基本情報

所属

• 理学研究院　化学部門　有機・生命化学分野

職名

• 准教授

学位

• 博士（理学）（北海道大学）

ホームページURL

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

科研費研究者番号

• 40750881

ORCID ID

•  https://orcid.org/0000-0002-6118-5091

J-Global ID

• 201501061206236480

研究キーワード

• 自然免疫   阻害剤   好中球   ペプチド化学   ホスファターゼ   細胞分化   生物化学   

研究分野

• ライフサイエンス / 分子生物学
• ナノテク・材料 / 生体化学
• ライフサイエンス / 機能生物化学
• ライフサイエンス / 細胞生物学

担当教育組織

•  学士課程　理学部
•  修士課程　総合化学院
•  博士課程　総合化学院

職歴

• 2020年04月 - 現在 北海道大学 大学院理学研究院化学部門 准教授
• 2014年05月 - 2020年03月 北海道大学 大学院理学研究院化学部門 助教
• 2012年05月 - 2014年04月 National Institutes of Health, National Institute of Child Health and Human Development, Visiting Fellow
• 2011年04月 - 2012年04月 京都大学 大学院工学研究科 特定研究員
• 2008年04月 - 2011年03月 北海道大学 大学院理学院 日本学術振興会特別研究員（DC1）

学歴

• 2007年10月 - 2010年09月   北海道大学   大学院理学院   化学専攻 博士後期課程
• 2006年04月 - 2007年09月   北海道大学   大学院理学院   化学専攻 修士課程
• 2002年04月 - 2006年03月   北海道大学   理学部   化学科

所属学協会

• 日本プロテインホスファターゼ研究会   日本生化学会   日本ペプチド学会   日本化学会   

研究活動情報

論文

• A single-cell genomic strategy for alternative transcript start sites identification.

  Yanling Peng, Qitong Huang, Danli Liu, Siyuan Kong, Rui Kamada, Keiko Ozato, Yubo Zhang, Jun Zhu

  Biotechnology journal 19 3 e2300516  2024年03月 

  Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-β stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-β stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.
• 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 [査読有り]
   

  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 [査読有り]
   

  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 28 13 2023年07月 [査読有り]
   

  Biologically derived hydrogels have attracted attention as promising polymers for use in biomedical applications because of their high biocompatibility, biodegradability, and low toxicity. Elastin-mimetic polypeptides (EMPs), which contain a repeated amino acid sequence derived from the hydrophobic domain of tropoelastin, exhibit reversible phase transition behavior, and thus, represent an interesting starting point for the development of biologically derived hydrogels. In this study, we succeeded in developing functional EMP-conjugated hydrogels that displayed temperature-responsive swelling/shrinking properties. The EMP-conjugated hydrogels were prepared through the polymerization of acrylated EMP with acrylamide. The EMP hydrogel swelled and shrank in response to temperature changes, and the swelling/shrinking capacity of the EMP hydrogels could be controlled by altering either the amount of EMP or the salt concentration in the buffer. The EMP hydrogels were able to select a uniform component of EMPs with a desired and specific repeat number of the EMP sequence, which could control the swelling/shrinking property of the EMP hydrogel. Moreover, we developed a smart hydrogel actuator based on EMP crosslinked hydrogels and non-crosslinked hydrogels that exhibited bidirectional curvature behavior in response to changes in temperature. These thermally responsive EMP hydrogels have potential use as bio-actuators for a number of biomedical applications.
• 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 136 12 2023年05月22日 [査読有り]
   

  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 [査読有り]
   

  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 [査読有り]
   

  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 [査読有り][通常論文]
  
• 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03日 [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.
• 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.
• The tetramerization domain of the tree shrew p53 protein displays unique thermostability despite sharing high sequence identity with the human p53 protein.

  Natsumi Nakagawa, Shuya Sakaguchi, Takao Nomura, Rui Kamada, James G Omichinski, Kazuyasu Sakaguchi

  Biochemical and biophysical research communications 521 3 681 - 686 2020年01月15日 [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 protein phosphatase PPM1D enhances retinoic acid-induced differentiation in human embryonic carcinoma cell line.

