R RUDERSDORF, HM GREY, R DEMARS

  SCIENCE 258 5089 1801 - 1804 1992年12月 [査読有り][通常論文]
   

  Class II major histocompatibility complexes bind peptides in an endosome-like compartment. When the class II null cell line 721.174 was transfected with class II DR3 genes, DR molecules were produced in normal amounts. However, the DR molecules were abnormally conformed and unstable because deletion of an antigen-processing gene had impaired intracellular formation of most class II-peptide complexes. Yet, 70 percent of the DR molecules still bore peptides, 80 percent of which were 21- to 24-amino acid fragments of the class II-associated invariant chain. These peptides were rare on DR3 from control cells. Thus, a defect in the main antigen-processing pathway revealed a process in which DR molecules bind long peptides derived from proteins present in the same compartment.
• HUMAN DNA-ACTIVATED PROTEIN-KINASE PHOSPHORYLATES SERINE-15 AND SERINE-37 IN THE AMINO-TERMINAL TRANSACTIVATION DOMAIN OF HUMAN P53

  SP LEESMILLER, K SAKAGUCHI, SJ ULLRICH, E APPELLA, CW ANDERSON

  MOLECULAR AND CELLULAR BIOLOGY 12 11 5041 - 5049 1992年11月 [査読有り][通常論文]
   

  Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.
• THYMOCYTE COSTIMULATING ANTIGEN IS CD26 (DIPEPTIDYLPEPTIDASE-IV) - COSTIMULATION OF GRANULOCYTE, MACROPHAGE, AND T-LINEAGE CELL-PROLIFERATION VIA CD26

  LA BRISTOL, K SAKAGUCHI, E APPELLA, D DOYLE, L TAKACS

  JOURNAL OF IMMUNOLOGY 149 2 367 - 372 1992年07月 [査読有り][通常論文]
   

  The rat thymocyte costimulating protein appears to be involved in the regulation of CD4-/CD8- thymocyte proliferation in vitro. We show that thymocyte costimulating protein is dipeptidyl peptidase IV (E.C.N.3.4.14.5) also known as CD26. Some bone marrow cells as well as CD4-/CD8- and, and to a lesser extent, more differentiated T cells respond by proliferating to a CD26 specific mAb-mediated costimulus that does not influence dipeptidyl dipeptidase IV enzyme activity. This suggests the existence of a CD26-linked regulatory mechanism of proliferation that is operational on granulocyte- and macrophage-lineage cells and throughout T cell development.
• PEPTIDES PRESENTED TO THE IMMUNE-SYSTEM BY THE MURINE CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULE-I-A(D)

  DF HUNT, H MICHEL, TA DICKINSON, J SHABANOWITZ, AL COX, K SAKAGUCHI, E APPELLA, HM GREY, A SETTE

  SCIENCE 256 5065 1817 - 1820 1992年06月 [査読有り][通常論文]
   

  Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-A(d). Sequences for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides were 16 to 18 residues long, had ragged NH2- and COOH-termini, and contained a six-residue binding motif that was variably placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules can be open at both ends.
• INTERNALIZATION OF THE UROKINASE-PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 COMPLEX IS MEDIATED BY THE UROKINASE RECEPTOR

  D OLSON, J POLLANEN, G HOYERHANSEN, E RONNE, K SAKAGUCHI, TC WUN, E APPELLA, K DANO, F BLASI

  JOURNAL OF BIOLOGICAL CHEMISTRY 267 13 9129 - 9133 1992年05月 [査読有り][通常論文]
   

  The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4-degrees-C uPA fluorescence was visible at the cell surface, shift of the temperature to 37-degrees-C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.
• 3-DIMENSIONAL SOLUTION STRUCTURE OF THE E3-BINDING DOMAIN OF THE DIHYDROLIPOAMIDE SUCCINYL TRANSFERASE CORE FROM THE 2-OXOGLUTARATE DEHYDROGENASE MULTIENZYME COMPLEX OF ESCHERICHIA-COLI

  MA ROBIEN, GM CLORE, JG OMICHINSKI, RN PERHAM, E APPELLA, K SAKAGUCHI, AM GRONENBORN

  BIOCHEMISTRY 31 13 3463 - 3471 1992年04月 [査読有り][通常論文]
   

  The three-dimensional solution structure of a 51-residue synthetic peptide comprising the dihydrolipoamide dehydrogenase (E3)-binding domain of the dihydrolipoamide succinyltransferase (E2) core Of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli has been determined by nuclear magnetic resonance spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is based on 630 approximate interproton distance and 101 torsion angle (phi, psi, chi-1) restraints. A total of 56 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions for residues 12-48 of the synthetic peptide is 1.24 angstrom for the backbone atoms, 1.68 angstrom for all atoms, and 1.33 angstrom for all atoms excluding the six side chains which are disordered at chi-1 and the seven which are disordered at chi-2; when the irregular partially disordered loop from residues 31 to 39 is excluded, the rms distribution drops to 0.77 angstrom for the backbone atoms, 1.55 angstrom for all atoms, and 0.89 angstrom for ordered side chains. Although proton resonance assignments for the N-terminal 11 residues and the C-terminal 3 residues were obtained, these two segments of the polypeptide are disordered in solution as evidenced by the absence of nonsequential nuclear Overhauser effects. The solution structure of the E3-binding domain consists of two parallel helices (residues 14-23 and 40-48), a short extended strand (24-26), a five-residue helical-like turn, and an irregular (and more disordered) loop (residues 31-39). This report presents the first structure of an E3-binding domain from a 2-oxo acid dehydrogenase complex. Related domains from the E2 chains of other 2-oxo acid dehydrogenase complexes are likely to be structurally analogous, given their marked sequence similarity and the presence of a number of conserved residues at pivotal positions. This is particularly true for the E. coli pyruvate dehydrogenase complex since the E3 component which is bound by its dihydrolipoamide acetyltransferase core is identical to that bound by the dihydrolipoamide succinyltransferase core of the 2-oxoglutarate dehydrogenase complex.
• RECEPTOR INTERACTIONS OF SYNTHETIC MORPHICEPTIN ANALOGS CONTAINING PHENYLALANINE HOMOLOGS IN POSITION-4

