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Master

Affiliation (Master)

  • Faculty of Science　Chemistry　Organic and Biological Chemistry

Affiliation (Master)

  • Faculty of Science　Chemistry　Organic and Biological Chemistry

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Profile and Settings

Profile and Settings

  • Name (Japanese)

    Sakaguchi

  • Name (Kana)

    Kazuyasu

  • Name

    200901070729888644

Alternate Names

Achievement

Research Interests

  • Inhibitor   Enzyme   Differentiation   Cell Cycle   Cancer   Dephospharylation   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   Biochemistry   

Research Areas

  • Nanotechnology/Materials / Biochemistry
  • Life sciences / Structural biochemistry
  • Life sciences / Functional biochemistry
  • Nanotechnology/Materials / Molecular biochemistry

Awards

  • 2010/10 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: Chuman, Y;Kurihashi, W;Mizukami, Y;Nashimoto, T;Yagi, H;Sakaguchi, K

Published Papers

  • Tatsuya Sakaguchi, Natsumi Nakagawa, Kenta Mine, Jose Isagani B. Janairo, Rui Kamada, James G. Omichinski, Kazuyasu Sakaguchi
    Biomimetics 8 (8) 606 - 606 2023/12/14 
    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.
  • Shuya Sakaguchi, Natsumi Nakagawa, Haytham M Wahba, Junya Wada, Rui Kamada, James G Omichinski, Kazuyasu Sakaguchi
    International journal of molecular sciences 24 (23) 2023/11/22 
    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.
  • Rui Kamada, Hiromitsu Miyazaki, Jose Isagani Janairo, Yoshiro Chuman, Kazuyasu Sakaguchi
    Molecules 2023/07
  • Tatsuki Tsuruoka, Emiri Nakayama, Takuya Endo, Shingo Harashima, Rui Kamada, Kazuyasu Sakaguchi, Toshiaki Imagawa
    Journal of cell science 2023/05/22 
    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.
  • Itsumi Tani, Yui Oikawa, Seiyo Doi, Jose Isagani B Janairo, Rui Kamada, Kazuyasu Sakaguchi
    Biochemical and biophysical research communications 642 35 - 40 2023/01/29 
    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.
  • Itsumi Tani, Yui Oikawa, Seiyo Doi, Jose Isagani B. Janairo, Rui Kamada, Kazuyasu Sakaguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 642 35 - 40 0006-291X 2023/01 
    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.
  • Rui Kamada, Sae Uno, Nozomi Kimura, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi
    International Journal of Molecular Sciences 23 (19) 12046 - 12046 2022/10/10 
    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.
  • Rui Kamada, Shuya Sakaguchi, Shoma Kura, Kaori Takamatsu, Tetsuya Yamamoto, Toshiaki Imagawa, Kazuyasu Sakaguchi
    Chemistry Letters 51 (10) 1029 - 1032 0366-7022 2022/10/05
  • 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 0006-291X 2021/12 [Refereed]
     
    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.
  • 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 2021/11/30 
    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.
  • Rui Kamada, Fuki Kudoh, Shogo Ito, Itsumi Tani, Jose Isagani B. Janairo, James G. Omichinski, Kazuyasu Sakaguchi
    Pharmacology & Therapeutics 215 107622 - 107622 0163-7258 2020/11 [Refereed][Invited]
     
    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.
  • 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 2020/05/05 
    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.
  • Xavier H. Mascle, Christina Gagnon, Haytham M. Wahba, Mathieu Lussier-Price, Laurent Cappadocia, Kazuyasu Sakaguchi, James G. Omichinski
    Structure 28 (2) 157 - 168.e5 0969-2126 2020/02
  • Nakagawa N, Sakaguchi S, Nomura T, Kamada R, Omichinski JG, Sakaguchi K
    Biochemical and biophysical research communications 521 (3) 681 - 686 0006-291X 2019/11 [Refereed][Not invited]
     
    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.
  • Rui Kamada, Nozomi Kimura, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi
    PLOS ONE 14 (2) e0212682  2019/02 [Refereed][Not invited]
     