  Sari Ogasawara, Yoshiro Chuman, Takahiro Michiba, Rui Kamada, Toshiaki Imagawa, Kazuyasu Sakaguchi

  Journal of biochemistry 165 6 471 - 477 2019年06月01日 [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.
• 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 [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.
• Interferon stimulation creates chromatin marks and establishes transcriptional memory

  Kamada, R, Yang, W, Zhang, Y, Patel, MC, Yang, Y, Ouda, R, Dey, A, Wakabayashi, Y, Sakaguchi, K, Fujita, T, Tamura, T, Zhu, J, Ozato, K

  Proc. Natl. Acad. Sci. U.S.A. 115 39 E9162 - E9171 2018年09月 [査読有り][通常論文]
   

  Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNβ stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNβ-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.
• 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 & Peptide Letters 25 1 4 - 14 Bentham Science Publishers Ltd. 2018年04月18日 [査読無し]
  
• 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 

  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.
• 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.
• 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.
• On the role of H3. 3 in retroviral silencing

  Wolf, Gernot, Rebollo, Rita, Karimi, Mohammad M, Ewing, Adam D, Kamada, Rui, Wu, Warren, Wu, Brenda, Bachu, Mahesh, Ozato, Keiko, Faulkner, Geoffrey J, others

  Nature 548 7665 2017年
• Synergic Strategies for the Enhanced Self-Assembly of Biomineralization Peptides for the Synthesis of Functional Nanomaterials.

  Janairo, JIB, Sakaguchi, T, Mine, K, Kamada, R, Sakaguchi, K

  Protein Pept. Lett. 2017年 [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.
• 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.
• 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.
• Tetramer Formation of Tumor Suppressor Protein p53: Structure, Function, and Applications

  Rui Kamada, Yu Toguchi, Takao Nomura, Toshiaki Imagawa, Kazuyasu Sakaguchi

  BIOPOLYMERS 106 4 598 - 612 2016年07月 [査読有り][招待有り]
   

  Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal, and reprogramming of differentiated cells into stem cells, immune system, and metastasis. More than 50% of human tumors have TP53 gene mutations, and most of them are missense mutations that presumably reduce tumor suppressor activity of p53. This review focuses on the role of the tetramerization (oligomerization), which is modulated by the protein concentration of p53, posttranslational modifications, and/or interactions with its binding proteins, in regulating the tumor suppressor function of p53. Functional control of p53 by stabilizing or inhibiting oligomer formation and its bio-applications are also discussed. (C) 2015 Wiley Periodicals, Inc.
• 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.
• 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.
• Specific Detection and Imaging of Enzyme Activity by Signal-Amplifiable Self-Assembling F-19 MRI Probes

  Kazuya Matsuo, Rui Kamada, Keigo Mizusawa, Hirohiko Imai, Yuki Takayama, Michiko Narazaki, Tetsuya Matsuda, Yousuke Takaoka, Itaru Hamachi

  CHEMISTRY-A EUROPEAN JOURNAL 19 38 12875 - 12883 2013年09月 [査読有り][通常論文]
   

  Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling (FNMR)-F-19/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are silent when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these F-19 probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by (FNMR)-F-19 spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by (FMRI)-F-19, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on F-19 probes may serve as a screening platform for the activity of MMPs.
• 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.
• Tetramer Stability and Functional Regulation of Tumor Suppressor Protein

  Kamada, Rui

  2012年
• Inhibition of the Transcriptional Activity of p53 Through Hetero-Oligomerization

  Kamada, Rui

  Tetramer Stability and Functional Regulation of Tumor Suppressor Protein p53 61 - 70 2012年
• Stabilization of Mutant Tetrameric Structures by Calixarene Derivatives

  Kamada, Rui

  Tetramer Stability and Functional Regulation of Tumor Suppressor Protein p53 45 - 59 2012年
• Quantitative Analysis for p53 Tetramerization Domain Mutants Reveals a Low Threshold for Tumor Suppressor Inactivation