  K SAKAGUCHI, T COSTA, H SAKAMOTO, Y SHIMOHIGASHI

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 65 4 1052 - 1056 1992年04月 [査読有り][通常論文]
   

  A series of phenylalanine homologs with elongated phenylalkyl side chains (R = -(CH2)n-C6H5, n = 1-4) have been incorporated into opioid peptide morphiceptin at position 4 in order to elucidate a role of the amino acid residue in the molecular mechanism of receptor activation. The receptor specificity and selectivity of peptides synthesized were examined in the radio-ligand receptor binding assays using tritiated DAGO- and DADLE-enkephalins. [Phe4]Morphiceptin was most potent for the mu-receptors and showed the most pronounced mu/delta-receptor selectivity with a specific ratio of 410. Its mu-affinity was about three times stronger than morphiceptin. All analogs showed very similar CD spectra, suggesting that these peptides have a similar backbone conformation. The results strongly suggest that, besides the backbone conformation, the side-chain aromatic rings at positions 1 (Tyr), 3 (Phe), and 4 (Phe) array in a specific stereoorientation and this array is important to activate mu-receptors. Analogs with longer phenylalkyl side chains appeared not to retain such an arrangement due to the steric hindrance caused by the presence of more than two methylene groups.
• HLA-A2.1-ASSOCIATED PEPTIDES FROM A MUTANT-CELL LINE - A 2ND PATHWAY OF ANTIGEN PRESENTATION

  RA HENDERSON, H MICHEL, K SAKAGUCHI, J SHABANOWITZ, E APPELLA, DF HUNT, VH ENGELHARD

  SCIENCE 255 5049 1264 - 1266 1992年03月 [査読有り][通常論文]
   

  Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.
• CHARACTERIZATION OF PEPTIDES BOUND TO THE CLASS-I MHC MOLECULE HLA-A2.1 BY MASS-SPECTROMETRY

  DF HUNT, RA HENDERSON, J SHABANOWITZ, K SAKAGUCHI, H MICHEL, N SEVILIR, AL COX, E APPELLA, VH ENGELHARD

  SCIENCE 255 5049 1261 - 1263 1992年03月 [査読有り][通常論文]
   

  Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography - electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.
• High-resolution structure of a double zinc finger from the human enhancer binding protein MPB-1 in solution

  Omichinski James G, Sakaguchi Kazuyasu, Clore G Marius, Gronenborn Angela M, Appella Ettore

  Journal of Protein Chemistry 11 4 408  Springer 1992年 [査読有り][通常論文]
  
• SPECIFIC DNA-BINDING TO A MAJOR HISTOCOMPATIBILITY COMPLEX ENHANCER SEQUENCE BY A SYNTHETIC 57-RESIDUE DOUBLE ZINC FINGER PEPTIDE FROM A HUMAN ENHANCER BINDING-PROTEIN

  K SAKAGUCHI, E APPELLA, JG OMICHINSKI, GM CLORE, AM GRONENBORN

  JOURNAL OF BIOLOGICAL CHEMISTRY 266 11 7306 - 7311 1991年04月 [査読有り][通常論文]
   

  Two 57-residue peptides containing one pair of "zinc fingers" from a human enhancer binding protein were prepared by solid-phase peptide synthesis. One peptide (MBP-DF) contained the native sequence, while the second peptide ([Abu11]MBP-DF) has an alpha-aminobutyric acid residue substituted for a nonconserved cysteine residue at position 11. The peptides were characterized by several chemical and physical methods, and their DNA binding properties were evaluated using gel retardation experiments. Spectroscopic studies demonstrated that addition of metal ions such as zinc and cobalt resulted in specific conformational changes in both peptides, indicating that cysteine-11 does not appear to be involved in metal chelation. One-dimensional H-1 NMR studies indicate that a stable folded structure is formed upon addition of zinc, and the chemical shift pattern is consistent with that previously observed for one constituent single finger (Omichinski, J., Clore, G. M., Appella, E., Sakaguchi, K., and Gronenborn, A. M. (1990) Biochemistry 29, 9324-9334). Gel retardation experiments demonstrate that the peptides are capable of interacting with a 15-mer oligonucleotide comprising a portion of the major histocompatibility complex enhancer sequence and that the interaction is zinc-dependent. The dissociation constant for the [Abu11]MBP-DF peptide is 1.4 x 10(-7) M with maximal binding occurring at a zinc-to-peptide ratio of 2 to 1. The binding specificity observed with respect to related enhancer sequences exhibits the same relative order as noted previously for the whole protein. Studies with point mutants of the major histocompatibility complex enhancer binding sequence indicate that the last GC base pair in a four-guanine stretch plays a pivotal role in the interaction between the peptide and DNA.
• HIGH-RESOLUTION 3-DIMENSIONAL STRUCTURE OF A SINGLE ZINC FINGER FROM A HUMAN ENHANCER BINDING-PROTEIN IN SOLUTION

  JG OMICHINSKI, GM CLORE, E APPELLA, K SAKAGUCHI, AM GRONENBORN

  BIOCHEMISTRY 29 40 9324 - 9334 1990年10月 [査読有り][通常論文]
  
• Opioid Activities of Morphiceptin Analogs Derived from Human $β$-Casein

  Sakaguchi Kazuyasu, Sakamoto Hiroshi, Tsubaki Yoshiko, Waki Michinori, Costa Tommaso, Shimohigashi Yasuyuki

  Bulletin of the Chemical Society of Japan 63 6 1753 - 1757 The Chemical Society of Japan 1990年06月 [査読有り][通常論文]
  
• CHARACTERIZATION OF TACHYKININ RECEPTORS IN ENDOTHELIAL-CELLS OF PORCINE ARTERY

  R SAITO, H KONISHI, Y TAKANO, S NONAKA, K SAKAGUCHI, Y SHIMOHIGASHI, HO KAMIYA

  NEUROSCIENCE LETTERS 110 3 337 - 342 1990年03月 [査読有り][通常論文]
  
• Opioid activities of morphiceptin-like peptides latent in various natural proteins.