    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.
  • J. Biochem
    J. Biochem. 165 (6) 471 - 477 2019 [Refereed][Not invited]
     
    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 2018/09 [Refereed][Not invited]
  • 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 2018/06/26 [Refereed][Not invited]
     
    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.
  • Jose Isagani B. Janairo, Kazuyasu Sakaguchi
    Materials Science Forum 928 77 - 82 1662-9752 2018 
    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.
  • Yu Toguchi, Rui Kamada, Madoka Kanno, Toshiaki Imagawa, Kazuyasu Sakaguchi
    Chemistry Letters 47 (2) 217 - 220 1348-0715 2018 [Refereed][Not invited]
     
    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.
  • Jose Isagani B. Janairo, Tatsuya Sakaguchi, Kenta Mine, Rui Kamada, Kazuyasu Sakaguchi
    Protein and Peptide Letters 25 (1) 4 - 14 1875-5305 2018/01/01 [Refereed][Invited]
     
    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.
  • Rui Kamada, Fuki Kudoh, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi
    JOURNAL OF BIOCHEMISTRY 162 (4) 303 - 308 0021-924X 2017/10 [Refereed][Not invited]
     
    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.
  • Rui Kamada, Natsumi Nakagawa, Taiji Oyama, Kazuyasu Sakaguchi
    JOURNAL OF PEPTIDE SCIENCE 23 (7-8) 644 - 649 1075-2617 2017/07 [Refereed][Not invited]
     
    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.
  • Tatsuya Sakaguchi, Jose Isagani B. Janairo, Mathieu Lussier-Price, Junya Wada, James G. Omichinski, Kazuyasu Sakaguchi
    SCIENTIFIC REPORTS 7 (1) 1400  2045-2322 2017/05 [Refereed][Not invited]
     
    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.
  • Kamada, R, Toguchi, Y, Nomura, T, Sakaguchi, K
    Biopolymers: Peptide Science 106 (4) 598 - 612 2016/11 [Refereed][Invited]
  • Yuuki Kozakai, Rui Kamada, Junya Furuta, Yuhei Kiyota, Yoshiro Chuman, Kazuyasu Sakaguchi
    SCIENTIFIC REPORTS 6 33272  2045-2322 2016/09 [Refereed][Not invited]
     
    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.
  • Hiroki Sakai, Ken Watanabe, Fuki Kudoh, Rui Kamada, Yoshiro Chuman, Kazuyasu Sakaguchi
    SCIENTIFIC REPORTS 6 31993  2045-2322 2016/08 [Refereed][Not invited]
     
    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.
  • Rui Kamada, Fumi Tano, Fuki Kudoh, Nozomi Kimura, Yoshiro Chuman, Ayumi Osawa, Kosuke Namba, Keiji Tanino, Kazuyasu Sakaguchi
    PLOS ONE 11 (8) e0160625  1932-6203 2016/08 [Refereed][Not invited]
     
    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.
  • Junya Wada, Hiromitsu Miyazaki, Rui Kamada, Kazuyasu Sakaguchi
    CHEMISTRY LETTERS 45 (2) 185 - 187 0366-7022 2016/02 [Refereed][Not invited]
     
    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 [Refereed][Not invited]
  • Sari Ogasawara, Yuhei Kiyota, Yoshiro Chuman, Ayano Kowata, Fumihiko Yoshimura, Keiji Tanino, Rui Kamada, Kazuyasu Sakaguchi
    BIOORGANIC & MEDICINAL CHEMISTRY 23 (19) 6246 - 6249 0968-0896 2015/10 [Refereed][Not invited]
     
    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.
  • Tatsuya Sakaguchi, Kenta Mine, Fuki Kudoh, Rui Kamada, Kazuyasu Sakaguchi
    CHEMISTRY LETTERS 44 (3) 327 - 329 0366-7022 2015/03 [Refereed][Not invited]
     
    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.
  • 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 2041-6520 2015 [Refereed][Not invited]
     