  Kamada, Rui

  Tetramer Stability and Functional Regulation of Tumor Suppressor Protein p53 13 - 43 2012年
• Probing Phenylalanine Environments in Oligomeric Structures with Pentafluorophenylalanine and Cyclohexylalanine

  Takao Nomura, Rui Kamada, Issaku Ito, Koichi Sakamoto, Yoshiro Chuman, Koichiro Ishimori, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi

  BIOPOLYMERS 95 6 410 - 419 2011年06月 [査読有り][通常論文]
   

  Stabilization of protein structures and protein protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F(5)Phe) and cyclohexylalanine (Cha). In this study, substitution of F(5)Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53 p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F(5)Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of similar to 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p.5.3 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F(5)Phe is highly suitable for probing the environments of Phe residues. (C) 2011 Wiley Periodicals, Inc. Biopolymers 95: 410-419, 2011.
• Cancer-associated p53 Tetramerization Domain Mutants QUANTITATIVE ANALYSIS REVEALS A LOW THRESHOLD FOR TUMOR SUPPRESSOR INACTIVATION

  Rui Kamada, Takao Nomura, Carl W. Anderson, Kazuyasu Sakaguchi

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 1 252 - 258 2011年01月 [査読有り][通常論文]
   

  The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely (Delta T(m) = 4.8 similar to -46.8 degrees C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.
• 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.
• 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.
• Evaluation of transcriptional activity of p53 in individual living mammalian cells

  Toshiaki Imagawa, Tomoko Terai, Yoshifumi Yamada, Rui Kamada, Kazuyasu Sakaguchi

  ANALYTICAL BIOCHEMISTRY 387 2 249 - 256 2009年04月 [査読有り][通常論文]
   

  To estimate the transcriptional activity of p53 in individual living mammalian cells, we constructed the enhanced green fluorescent protein-red fluorescent protein (EGFP-DsRed) reporter system with the EGFP-p53 expression vector and the reporter plasmid, which carried a p53-dependent promoter. The expression level and transcriptional activity of EGFP-p53 were determined simultaneously by green and red fluorescence signals, respectively. In this system, we could target only the cells expressing p53 at endogenous levels, as observed in UV- or adriamycin-stimulated A549 cells. Using this system, we investigated the transcriptional activity of mutant p53s in tetramerization domain. Transcriptional activities were nearly abolished by seven mutations and significantly reduced in several mutant p53s. However, under overexpression conditions, the latter mutant p53s showed activity similar to that observed in wild-type p53. These results indicated the importance of physiological concentration for p53 proteins in cells so as to analyze their activities. Fluorescence intensity distribution analysis indicated that the mutant p53s lacking transcriptional activity presented as monomer forms in the cellular extract. In most of the mutant p53s, the decrease in transcriptional activity correlated with an increase in the fraction of monomers. This reporter system can be used for estimating the transcriptional activity of mutant p53s without contribution of the cells overexpressing p53. (C) 2009 Elsevier Inc. All rights reserved.
• Oxidation of Methionine Residue at Hydrophobic Core Destabilizes p53 Tetrameric Structure

  Takao Nomura, Rui Kamada, Issaku Ito, Yoshiro Chuman, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi

  BIOPOLYMERS 91 1 78 - 84 2009年01月 [査読有り][通常論文]
   