  Shimohigashi Y, Sakaguchi K, Sakamoto H, Waki M, Costa T

  Peptide research 3 5 216  1990年 [査読有り][通常論文]
  
• Design and synthesis of dimeric analogs of neurokinin A and B: effect of dimerization of COOH-terminal heptapeptides on receptor selection.

  Sakaguchi K, Kodama H, Matsumoto H, Yoshida M, Takano Y, Kamiya H, Waki M, Shimohigashi Y

  Peptide research 2 5 345  1989年 [査読有り][通常論文]
  
• STUDIES OF PEPTIDE ANTIBIOTICS .48. CONFORMATIONALLY STABILIZED GRAMICIDIN S-ANALOG CONTAINING DEHYDROPHENYLALANINE IN PLACE OF D-PHENYLALANINE4,4' - SYNTHESIS AND ANTIMICROBIAL ACTIVITY

  S IMAZU, Y SHIMOHIGASHI, H KODAMA, K SAKAGUCHI, M WAKI, T KATO, N IZUMIYA

  INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH 32 4 298 - 306 1988年10月 [査読有り][通常論文]
  
• DIMERIZATION OF NEUROKININ-A AND NEUROKININ-B COOH-TERMINAL HEPTAPEPTIDE FRAGMENTS ENHANCED THE SELECTIVITY FOR TACHYKININ RECEPTOR SUBTYPES

  H KODAMA, Y SHIMOHIGASHI, K SAKAGUCHI, M WAKI, Y TAKANO, A YAMADA, Y HATAE, H KAMIYA

  EUROPEAN JOURNAL OF PHARMACOLOGY 151 2 317 - 320 1988年07月 [査読有り][通常論文]
  
• BIOLOGICAL EVALUATION OF DIMERIC ANALOGS OF TACHYKININ PEPTIDES

  Y SHIMOHIGASHI, H KODAMA, K SAKAGUCHI, T KATO, M WAKI, H KAMIYA, A YAMADA, Y HIGUCHI, Y TAKANO

  REGULATORY PEPTIDES 22 1-2 172 - 172 1988年07月 [査読有り][通常論文]
  

MISC

• 自然免疫を担うインターフェロン経路における「転写記憶」の発見とその制御機構解明

  鎌田瑠泉, 坂口和靖, 尾里啓子 実験医学 37 (3) 415 -418 2019年02月 [査読無し][招待有り]
  
• EVOLUTION OF THE TETRAMERIZATION DOMAIN IN TUMOR SUPPRESSOR PROTEIN P53

  N. Nakagawa, J. Wada, R. Kamada, T. Nomura, T. Imagawa, K. Sakaguchi JOURNAL OF PEPTIDE SCIENCE 22 S22 -S22 2016年09月 [査読無し][通常論文]
  
• ARGININE METHYLATION OF TETRAMERIZATION DOMAIN DESTABILIZES THE TETRAMERIC STRUCTURE OF TUMOR SUPPRESSOR PROTEIN P53

  R. Kamada, N. Nakagawa, J. Wada, K. Sakaguchi JOURNAL OF PEPTIDE SCIENCE 22 S128 -S128 2016年09月 [査読無し][通常論文]
  
• THE EFFECTS OF P53 TETRAMERIZATION DOMAIN MUTANTS FOUND IN LI-FRAUMENI SYNDROME ON HETERO-OLIGOMER FORMATION AND TRANSCRIPTIONAL ACTIVITY

  Y. Toguchi, M. Kanno, R. Kamada, T. Imagawa, K. Sakaguchi JOURNAL OF PEPTIDE SCIENCE 22 S86 -S88 2016年09月 [査読無し][通常論文]
  
• 癌原遺伝子産物プロテインホスファターゼによる細胞癌化機構と阻害剤開発

  小笠原紗里, 鎌田瑠泉, 坂口和靖 化学工業 67 61 -66 2016年 [査読無し][招待有り]
  
• The PPM1D encoded phosphatase Wip1 is a novel oncogene and potential therapeutic target in neuroblastoma and medulloblastoma

  Jelena Miloseyic, Diana Treis, Malin Wickstrom, Susanne Fransson, Nina Eissler, Baldur Syeinbjornsson, Ninib Baryawno, Keiji Tanino, Galina Selivanova, Kazuyasu Sakaguchi, Tommy Martinsson, John Inge Johnsen, Per Kogner CANCER RESEARCH 75 2015年08月 [査読無し][通常論文]
  
• 多量体化と配向化を基盤としたバイオミネラリゼーションペプチドによる銀ナノ構造制御

  坂口達也, 峯健太, 工藤風樹, 鎌田瑠泉, 坂口和靖 日本生化学会大会(Web) 88th 2015年
• がん原遺伝子産物PPM1Dの細胞がん化機構および創薬を指向した阻害剤

  鎌田瑠泉, 中馬吉郎, 小境夕紀, 坂口和靖 生化学 87 531 -538 2015年 [査読有り][招待有り]
  
• Analysis of Substrate Preference of Human PPM1 Type Ser/Thr Phosphatases

  SHIRAHATA Yukiko, CHUMAN Yoshiro, IWAMURO Ryo, JANAIRO Jose isagani B., KIYOTA Yuhei, YAGI Hiroaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2012 403 -404 2013年03月01日
• Silver Nanocrystals Formed by Oligomeric Biomineralization Peptide via p53 Tetramerization Domain