    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.
  • 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 0969-2126 2015/01 [Refereed][Not invited]
     
    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.
  • Kamada R, Chuman Y, Kozakai Y, Sakaguchi K
    Seikagaku. The Journal of Japanese Biochemical Society 日本生化学会 87 (5) 531 - 538 0037-1017 2015 [Refereed][Not invited]
  • Yuuki Kozakai, Rui Kamada, Yuhei Kiyota, Fumihiko Yoshimura, Keiji Tanino, Kazuyasu Sakaguchi
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 (24) 5593 - 5596 0960-894X 2014/12 [Refereed][Not invited]
     
    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.
  • Jose Isagani B. Janairo, Kazuyasu Sakaguchi
    CHEMISTRY LETTERS 43 (8) 1315 - 1317 0366-7022 2014/08 [Refereed][Not invited]
     
    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.
  • Jose Isagani B. Janairo, Tatsuya Sakaguchi, Kenji Hara, Atsushi Fukuoka, Kazuyasu Sakaguchi
    CHEMICAL COMMUNICATIONS 50 (66) 9259 - 9262 1359-7345 2014/08 [Refereed][Not invited]
     
    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.
  • 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 1616-301X 2013/10/18 [Refereed][Not invited]
     
    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.
  • Yoshiro Chuman, Mariko Ueyama, Satoshi Sano, Fei Wu, Yuhei Kiyota, Takayoshi Higashi, Satoshi Osada, Kazuyasu Sakaguchi
    CHEMISTRY LETTERS 42 (8) 833 - 835 0366-7022 2013/08 [Refereed][Not invited]
     
    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.
  • Junya Wada, Rui Kamada, Toshiaki Imagawa, Yoshiro Chuman, Kazuyasu Sakaguchi
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 22 (8) 2780 - 2783 0960-894X 2012/04 [Refereed][Not invited]
     
    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.
  • Kozakai Yuuki, Kiyota Yuhei, Yagi Hiroaki, Chuman Yoshiro, Sakaguchi Kazuyasu
    Abstracts for Annual Meeting of Japanese Proteomics Society 日本プロテオーム学会（日本ヒトプロテオーム機構） 2012 138 - 138 2012
  • Yagi Hiroaki, Chuman Yoshiro, Kozakai Yuuki, Imagawa Toshiaki, Takahashi Yu, Yoshimura Fumihiko, Tanino Keiji, Sakaguchi Kazuyasu
    Bioorganic & Medicinal Chemistry Letters 22 (1) 729 - 732 0960-894X 2012 [Refereed][Not invited]
  • Yoshiro Chuman, Kanako Iizuka, Takeshi Honda, Hitoshi Onoue, Yasuyuki Shimohigashi, Kazuyasu Sakaguchi
    JOURNAL OF BIOCHEMISTRY 150 (3) 319 - 325 0021-924X 2011/09 [Refereed][Not invited]
     
    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.
  • Han Ying, Noguchi Hidenori, Kazuyasu Sakaguchi, Uosaki Kohei
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 241 0065-7727 2011/03/27 [Refereed][Not invited]
  • Sakai Hiroki, Watanabe Ken, Chuman Yoshiro, Uosaki Kohei, Sakaguchi Kazuyasu
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 241 0065-7727 2011/03/27 [Refereed][Not invited]
  • Kamada, R, Nomura, T, Anderson, C. W, Sakaguchi, K
    J. Biol. Chem. 286 252 - 258 2011 [Refereed][Not invited]
  • Nomura, T, Kamada, R, Ito, I, Sakamoto, K, Chuman, Y, Ishimori, K, Shimohigashi, Y, Sakaguchi, K
    Biopolymers 95 (6) 410 - 419 2011 [Refereed][Not invited]
  • Yagi Hiroaki, Chuman Yoshiro, Yoshimura Fumihiko, Tanino Keiji, Sakaguchi Kazuyasu
    Abstracts of Papers of the American Chemical Society 241 2011 [Refereed][Not invited]
  • 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 0022-2623 2010/08 
    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.
  • Rui Kamada, Wataru Yoshino, Takao Nomura, Yoshiro Chuman, Toshiaki Imagawa, Takanori Suzuki, Kazuyasu Sakaguchi
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 (15) 4412 - 4415 0960-894X 2010/08 [Refereed][Not invited]
     