  The tumor suppressor protein p53 is a tetrameric phosphoprotein that. induces cell cycle, development, and differentiation by regulating the expression of target genes. The tetramerization of p53 is essential for its tumor suppressor functions. It has been known that oxidation of proteins affects their structure and function. A methionine residue (Met340) is located at the hydrophobic core tit p53 tetramerization domain. Here, we demonstrated that Met340 residue can be oxidized to methionine sulfoxide under oxidative conditions and investigated effects of-the oxidation of p53 tetramerization domain oil its stability and oligomerization state by CD measurement and gel filtration. The oxidation of Met340 drastically induced destabilization of the p53 tetramer by 22.8 kJ/mol of Delta Delta G (TM), while retaining the identical conformation as that of the wild-type peptide. Trypsin digestion experiments also showed that oxidation of Met340 allowed the peptide to form locally loose structure and become more sensitive to enzyme degradation. Tit(tetrameric structure may be destabilized because the oxidation of Met340 induces charge repulsion and/or steric hindrance between the sulfoxide groups. These results taken together suggested that oxidation of methionine residues tit the p53 protein might lie one of the inactivation mechanism of p53 transcriptional function under conditions of oxidative stress. (C) 2008 Periodicals, Inc. Biopolymers 91: 78-84, 2009.
• Effects of Tumor-Associated Mutations in the p53 Tetramerization Domain on Oligomerization State and Transcriptional Activity

  Rui Kamada, Tomoko Terai, Takao Nomura, Yoshiro Chuman, Toshiaki Imagawa, Kazuyasu Sakaguchi

  PEPTIDES FOR YOUTH 611 567 - 568 2009年 [査読有り][通常論文]
  
• J. Peptide Res. J. Peptide Res. 53, 507-517, 1999

  KAMADA, Rui, FUKUDA, Kouichiro, MIYAZAKI, Hiromitsu, NOMURA, Takao, CHUMAN, Yoshiro, IMAGAWA, Toshiaki, TANINO, Keiji, SAKAGUCHI, Kazuyasu
• Nat. Struc. Biol. Nat. Struc. Biol. 2, 386-391, 1995

  KAMADA, Rui, TERAI, Tomoko, NOMURA, Takao, IMAGAWA, Toshiaki, SAKAGUCHI, Kazuyasu
• Nat. Struc. Biol. Nat. Struc. Biol. 2, 386-391, 1995

  WADA, Junya, KAMADA, Rui, CHUMAN, Yoshiro, IMAGAWA, Toshiaki, SAKAGUCHI, Kazuyasu
• Peptide Science 2005, 2006

  MIYAZAKI, Hiromitsu, KAMADA, Rui, NOMURA, Takao, CHUMAN, Yoshiro, IMAGAWA, Toshiaki, SAKAGUCHI, Kazuyasu
• Nat. Struc. Biol. Nat. Struc. Biol. 2, 386-391, 1995

  NOMURA, Takao, KAMADA, Rui, CHUMAN, Yoshiro, SAKAGUCHI, Kazuyasu

書籍

• Tetramer Stability and Functional Regulation of Tumor Suppressor Protein p53

  Kamada, R (担当:共著)
  Springer 2012年

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  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
• 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
• 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

受賞

• 2022年03月 2021年度 日本化学会 女性化学者奨励賞
   

  受賞者: 鎌田 瑠泉
• 2020年12月 第14回プロテインホスファターゼ国際カンファレンス Young Investigator Award in ICPP2020 (Best Presentation)
  
• 2019年03月 日本生化学会北海道支部 平成３１年度若手奨励賞
   

  受賞者: 鎌田 瑠泉
• 2016年11月 日本プロテインホスファターゼ研究会 奨励賞
   

  受賞者: 鎌田 瑠泉
• 2011年03月 北海道大学 大塚賞
   

  受賞者: 鎌田 瑠泉
• 2010年11月 日本ペプチド学会『第46回ペプチド討論会』 Poster Award
   『Inhibition of p53 by introducing tetramerization domain peptide fused with polyvalent cationic sequence into cells』 

  受賞者: 鎌田 瑠泉

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
• 好中球サブセット分化制御を介したがん免疫制御機構の解明

  武田科学振興財団：

  研究期間 : 2023年07月 

  代表者 : 鎌田 瑠泉
• 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 基盤研究(B)

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 坂口 和靖, 鎌田 瑠泉

   

  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：挑戦的研究（萌芽）

  研究期間 : 2019年06月 -2021年03月 

  代表者 : 坂口 和靖

   