  SAKAGUCHI Tatsuya, JANAIRO Jose Isagani B., CHUMAN Yoshiro, HARA Kenji, FUKUOKA Atsushi, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2012 57 -58 2013年03月01日
• p53四量体形成ドメインを介したバイオミネラリゼーションペプチドの立体配置による銀ナノ結晶形成

  坂口達也, JANAIRO Jose Isagani, 中馬吉郎, 原賢二, 福岡淳, 坂口和靖 日本化学会講演予稿集 93rd (3) 2013年
• Probing the Differential Phosphothreonine and Metal Selectivity of Human PPM1 Phosphatase Family

  JANAIRO Jose Isagani, IWAMURO Ryo, KAYA Shunji, YAGI Hiroaki, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 245 -246 2012年03月01日
• Analysis of Interaction between p53-Inducible Protein Phosphatase PPM1D and Nucleolar Protein

  KOZAKAI Yuuki, YAGI Hiroaki, TEDUKA Youhei, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 43 -44 2012年03月01日
• Formation of Functionalized Nanowires Using Structure-Controllable Amyloid Peptide

  SAKAI Hiroki, WATANABE Ken, CHUMAN Yoshiro, MASUDA Takuya, UOSAKI Kohei, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 23 -24 2012年03月01日
• Repression of p53 Transcriptional Activity by Inhibitory Peptide via Heterotetramerization

  WADA Junya, KAMADA Rui, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 301 -302 2012年03月01日
• Nanostructure Formed by Biomineralization Peptide Oligomerized via p53 Tetramerization

  SAKAGUCHI Tatsuya, KAMADA Rui, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 345 -346 2012年03月01日
• Involvement of PPM1D Specific Pro-Loop in Regulatory Mechanism of PPM1D Phosphatase Activity

  YAGI Hiroaki, CHUMAN Yoshiro, KOZAKAI Yuuki, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 373 -374 2012年03月01日
• p53四量体形成ドメインを介した多量体化バイオミネラリゼーションペプチドによる銀粒子形成

  坂口達也, 鎌田瑠泉, 中馬吉郎, 坂口和靖 日本化学会講演予稿集 92nd (3) 2012年
• Formation Process and Solvent-Dependent Structure of a Polyproline Self-Assembled Monolayer on a Gold Surface

  Ying Han, Hidenori Noguchi, Kazuyasu Sakaguchi, Kohei Uosaki LANGMUIR 27 (19) 11951 -11957 2011年10月 [査読無し][通常論文]
   

  The formation process and structure of a self-assembled monolayer (SAM) of lipoic-acid-terminated polyproline on a gold surface in aqueous solution were investigated by several techniques. The amount of polyproline molecules on the gold surface was determined from the area of the reductive desorption peak, and orientation and thickness of the polyproline SAM were determined in situ by attenuated total reflection infrared (ATR-IR) spectroscopy and ellipsometry. The kinetics of the polyproline SAM formation process were discussed on the basis of these results. The in situ IR study confirmed that the conformation of the polyproline SAM was changed by changing the solvent from water to methanol and methanol to water, as is the case for polyproline dissolved in solution.
• Effect of Peptide Oligomerization Mediated by p53 Tetramerization Domain on Biomineralization Activity

  SAKAGUCHI Tatsuya, KAMADA Rui, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 269 -269 2011年03月01日
• Controlling Initiation Position of Amyloid Nanowire Using a Gold Nanoparticle

  SAKAI Hiroki, WATANABE Ken, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 276 -276 2011年03月01日
• Dephosphorylation of Nucleolar Protein Nucleophosmin by Protein Phosphatase PPM1D

  KOZAKAI Yuuki, YAGI Hiroaki, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 92 -92 2011年03月01日
• Repression of p53 transcriptional activity by p53 tetramerization domain peptide containing PTD and NLS domain

  WADA Junya, KAMADA Rui, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 95 -95 2011年03月01日
• Low Threshold of Destabilization for Loss of Tumor Suppressor Activity of p53 : A Quantitative Analysis of p53 Tetramerization Domain Mutants

  KAMADA Rui, NOMURA Takao, CHUMAN Yoshiro, IMAGAWA Toshiaki, ANDERSON Carl W., SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 149 -149 2011年03月01日
• p53四量体形成ドメインを介した空間配向制御によるバイオミネラリゼーションの活性増強と構造制御

  坂口達也, 鎌田瑠泉, 野村尚生, 中馬吉郎, 今川敏明, 坂口和靖 日本化学会講演予稿集 90th (3) 2010年
• p53四量体形成ドメインとバイオミネラリゼーションペプチドの融合による銀粒子の構造制御

  坂口達也, 鎌田瑠泉, 野村尚生, 中馬吉郎, 今川敏明, 坂口和靖 日本化学会北海道支部夏季研究発表会講演要旨集 2010 2010年
• 赤外分光法によるペプチド固相合成過程におけるペプチド構造のその場追跡

  野口秀典, 野口秀典, 安達龍彦, 坂口和靖, 魚崎浩平, 魚崎浩平 日本化学会講演予稿集 90th (3) 2010年
• がん抑制タンパク質p53の四量体形成

  鎌田瑠泉, 坂口和靖 生化学 82 (7) 484 -493 2010年 [査読有り][招待有り]
  
• Extremely Long Nanofibrils of Functionalized Structure-Controlled-Amyloid Peptides Derived from Transthyretin

  SAKAI Hiroki, ASANOMI Yuya, KOBAYASHI Yumiko, CHEN Xinjiang, CHUMAN Yoshiro, MASUDA Takuya, UOSAKI Kohei, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2008 465 -466 2009年03月01日
• PPM1D430, a Novel Alternative Splicing Variant of the Human PPM1D

  CHUMAN Yoshiro, KURIHASHI Wataru, MIZUKAMI Yohei, NASHIMOTO Takehiro, YAGI Hiroaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2008 531 -532 2009年03月01日
• Thermal Stability of p53 Tetramerization Domain Peptides Derived from Various Species