    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.
  • Yuya Asanomi, Yumiko Kobayashi, Hiroki Sakai, Takuya Masuda, Xinjiang Chen, Yoshiro Chuman, Kohei Uosaki, Kazuyasu Sakaguchi
    PROTEIN AND PEPTIDE LETTERS Bentham Science Publishers 17 (4) 458 - 463 0929-8665 2010/04 [Refereed][Not invited]
     
    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.
  • OSADA S, SANO S, KODAMA H, UEYAMA M, CHUMAN Y, SAKAGUCHI K
    Bioorg. Med. Chem. 18 (2) 605 - 611 0968-0896 2010 [Refereed][Not invited]
     
    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.
  • Rui Kamada, Kazuyasu Sakaguchi
    Seikagaku 82 (6) 484 - 493 0037-1017 2010 [Refereed][Not invited]
  • 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 1538-4101 2009/10 [Refereed][Not invited]
     
    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.
  • Imagawa, T, Terai, T, Yamada, Y, Kamada, R, Sakaguchi, K
    Anal. Biochem. 387 249 - 256 2009 [Refereed][Not invited]
  • Kamada, R, Terai, T, Nomura, T, Chuman, Y, Imagawa, T, Sakaguchi, K
    Adv. Exp. Med. Biol. 611 567 - 568 2009 [Refereed][Not invited]
  • NOMURA Takao, KAMADA Rui, ITO Issaku, CHUMAN Yoshiro, SHIMOHIGASHI Yasuyuki, SAKAGUCHI Kazuyasu
    Biopolymers 91 (1) 78 - 84 0006-3525 2009 [Refereed][Not invited]
  • Yoshiro Chuman, Wataru Kurihashi, Yohei Mizukami, Takehiro Nashimoto, Hiroaki Yagi, Kazuyasu Sakaguchi
    JOURNAL OF BIOCHEMISTRY 145 (1) 1 - 12 0021-924X 2009/01 [Refereed][Not invited]
     
    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.
  • 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 0022-2836 2008 [Refereed][Not invited]
  • Chuman, Y, Yagi, H, Fukuda, T, Nomura, T, Matsukizono, M, Shimohigashi, Y, Sakaguchi K
    Protein Pept. Lett. 15 938 - 948 2008 [Refereed][Not invited]
  • 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 [Refereed][Not invited]
  • 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 0021-9967 2006/11 [Refereed][Not invited]
     
    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.
  • Higashimoto Y, Asanomi Y, Takakusagi S, Lewis MS, Uosaki K, Durell SR, Anderson CW, Appella E, Sakaguchi K
    Biochemistry 45 (6) 1608 - 1619 0006-2960 2006/02 [Refereed][Not invited]
  • K Abe, S Kaya, K Taniguchi, Y Hayashi, T Imagawa, M Kikumoto, K Oiwa, K Sakaguchi
    JOURNAL OF BIOCHEMISTRY 138 (3) 293 - 301 0021-924X 2005/09 [Refereed][Not invited]
     
    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.
  • T Imagawa, T Yamamoto, S Kaya, K Sakaguchi, K Taniguchi
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (19) 18736 - 18744 0021-9258 2005/05 [Refereed][Not invited]
     
    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+.
  • ASANOMI Yuya, TAKAKUSAGI Satoru, CHUMAN Yoshiro, KAYA Shunji, IMAGAWA Toshiaki, UOSAKI Kohei, SAKAGUCHI Kazuyasu
    Peptide Science 2004 (0) 313 - 316 1344-7661 2005/03 [Not refereed][Not invited]
  • 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 0929-5666 2003 [Refereed][Not invited]
     