  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。
• 好中球サブセット分化・機能制御におけるPPM1Dの新規機能

  北海道大学：若手研究加速事業

  研究期間 : 2019年05月 -2020年03月 

  代表者 : 鎌田 瑠泉
• プロテインホスファターゼPPM1Dの好中球サブセット分化・成熟における新規機能

  公益財団法人 秋山記念生命科学振興財団：研究助成＜奨励＞

  研究期間 : 2019年04月 -2020年03月 

  代表者 : 鎌田 瑠泉
• p53誘導性ホスファターゼPPM1Dによる好中球分化と免疫応答制御機構の解明

  文部科学省：科学研究費補助金（若手研究(B)）

  研究期間 : 2017年04月 -2020年03月 

  代表者 : 鎌田 瑠泉

   

  癌抑制タンパク質p53誘導性Ser/ThrホスファターゼPPM1Dは、免疫系細胞の分化や免疫応答において重要な機能を有していることが示唆されている。しかしながら、免疫細胞分化・免疫応答におけるPPM1Dの分子制御機構はいまだ明らかとなっておらず、その解明が強く求められている。本研究では、PPM1Dが好中球分化および分化後の機能制御に関与していることを明らかにした。さらに、PPM1Dの各スプライスバリアント過剰発現細胞株を作製し、PPM1DスプライスバリアントPPM1D605, PPM1D430の好中球分化および機能成熟における機能を初めて明らかにすることに成功した。
• 脂肪滴形成・融合・分解の制御機構解明を目指した 新規蛍光プローブの開発

  豊田理化学研究所：豊田理研スカラー

  研究期間 : 2018年04月 -2019年03月 

  代表者 : 鎌田 瑠泉
• 自然免疫細胞分化におけるSer/ThrホスファターゼPPM1Dの機能解明

  ノーステック財団：

  研究期間 : 2017年08月 -2018年03月 

  代表者 : 鎌田 瑠泉
• 四量体構造-機能相関解析に基づく癌抑制タンパク質p53の機能修復

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  研究期間 : 2008年 -2010年 

  代表者 : 鎌田 瑠泉

   

  癌抑制タンパク質p53は様々な遺伝毒性ストレスに応答して活性化し、下流の種々の標的遺伝子の転写を活性化させて細胞周期停止・アポトーシスを誘導する。四量体形成はp53のDNA結合、翻訳後修飾、安定化に必須である。p53の変異はヒト悪性腫瘍で最も多く認められる異常であり、癌遺伝子治療の重要なターゲットの一つである。本研究では、四量体形成ドメインおよびDNA結合ドメインに変異を持つ変異型p53の機能回復を目指し、『四量体形成ドメイン変異型p53の構造を安定化させる新規化合物の開発』および『DNA結合ドメインの変異に対する非ヘテロオリゴマー形成性の改良型p53の開発』を目的としている。 前年度までに、二種のカリックス[6]アレーン誘導体を合成し、イミダゾリル基を持つイミダゾールカリックス[6]アレーンがp53-R337H変異体の四量体構造を安定化させることを明らかにしている。細胞内p53転写活性を解析する蛍光レポーターシステムを用い、カリックス[6]アレーン誘導体がp53の転写活性に及ぼす効果を解析した。その結果、四量体構造の安定化効果を示すイミダゾールカリックス[6]アレーンにより細胞内のp53-R337Hの転写活性を増加させることに成功した。また、R337H四量体構造の安定化を示さなかったピラゾールカリックス[6]アレーンでは転写活性に変化が見られなかったことから、イミダゾールカリックス[6]アレーンによる転写活性の増加は四量体構造の安定化を介した活性増加であることが示唆された。このカリックスアレーン誘導体による転写活性の増加は、p53の四量体構造を安定化した事による機能修復の初めての例である。

教育活動情報

主要な担当授業

大学運営

委員歴

• 2022年01月 - 現在   日本プロテインホスファターゼ研究会 世話人会
• 2018年01月 - 2021年03月   日本ペプチド学会   編集委員