  NOMURA Takao, KAMADA Rui, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2008 35 -36 2009年03月01日
• Identification of Novel Chemical Inhibitors for p53-Inducible Protein Phosphatase PPM1D

  YAGI Hiroaki, CHUMAN Yoshiro, YOSHIMURA Fumihiko, TANINO Keiji, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2008 385 -386 2009年03月01日
• Structural Photomodulation of Peptides with Azobenzoate Derivative in Peptide Backbone

  KAMADA Rui, FUKUDA Kouichiro, MIYAZAKI Hiromitsu, NOMURA Takao, CHUMAN Yoshiro, IMAGAWA Toshiaki, TANINO Keiji, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 447 -448 2008年03月01日
• Correlation between Expression Level in E. coli and Stability of Oligomeric Structure of the p53 Tetramerization Domain

  MIYAZAKI Hiromitsu, KAMADA Rui, NOMURA Takao, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 317 -318 2008年03月01日
• Screening of Peptides to Stabilize the p53 Tetramer Formation from Phage Displayed Library

  NOMURA Takao, KAMADA Rui, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 315 -316 2008年03月01日
• Correlation of Oligomeric Structure and Transcriptional Activity of p53 Mutants

  KAMADA Rui, TERAI Tomoko, NOMURA Takao, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2007 69 -70 2008年03月01日
• Function of Basic Loop in Substrate Recognition of Protein Phosphatase PPM1D

  CHUMAN Yoshiro, FUKUDA Tomohiko, YAGI Hiroaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 17 -18 2007年03月01日
• Stabilization of p53 Tetrameric Structure by Polyol Compounds

  NOMURA Takao, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 131 -132 2007年03月01日
• Thermodynamic Stability of Mutant p53 Tetramerization Domain Peptides Associated with Human Tumors

  KAMADA Rui, TERAI Tomoko, NOMURA Takao, CHUMAN Yoshiro, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 293 -294 2007年03月01日
• Effects of Tumor-Associated mutations in the p53 tetramerization domain on oligomerization state and transcriptional activity

  R. Kamada, T. Terai, T. Nomura, Y. Chuman, T. Imagawa, K. Sakaguchi BIOPOLYMERS 88 (4) 626 -626 2007年 [査読無し][通常論文]
  
• Effects of Mutation in the β-Strand Region of p53 Tetramerizaion Domain on Oligomeric Structure and Stability

  KAMADA Rui, TERAI Tomoko, NOMURA Takao, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 267 -270 2006年03月01日
• Fibrillar and Granular Aggregation of G334V Mutant p53 Tetramer Peptide

  ASANOMI Yuya, HIGASHIMOTO Yuichiro, CHUMAN Yoshiro, KAYA Shunji, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 397 -398 2006年03月01日
• Analysis of Stabilization by Substitution of Cyclohexylalanine for Phe341 in Hydrophobic Core of p53 Tetramer

  NOMURA Takao, SAKAMOTO Koichi, KASAI Yusuke, CHUMAN Yoshiro, ISHIMORI Koichiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 399 -402 2006年03月01日
• Characterization and Application of Monoclonal Antibody Specific for Ser(P)-Gln Phosphorylation Motif

  IIZUKA Kanako, CHUMAN Yoshiro, HONDA Takeshi, ONOUE Hitoshi, SHIMOHIGASHI Yasuyuki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 499 -502 2006年03月01日
• Substrate Recognition of PP2C Family Phosphatase PPM1D

  FUKUDA Tomohiko, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2005 503 -504 2006年03月01日
• New evidence for ATP binding induced catalytic subunit interactions in pig kidney Na/K-ATPase.

  Tanoue, K, Kaya, S, Hayashi, Y, Abe, K, Imagawa, T, Taniguchi, K, Sakaguchi, K Journal of Biochemistry 140 599-607 2006年 [査読無し][通常論文]
  
• The third Na+ binding pocket is located near the amino acid residues Thr-774 and Gln-923 in rat Na,K-ATPase.

  T Imagawa, T Yamamoto, S Kaya, K Sakaguchi, K Taniguchi JOURNAL OF GENERAL PHYSIOLOGY 126 (1) 19A -19A 2005年07月 [査読無し][通常論文]
  
• High and low affinity ATP effects on both NaE1 and KE2 of Na/K-ATPase studied by substrate analogues.

  K Tanoue, S Kaya, K Abe, T Imagawa, Y Hayashi, K Sakaguchi, K Taniguchi JOURNAL OF GENERAL PHYSIOLOGY 126 (1) 41A -42A 2005年07月 [査読無し][通常論文]
  
• Interaction region between catalytic subunits during ATP hydrolysis in oligomeric Na/K-ATPase.

  S Kaya, T Imagawa, K Sakaguchi, K Taniguchi JOURNAL OF GENERAL PHYSIOLOGY 126 (1) 37A -38A 2005年07月 [査読無し][通常論文]
  
• K+-induced change in oligomeric assembly of C12E8-solubilized pig gastric H/K-ATPase observed by single-molecule detection techniques.

  K Abe, S Kaya, K Sakaguchi, T Imagawa, Y Hayashi, K Taniguchi JOURNAL OF GENERAL PHYSIOLOGY 126 (1) 34A -35A 2005年07月 [査読無し][通常論文]
  
• Effects of Mutation and Modification on Tetramer Formation of Tumor Suppressor Protein p53

  SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2004 17 -20 2005年03月01日
• DNA Binding of Peptide with Single Helix-hairpin-Helix Motif

  YAMAMOTO Tetsuya, CHUMAN Yoshiro, KAYA Shunji, IMAGAWA Toshiaki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2004 517 -520 2005年03月01日
• Stability and Folding Mechanism for Trp-contaning Peptides of p53 Tetramerization Domain

  KASAI Yusuke, KAYA Shunji, CHUMAN Yoshiro, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2004 419 -422 2005年03月01日
• Characteraization of Superstable Variant Peptide of Human p53 Tetramerization Domain