    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.
  • 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 0270-7306 2002/11 [Refereed][Not invited]
     
    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.
  • M Kawano, K Okada, T Honda, T Nose, K Sakaguchi, T Costa, Y Shimohigashi
    JOURNAL OF PEPTIDE SCIENCE 8 (10) 561 - 569 1075-2617 2002/10 [Refereed][Not invited]
     
    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.
  • Higashimoto Y, Saito S, Tong XH, Hong A, Sakaguchi K, Appella E, Anderson CW
    The Journal of biological chemistry 275 (30) 23199 - 23203 0021-9258 2000/07 [Refereed][Not invited]
  • K Sakaguchi, S Saito, Y Higashimoto, S Roy, CW Anderson, E Appella
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 (13) 9278 - 9283 0021-9258 2000/03 [Refereed][Not invited]
     
    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).
  • 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 0173-0835 2000/02 [Refereed][Not invited]
     
    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.
  • DV Bulavin, S Saito, MC Hollander, K Sakaguchi, CW Anderson, E Appella, AJ Fornace
    EMBO JOURNAL 18 (23) 6845 - 6854 0261-4189 1999/12 [Refereed][Not invited]
     
    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.
  • 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 0014-5793 1999/11 [Refereed][Not invited]
     
    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.
  • 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 0006-4971 1999/10 [Refereed][Not invited]
     
    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.
  • 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 0021-9258 1999/04 [Refereed][Not invited]
  • 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 1420-682X 1999/03 [Refereed][Not invited]
     
    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.
  • Y Higashimoto, MS Lewis, P Kennedy, AM Gronenborn, GM Clore, E Appella, K Sakaguchi
    PEPTIDE SCIENCE - PRESENT AND FUTURE 303 - 305 1999 [Refereed][Not invited]
  • 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 0022-1007 1998/12 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1998/12 [Refereed][Not invited]
     
    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.
  • CW Anderson, E Appella, K Sakaguchi
    JOURNAL OF PROTEIN CHEMISTRY 17 (6) 527 - 527 0277-8033 1998/08 [Refereed][Not invited]
  • CA Pise-Masison, M Radonovich, K Sakaguchi, E Appella, JN Brady
    JOURNAL OF VIROLOGY 72 (8) 6348 - 6355 0022-538X 1998/08 [Refereed][Not invited]
     
    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.
  • Munoz-Barroso, I, S Durell, K Sakaguchi, E Appella, R Blumenthal
    JOURNAL OF CELL BIOLOGY 140 (2) 315 - 323 0021-9525 1998/01 [Refereed][Not invited]
     
    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.
  • ML Cai, Y Huang, K Sakaguchi, GM Clore, AM Gronenborn, R Craigie
    JOURNAL OF BIOMOLECULAR NMR 11 (1) 97 - 102 0925-2738 1998/01 [Refereed][Not invited]
     
    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.
  • JD Siliciano, CE Canman, Y Taya, K Sakaguchi, E Appella, MB Kastan
    GENES & DEVELOPMENT 11 (24) 3471 - 3481 0890-9369 1997/12 [Refereed][Not invited]
     
    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.
  • AC Bergman, C Linder, K Sakaguchi, M StenLinder, AA Alaiya, B Franzen, MC Shoshan, T Bergman, B Wiman, G Auer, E Appella, H Jornvall, S Linder
    FEBS LETTERS Wiley Online Library 417 (1) 17 - 20 0014-5793 1997/11 [Refereed][Not invited]
     
    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.
  • K Sakaguchi, H Sakamoto, MS Lewis, CW Anderson, JW Erickson, E Appella, D Xie
    BIOCHEMISTRY 36 (33) 10117 - 10124 0006-2960 1997/08 [Refereed][Not invited]
     
    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.
  • 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 0027-8424 1997/06 [Refereed][Not invited]
     
    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.
  • 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 0264-410X 1997/03 [Refereed][Not invited]
     
    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.
  • Tsai, V, S Southwood, J Sidney, K Sakaguchi, Y Kawakami, E Appella, A Sette, E Celis
    JOURNAL OF IMMUNOLOGY 158 (4) 1796 - 1802 0022-1767 1997/02 [Refereed][Not invited]
     
    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.
  • 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 0367-8377 1996/11 [Refereed][Not invited]
     
    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.
  • B Fineschi, K Sakaguchi, E Appella, J Miller
    JOURNAL OF IMMUNOLOGY 157 (8) 3211 - 3215 0022-1767 1996/10 [Refereed][Not invited]
     
    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.
  • Sakaguchi Kazuyasu, Roller Peter P, Appella Ettore
    Genetic engineering Springer, Boston, MA 249  1996 [Refereed][Not invited]
  • 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 0022-1767 1995/08 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1995/06 [Refereed][Not invited]
     
    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.
  • RT CLUBB, JG OMICHINSKI, K SAKAGUCHI, E APPELLA, AM GRONENBORN, GM CLORE
    PROTEIN SCIENCE 4 (5) 855 - 862 0961-8368 1995/05 [Refereed][Not invited]
     
    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.
  • GM CLORE, J ERNST, R CLUBB, JG OMICHINSKI, WMP KENNEDY, K SAKAGUCHI, E APPELLA, AM GRONENBORN
    NATURE STRUCTURAL BIOLOGY 2 (4) 321 - 333 1072-8368 1995/04 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1995/04 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1995/03 [Refereed][Not invited]
     
    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.
  • 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 0277-8033 1994/07 [Refereed][Not invited]
  • 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 0277-8033 1994/07 [Refereed][Not invited]
  • Y KAWAKAMI, S ELIYAHU, K SAKAGUCHI, PF ROBBINS, L RIVOLTINI, YANNELLI, JR, E APPELLA, SA ROSENBERG
    JOURNAL OF EXPERIMENTAL MEDICINE 180 (1) 347 - 352 0022-1007 1994/07 [Refereed][Not invited]
     
    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.
  • 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 0027-8424 1994/07 [Refereed][Not invited]
     
    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.
  • GM CLORE, JG OMICHINSKI, K SAKAGUCHI, N ZAMBRANO, H SAKAMOTO, E APPELLA, AM GRONENBORN
    SCIENCE 265 (5170) 386 - 391 0036-8075 1994/07 [Refereed][Not invited]
     
    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.
  • K SAKAGUCHI, H KODAMA, Y OGINO, T COSTA, T NOSE, Y SHIMOHIGASHI
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 67 (6) 1659 - 1663 0009-2673 1994/06 [Refereed][Not invited]
     
    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.
  • 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 0027-8424 1994/05 [Refereed][Not invited]
     
    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.
  • CM BURNS, K SAKAGUCHI, E APPELLA, JD ASHWELL
    JOURNAL OF BIOLOGICAL CHEMISTRY 269 (18) 13594 - 13600 0021-9258 1994/05 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1994/04 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1994/03 [Refereed][Not invited]
     
    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.
  • DS HIPPS, LC PACKMAN, MD ALLEN, C FULLER, K SAKAGUCHI, E APPELLA, RN PERHAM
    BIOCHEMICAL JOURNAL 297 (1) 137 - 143 0264-6021 1994/01 [Refereed][Not invited]
     
    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.
  • 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 0027-8424 1993/11 [Refereed][Not invited]
     
    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.
  • 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 0022-1767 1993/09 [Refereed][Not invited]
     
    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.
  • PC DRISCOLL, JD ALTMAN, JJ BONIFACE, K SAKAGUCHI, PA REAY, JG OMICHINSKI, E APPELLA, MM DAVIS
    JOURNAL OF MOLECULAR BIOLOGY 232 (2) 342 - 350 0022-2836 1993/07 [Refereed][Not invited]
  • 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 0027-8424 1993/07 [Refereed][Not invited]
     
    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.
  • 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 0027-8424 1993/06 [Refereed][Not invited]
     