  NOMURA Takao, ITO Issaku, CHUMAN Yoshiro, SHIMOHIGASHI Yasuyuki, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2004 423 -426 2005年03月01日
• δ‐オピオイド受容体のチオールジスルフィド交換反応による特異的親和標識

  磯崎要, 深堀英彦, 岡田一志, 白須直人, 本田健, 野瀬健, 坂口和靖, COSTA T, 下東康幸 生化学 76 (3) 319 2004年03月25日 [査読無し][通常論文]
  
• 概日リズムペースメーカーホルモン・PDFの局在部位解析を目的とした部位特異的抗体の作製

  本田健, 中馬吉郎, 浅井大輔, 野瀬健, 坂口和靖, 尾上均, 冨永佳也, 下東康幸, 下東美樹 生化学 76 (3) 318 2004年03月25日 [査読無し][通常論文]
  
• Destabilization and Oligomer Formation of p53 Tetramer Mutant Associated with Human Lung Carcinoma

  HIGASHIMOTO Yuichiro, ASANOMI Yuya, LEWIS Marc S., APPELLA Ettore, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 17 -20 2004年03月01日
• Mutant p53 Peptide Forms Amyloid under Physiological Condition

  ASANOMI Yuya, HIGASHIMOTO Yuichiro, IMAGAWA Toshiaki, KAYA Shunji, APPELLA Ettore, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 379 -382 2004年03月01日
• Binding Specificity of HhH Motif Peptide that Recognizes DNA Structure

  YAMAMOTO Tetsuya, IMAGAWA Toshiaki, KAYA Shunji, SAKAGUCHI Kazuyasu Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 311 -314 2004年03月01日
• Specific Affinity Labeling of Cys60 in the δ Opioid Receptor by Npys-containing Dynorphin A Analog

  ISOZAKI Kaname, FUKAHORI Hidehiko, OKADA Kazushi, SHIRASU Naoto, HONDA Takeshi, NOSE Takeru, SAKAGUCHI Kazuyasu, COSTA Tommaso, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 273 -276 2004年03月01日
• Hetero-Oligomer Formation of Peptides Derived from Tetramerization Domain in Tumor Suppressor Protein p53

  SAKAGUCHI Kazuyasu, MENO Takashi, ASANOMI Yuya, NOSE Takeru, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 307 -310 2004年03月01日
• Presence of two different ATP effects on both Na-bound and Rb-occluded Na/K-ATPase

  K Taniguchi, K Tanoue, K Abe, S Kaya, T Imagawa, K Sakaguchi BIOPHYSICAL JOURNAL 86 (1) 192A -192A 2004年01月 [査読無し][通常論文]
  
• Localization Profiles of the Precursor Protein of Circadian Rhythm Pacemaker Hormone PDF in Gryllus Brain

  HONDA Takeshi, CHUMAN Yoshiro, MATSUSHIMA Ayami, ASAI Daisuke, NOSE Takeru, SAKAGUCHI Kazuyasu, ONOUE Hitoshi, TOMINAGA Yoshiya, SHIMOHIGASHI Yasuyuki, SHIMOHIGASHI Miki Peptide science : proceedings of the ... Japanese Peptide Symposium 2002 433 -436 2003年02月01日
• Phosphorylation Site-Specific Monoclonal Antibody Recognizing Ser(P)-Gln Sequence

  SAKAGUCHI Kazuyasu, HONDA Takeshi, ONOUE Hitoshi, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2002 439 -442 2003年02月01日
• Specific Affinity Labeling of Delta Opioid Receptors via Thiol-Disulfide Exchange Reaction

  FUKAHORI Hidehiko, SHIRASU Naoto, NOSE Takeru, SAKAGUCHI Kazuyasu, COSTA Tommaso, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2002 257 -260 2003年02月01日
• Structure-Activity Studies on the Nociceptin Peptidic Antagonist Ac-RYYRIK-NH_2

  KAWANO Michiaki, HONDA Takeshi, OKADA Kazushi, CHUMAN Yoshiro, NOSE Takeru, SAKAGUCHI Kazuyasu, COSTA Tommaso, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2001 185 -188 2002年03月01日
• Structural Characterization of Phe Residues in the p53 Tetramerization Domain

  SAKAGUCHI Kazuyasu, ITO Issaku, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2001 135 -138 2002年03月01日
• Characteristic Intra- and Intermolecular Interactions of Dipeptide-Chymotrypsin Complex as Specific Structural Essentials for Enzyme Inhibition

  KOMADA Akira, YAMAUCHI Yasuko, IKESUE Koichi, FUJITA Tsugumi, NOSE Takeru, SAKAGUCHI Kazuyasu, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2001 179 -182 2002年03月01日
• ノシセプチンのアンタゴニスト・Ac‐RYYRIK‐NH₂のORL1受容体結合における構造活性相関

  河野道昭, 本田健, 岡田一志, 中馬吉郎, 野瀬健, 坂口和靖, COSTA T, 下東康幸 生化学 73 (11) 1376 2001年11月25日 [査読無し][通常論文]
  
• 鎮痛ペプチド・エンドモルフィン2Phe‐Phe残基の(F₅)Phe‐(F₅)Pheジペプチド置換による生物活性の変化

  本田健, 岡田一志, 藤田亜美, 野瀬健, 坂口和靖, COSTA T, 下東康幸 生化学 73 (8) 945 2001年08月25日 [査読無し][通常論文]
  
• ORL1ノシセプチン受容体アンタゴニスト・Ac‐RYYRIK‐NH₂の構造活性相

  河野道昭, 本田健, 岡田一志, 中馬吉郎, 野瀬健, 坂口和靖, COSTA T, 下東康幸 生化学 73 (8) 946 2001年08月25日 [査読無し][通常論文]
  
• ジペプチドH‐D‐Leu‐Phe‐NH‐Bzlのキモトリプシン阻害における疎水相互作用およびπ‐πスタッキング相互作用の重要性

  駒田彰, 藤田亜美, 野瀬健, 坂口和靖, 下東康幸 日本化学会講演予稿集 79th (2) 1027 2001年03月15日 [査読無し][通常論文]
  
• Preparation of the Antibody Which Discriminates the Conformations of Ligand-bound and Ligand-free Estrogen Receptors : Applications to the Quantitative Analysis of Ligand Bindings

  ASAI Daisuke, KOIZUMI Osamu, MOHRI Shirou, NAKAI Makoto, YAKABE Yoshikuni, SAKAGUCHI Kazuyasu, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2000 411 -412 2001年03月01日
• Destabilization of Tumor Suppressor Protein p53 Tetramer by Oxidation of Met340

  SAKAGUCHI Kazuyasu, ITO Issaku, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2000 277 -280 2001年03月01日
• Differential Receptor Binding Characteristics of the Phenylalanine Residues at Positions 3-4 of Endomorphin-2

  HONDA Takeshi, SHIGEHIRO Daiki, OKADA Kazushi, SHIRASU Naoto, CHUMAN Yoshiro, FUJITA Tsugumi, NOSE Takeru, SAKAGUCHI Kazuyasu, SHIMOHIGASHI Yasuyuki Peptide science : proceedings of the ... Japanese Peptide Symposium 2000 139 -142 2001年03月01日
• Stabilization of Tumor Suppressor Protein p53 through a Phosphorylation Cascade Mediated by DNA Damage

  SAKAGUCHI Kazuyasu, SAITO Shin'ichi, HIGASHIMOTO Yuichiro, ROY Siddhartha, ANDERSON Carl W., APPELLA Ettore Peptide science : proceedings of the ... Japanese Peptide Symposium 1999 45 -48 2000年03月01日
• DNA損傷によって誘起されるp53の18位スレオニンのリン酸化によるMDM2結合の阻害

  坂口 和靖, 斎藤 伸一, 東元 祐一郎, Roy Siddhartha, Anderson Carl W, Appella Ettore 生化学 71 (8) 759 -759 1999年08月 [査読無し][通常論文]
  
• Phosphorylation Influences The Association of p53 Tetramers

  SAKAGUCHI Kazuyasu, SAKAMOTO Hiroshi, XIE Dong, ERICKSON John W., LEWIS Marc S., ANDERSON Carl W., APPELLA Ettore Peptide chemistry : proceedings of the ... Symposium on Peptide Chemistry 1996 165 -168 1996年10月01日
• Chemical Synthesis of Phosphorylated Peptides of The Carboxy-Terminal Domain of Human p53 by A Segment Condensation Method

  SAKAMOTO Hiroshi, KODAMA Hiroaki, HIGASHIMOTO Yuichiro, KONDO Michio, LEWIS Marc S., ANDERSON Carl W., APPELLA Ettore, SAKAGUCHI Kazuyasu Peptide chemistry : proceedings of the ... Symposium on Peptide Chemistry 1996 85 -88 1996年10月01日
• SOLUTION STRUCTURE OF THE HUMAN P53 OLIGOMERIZATION DOMAIN BY THE USE OF SYNTHETIC PEPTIDES AND MULTIDIMENSIONAL NMR

  K SAKAGUCHI, H SAKAMOTO, MS LEWIS, H KODAMA, JG OMICHINSKI, AM GRONENBORN, GM CLORE, E APPELLA PEPTIDE CHEMISTRY 1994 105 -108 1995年 [査読無し][通常論文]
  
• Interhelical angles in the solution structure of the oligomerization domain of p53: Correction

  Clore,G. M, Omichinski,J. G, Sakaguchi,K, Zambrano,N, Sakamoto,H, Appella,E, Gronenborn,A. M Science 267 1515 -1516 1995年 [査読無し][通常論文]
  
• Specific sequences from the carboxy terminus of human p53 gene product form anti-parallel teramers in solution

  Sakamoto,H, Lewis,M. S, Kodama,H, Appella,E, Sakaguchi,K Proceedings of the National Academy of Sciences of the United States of America 91 8974 -8978 1994年 [査読無し][通常論文]
  
• STRUCTURE-ACTIVITY-RELATIONSHIPS OF A TETHERED PEPTIDE LIGAND OF THE HUMAN THROMBIN RECEPTOR

  K SAKAGUCHI, H KODAMA, Y OGINO, T COSTA, Y SHIMOHIGASHI PEPTIDE CHEMISTRY 1992 371 -372 1993年 [査読無し][通常論文]
  
• [4,4'-(Z)-DEHYDROPHENYLALANINE]GRAMICIDIN-S WITH STABILIZED BIOACTIVE CONFORMATION AND STRONG ANTIMICROBIAL ACTIVITY

  Y SHIMOHIGASHI, H KODAMA, S IMAZU, H HORIMOTO, K SAKAGUCHI, M WAKI, H UCHIDA, M KONDO, T KATO, N IZUMIYA FEBS LETTERS 222 (2) 251 -255 1987年10月 [査読無し][通常論文]
  

書籍等出版物

• Peptide Science 2011

  The Japanese Peptide Society 2012年
• 自然科学実験

  学術図書出版 2007年

所属学協会

• 日本プロテインホスファターゼ研究会   日本ケミカルバイオロジー学会   日本プロテオーム学会   American Chemical Society   日本生化学会   日本ペプチド学会   日本化学会   Japan Proteome Society   The Japanese Peptide Society   The Chemical Society of Japan   

共同研究・競争的資金等の研究課題

• 抗生物質に対する細菌の薬剤耐性獲得における新規機構の解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2023年06月 -2026年03月 

  代表者 : 坂口 和靖, 鎌田 瑠泉, 中川 夏美
• リボソームＲＮＡ潜在性ＯＲＦ由来機能性ポリペプチドによる細菌の生存危機防御機構

  日本学術振興会：科学研究費助成事業

  研究期間 : 2023年04月 -2026年03月 

  代表者 : 坂口 和靖, 鎌田 瑠泉, 中川 夏美
• 癌抑制タンパク質p53の一過的機能停止制御を介した新規ゲノム編集法の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 坂口 和靖, 鎌田 瑠泉, 中川 夏美