    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.
  • F FAZIOLI, WT WONG, SJ ULLRICH, K SAKAGUCHI, E APPELLA, PP DIFIORE
    ONCOGENE 8 (5) 1335 - 1345 0950-9232 1993/05 [Refereed][Not invited]
     
    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.
  • B GAO, M VIRMANI, E ROMM, E LAZARWESLEY, K SAKAGUCHI, E APPELLA, G KUNOS, L TAKACS
    GENE 126 (2) 287 - 288 0378-1119 1993/04 [Refereed][Not invited]
     
    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,
  • YN KALIA, SM BROCKLEHURST, DS HIPPS, E APPELLA, K SAKAGUCHI, RN PERHAM
    JOURNAL OF MOLECULAR BIOLOGY 230 (1) 323 - 341 0022-2836 1993/03 [Refereed][Not invited]
  • H MATSUMOTO, Y SHIMOHIGASHI, Y TAKANO, K SAKAGUCHI, H KAMIYA, M OHNO
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 66 (1) 196 - 204 0009-2673 1993/01 [Refereed][Not invited]
     
    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.
  • 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 0036-8075 1992/12 [Refereed][Not invited]
     
    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.
  • SP LEESMILLER, K SAKAGUCHI, SJ ULLRICH, E APPELLA, CW ANDERSON
    MOLECULAR AND CELLULAR BIOLOGY 12 (11) 5041 - 5049 0270-7306 1992/11 [Refereed][Not invited]
     
    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.
  • LA BRISTOL, K SAKAGUCHI, E APPELLA, D DOYLE, L TAKACS
    JOURNAL OF IMMUNOLOGY 149 (2) 367 - 372 0022-1767 1992/07 [Refereed][Not invited]
     
    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.
  • DF HUNT, H MICHEL, TA DICKINSON, J SHABANOWITZ, AL COX, K SAKAGUCHI, E APPELLA, HM GREY, A SETTE
    SCIENCE 256 (5065) 1817 - 1820 0036-8075 1992/06 [Refereed][Not invited]
     
    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.
  • 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 0021-9258 1992/05 [Refereed][Not invited]
     
    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.
  • MA ROBIEN, GM CLORE, JG OMICHINSKI, RN PERHAM, E APPELLA, K SAKAGUCHI, AM GRONENBORN
    BIOCHEMISTRY 31 (13) 3463 - 3471 0006-2960 1992/04 [Refereed][Not invited]
     
    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.
  • K SAKAGUCHI, T COSTA, H SAKAMOTO, Y SHIMOHIGASHI
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 65 (4) 1052 - 1056 0009-2673 1992/04 [Refereed][Not invited]
     
    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.
  • RA HENDERSON, H MICHEL, K SAKAGUCHI, J SHABANOWITZ, E APPELLA, DF HUNT, VH ENGELHARD
    SCIENCE 255 (5049) 1264 - 1266 0036-8075 1992/03 [Refereed][Not invited]
     
    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.
  • DF HUNT, RA HENDERSON, J SHABANOWITZ, K SAKAGUCHI, H MICHEL, N SEVILIR, AL COX, E APPELLA, VH ENGELHARD
    SCIENCE 255 (5049) 1261 - 1263 0036-8075 1992/03 [Refereed][Not invited]
     
    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.
  • Omichinski James G, Sakaguchi Kazuyasu, Clore G Marius, Gronenborn Angela M, Appella Ettore
    Journal of Protein Chemistry Springer 11 (4) 408  1992 [Refereed][Not invited]
  • K SAKAGUCHI, E APPELLA, JG OMICHINSKI, GM CLORE, AM GRONENBORN
    JOURNAL OF BIOLOGICAL CHEMISTRY 266 (11) 7306 - 7311 0021-9258 1991/04 [Refereed][Not invited]
     
    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.
  • JG OMICHINSKI, GM CLORE, E APPELLA, K SAKAGUCHI, AM GRONENBORN
    BIOCHEMISTRY 29 (40) 9324 - 9334 0006-2960 1990/10 [Refereed][Not invited]
  • K SAKAGUCHI, H SAKAMOTO, Y TSUBAKI, M WAKI, T COSTA, Y SHIMOHIGASHI
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN The Chemical Society of Japan 63 (6) 1753 - 1757 0009-2673 1990/06 [Refereed][Not invited]
  • R SAITO, H KONISHI, Y TAKANO, S NONAKA, K SAKAGUCHI, Y SHIMOHIGASHI, HO KAMIYA
    NEUROSCIENCE LETTERS 110 (3) 337 - 342 0304-3940 1990/03 [Refereed][Not invited]
  • Shimohigashi Y, Sakaguchi K, Sakamoto H, Waki M, Costa T
    Peptide research 3 (5) 216  1990 [Refereed][Not invited]
  • Sakaguchi K, Kodama H, Matsumoto H, Yoshida M, Takano Y, Kamiya H, Waki M, Shimohigashi Y
    Peptide research 2 (5) 345  1989 [Refereed][Not invited]
  • 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 0367-8377 1988/10 [Refereed][Not invited]
  • H KODAMA, Y SHIMOHIGASHI, K SAKAGUCHI, M WAKI, Y TAKANO, A YAMADA, Y HATAE, H KAMIYA
    EUROPEAN JOURNAL OF PHARMACOLOGY 151 (2) 317 - 320 0014-2999 1988/07 [Refereed][Not invited]
  • 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 0167-0115 1988/07 [Refereed][Not invited]

MISC

Books etc

  • Peptide Science 2011
    The Japanese Peptide Society 2012
  • 自然科学実験
    学術図書出版 2007

Association Memberships

  • 日本プロテインホスファターゼ研究会   JAPANESE SOCIETY FOR CHEMICAL BIOLOGY   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   

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/06 -2026/03 
    Author : 坂口 和靖, 鎌田 瑠泉, 中川 夏美
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/04 -2026/03 
    Author : 坂口 和靖, 鎌田 瑠泉, 中川 夏美
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2020/04 -2023/03 
    Author : 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。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/06 -2022/03 
    Author : Sakaguchi Kazuyasu
     
    In recent years, antibiotics have become increasingly important in medicine and society. In this study, we investigated the identification and function of polypeptides derived from rRNA sequences (r-peptides) to elucidate the "r-Peptide" hypothesis that prokaryotic rRNA, which is supposed to be a non-protein-coding RNA, intrinsically encodes genetic information, and is translated into polypeptides under special circumstances such as when survival is threatened. The identification and function of rRNA-derived polypeptides (r-peptides) were studied to elucidate the "r-Peptide" hypothesis. Bioinformatics analysis showed that the r-peptides had specific anti-bacterial activity and provided a mechanistic model for their action. In addition, mass spectrometry analysis suggested the expression of oxidized form of the r-peptide in the presence of the antibiotics.
  • 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
    Date (from‐to) : 2015 -2016 
    Author : 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）
    Date (from‐to) : 2012 -2014 
    Author : 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）
    Date (from‐to) : 2012 -2014 
    Author : 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
    Date (from‐to) : 2011 -2012 
    Author : 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）
    Date (from‐to) : 2009 -2011 
    Author : 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
    Date (from‐to) : 2009 -2010 
    Author : 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）
    Date (from‐to) : 2006 -2008 
    Author : 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
    Date (from‐to) : 2004 -2005 
    Author : 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
    Date (from‐to) : 2003 -2004 
    Author : 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）
    Date (from‐to) : 2002 -2004 
    Author : 坂口 和靖
  • Structure and Post-translational Modification of Tumor suppressor Protein p53 and its related proteins
    Date (from‐to) : 2003
  • 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）
    Date (from‐to) : 2000 -2001
  • 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）
    Date (from‐to) : 2001
  • 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）
    Date (from‐to) : 2000
  • Cell cycle control by protein phosphatase
    Date (from‐to) : 1999

Industrial Property Rights

  • Methods for Generating Phosphorylation Site-Specific Immunological Reagents
    US 6,309,863, B1

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