   

  ゲノム編集は、次世代の遺伝子治療法として大きな注目を集めている。しかしながら、CRISPR/Cas9においても、癌抑制タンパク質p53が変異・欠損している細胞に対して優先的にゲノム編集が起こり、治療後に細胞癌化の副作用の恐れがある。このため、有効な治療法が遺伝子治療のみである遺伝性疾患等の治療のために、より安全なゲノム編集法の開発が求められている。安全なゲノム編集のためには、細胞癌化の抑制機構経路の中心であるp53機能を『一過的』に停止制御することが必須である。本研究では、癌抑制タンパク質p53機能の時間的制御による新規ゲノム編集法の開発研究を実施する。すなわち、p53の機能発現に必須な四量体形成を介して、ゲノム編集するときのみp53活性を停止させ、効率的なゲノム編集を達成し、編集後にp53活性を回復させることにより細胞癌化を抑止可能な安全なゲノム編集法の開発を目指す。 初年度は、コイルドコイルペプチドによる機能性ペプチドの多量体化がその生理活性に及ぼす効果を制御することを見出した。今年度は、機能性ペプチドとして我々が発見した抗菌活性ペプチドr-Pep1を用い、その多量体化により大腸菌および哺乳細胞の増殖に対する効果を顕著に変化させることを見出した。さらに、一過的にp53の機能を阻害した際のゲノム編集効率を解析するため、ID-p53Tetペプチドの機能解析のためのp53活性およびゲノム編集効率を同時にモニターするレポーター系を確立した。 本研究における、多量体化を基盤とした新規機能性ペプチドの創成および癌抑制タンパク質p53のヘテロオリゴマー化を介した一過的機能阻害ペプチドID-p53Tetの開発により、安全かつ効率的なゲノム編集法の開発が強く期待される。
• 細菌リボソームRNAにコードされ生存危機ストレスにより翻訳される防御ポリペプチド

  日本学術振興会：科学研究費助成事業

  研究期間 : 2019年06月 -2022年03月 

  代表者 : 坂口 和靖, 鎌田 瑠泉

   

  近年、抗生剤の重要性が医学的・社会的にますます大きくなっている。本研究では、ノンコーディングRNAとされている原核生物リボソームRNA（rRNA）が遺伝子情報を内在的にコードし、生存危機回避などの特殊な状況においてポリペプチドに翻訳されるという『r-Peptide』仮説の解明を目指し、rRNA配列由来のポリペプチド（r-peptide）の同定と機能について研究を実施した。バイオインフォマティクス解析より得られたr-peptideが特異的な抗-抗菌活性を有し、その作用機序モデルを示した。さらに、質量分析解析により、抗生物質存在下において酸化型r-peptideの発現が示唆された。
• 配位金属制御に基づく新規な基質トラッピング法による癌抑制ホスファターゼ標的の探索

  科研費：挑戦的萌芽研究

  研究期間 : 2015年 -2016年 

  代表者 : 坂口 和靖
• 癌抑制タンパク質ｐ５３の多量体化と配向化を基盤とした生物イベント制御と機能解明

  科研費：基盤研究（B）

  研究期間 : 2012年 -2014年 

  代表者 : 坂口 和靖
• ＰＰＭ１Ｄホスファターゼ過剰発現癌細胞における染色体分配制御の破綻と分子基盤解明

  科研費：基盤研究（C）

  研究期間 : 2012年 -2014年 

  代表者 : 中馬 吉郎
• iPS細胞の樹立効率化のためのヘテロオリゴマーを介した癌抑制タンパク質p53阻害

  科研費：挑戦的萌芽研究

  研究期間 : 2011年 -2012年 

  代表者 : 坂口 和靖
• 腫瘍由来変異と進化に基づく癌抑制タンパク質p53四量体安定性と機能不全閾値の解明

  科研費：基盤研究（B）

  研究期間 : 2009年 -2011年 

  代表者 : 坂口 和靖
• E‐Life構築を目指したD型タンパク質翻訳系の開発

  科研費：挑戦的萌芽研究

  研究期間 : 2009年 -2010年 

  代表者 : 坂口 和靖
• がん抑制タンパク質p53四量体形成変異による不活性化機構の解明と機能修復剤の開発研

  科研費：基盤研究（B）

  研究期間 : 2006年 -2008年 

  代表者 : 坂口 和靖
• がん抑制タンパク質p53の四量体形成ドメインのフォールディング

  科研費：特定領域研究

  研究期間 : 2004年 -2005年 

  代表者 : 坂口 和靖
• 酸化ストレス解析のための特異的モノクローナル抗体作製法の開発

  科研費：萌芽研究

  研究期間 : 2003年 -2004年 

  代表者 : 坂口 和靖
• がん遺伝子治療用の非ヘテロ四量体形成性がん抑制タンパク質p53の設計と開発

  科研費：基盤研究（B）

  研究期間 : 2002年 -2004年 

  代表者 : 坂口 和靖
• Structure and Post-translational Modification of Tumor suppressor Protein p53 and its related proteins

  研究期間 : 2003年
• リン酸化モチーフ特異的抗体を用いたタンパク質リン酸化解析法の開発

  科研費：基盤研究（C）

  研究期間 : 2000年 -2001年
• p53誘導性ホスファターゼWip1の局在化とSH3蛋白質を介した細胞周期の制御

  科研費：特定領域研究（C）

  研究期間 : 2001年
• p53誘導性ホスファターゼWip1の局在化調節を介した細胞周期の制御機構の解明

  科研費：特定領域研究（C）

  研究期間 : 2000年
• Cell cycle control by protein phosphatase

  研究期間 : 1999年

産業財産権

• Methods for Generating Phosphorylation Site-Specific Immunological Reagents

  US 6,309,863, B1