Application of Mass Spectrometry to Protein Analysis, Proteomic Analysis of Signal Transduction (proteome, signal transduction, phosphorylation, mass spectrometry)
Book / Paper
Book:
1.
Hisaaki Taniguchi and Takako Taniguchi : 医用質量分析ガイドブック 質量分析によるプロテオミクス, 株式会社 診断と治療社, Tokyo, Dec. 2013.
2.
Hisaaki Taniguchi and Emiko Yamauchi : タンパク質の翻訳後修飾解析プロトコール 稲垣昌樹編, YODOSHA CO., LTD., Tokyo, Dec. 2005.
Emiko Yamauchi and Hisaaki Taniguchi : クローズアップ実験法, YODOSHA CO., LTD., Tokyo, Aug. 1997.
38.
Hisaaki Taniguchi : Capillary LC/MS Analysis of Posttranslational and Posttranscriptional Modifications of Proteins and Nucleic Acids, J Mass Spectrom Soc Japan, Tokyo, May 1996.
39.
吉田 和夫, Hisaaki Taniguchi and 杉浦 昌弘 : バイオ高性能機器導入·共同利用マニュアル, KYORITSU SHUPPAN.CO.,LTD, Tokyo, 1995.
40.
Hisaaki Taniguchi : 生体高分子の質量分析法, KYORITSU SHUPPAN.CO.,LTD, Tokyo, 1994.
Hisaaki Taniguchi and 今井 嘉郎 : チトクロームP-450による薬物代謝のメカニズム, 清至書院, Tokyo, Dec. 1983.
43.
Hisaaki Taniguchi and 今井 嘉郎 : 膜上における蛋白質間相互作用, The Biophysical Society of Japan, Tokyo, 1981.
Academic Paper (Judged Full Paper):
1.
Nori Sato, Takako Taniguchi, Yuichiro Goda, Hirofumi Kosaka, Kosaku Higashino, Toshinori Sakai, Shinsuke Katoh, Natsuo Yasui, Koichi Sairyo and Hisaaki Taniguchi : Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues., Journal of Proteome Research, Vol.15, No.12, 4709-4721, 2016.
(Summary)
Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.
Afzal Husain, A Nasim Begum, Takako Taniguchi, Hisaaki Taniguchi, Maki Kobayashi and Tasuku Honjo : Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability., Nature Communications, Vol.7, 2016.
(Summary)
Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.
Akihiro Sugawara, Nobuo Maita, Hiroaki Gouda, Tsuyoshi Yamamoto, Tomoyasu Hirose, Saori Kimura, Yoshifumi Saito, Hayato Nakano, Takako Kasai, Hirofumi Nakano, Kazuro Shiomi, Shuichi Hirono, Takeshi Watanabe, Hisaaki Taniguchi, Satoshi Ōmura and Toshiaki Sunazuka : Creation of Customized Bioactivity within a 14-Membered Macrolide Scaffold: Design, Synthesis, and Biological Evaluation Using a Family-18 Chitinase., Journal of Medicinal Chemistry, Vol.58, No.12, 4984-4997, 2015.
(Summary)
Argifin, a 17-membered pentapeptide, inhibits chitinase. As argifin has properties that render it unsuitable as a drug development candidate, we devised a mechanism to create the structural component of argifin that bestows the chitinase inhibition and introduce it into a 14-membered macrolide scaffold. Here we describe (1) the designed macrolide, which exhibits 200-fold more potent chitinase inhibition than argifin, (2) the binding modes of the macrolide with Serratia marcescens chitinase B, and (3) the computed analysis explaining the reason for derivatives displaying increased inhibition compared to argifin, the macrolide aglycone displaying inhibition in a nanomolar range. This promises a class of chitinase inhibitors with novel skeletons, providing innovative insight for drug design and the use of macrolides as adaptable, flexible templates for use in drug discovery research and development.
Kiwamu Hyodo, Takako Taniguchi, Yuki Manabe, Masanori Kaido, Kazuyuki Mise, Tatsuya Sugawara, Hisaaki Taniguchi and Tetsuro Okuno : Phosphatidic Acid produced by phospholipase d promotes RNA replication of a plant RNA virus., PLoS Pathogens, Vol.11, No.5, e1004909, 2015.
(Summary)
Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.
Masanori Kaido, Kazutomo Abe, Akira Mine, Kiwamu Hyodo, Takako Taniguchi, Hisaaki Taniguchi, Kazuyuki Mise and Tetsuro Okuno : GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement., PLoS Pathogens, Vol.10, No.11, e1004505, 2014.
(Summary)
The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.
Tomoyasu Hirose, Nobuo Maita, Hiroaki Gouda, Jun Koseki, Tsuyoshi Yamamoto, Akihiko Sugawara, Hirofumi Nakano, Shuichi Hirono, Kazuro Shiomi, Takeshi Watanabe, Hisaaki Taniguchi, K. Barry Sharpless, Satoshi Ōmura and Toshiaki Sunazuka : Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor, Proceedings of the National Academy of Sciences of the United States of America, Vol.110, No.40, 15892-15897, 2013.
(Summary)
The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed "in situ click chemistry," has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]-["click ligand"] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on "postclick" structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.
Nobuo Maita, Takahiro Tsukimura, Takako Taniguchi, Seiji Saito, Kazuki Ohno, Hisaaki Taniguchi and Hitoshi Sakuraba : Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module, Proceedings of the National Academy of Sciences of the United States of America, Vol.110, No.36, 14628-14633, 2013.
(Summary)
N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.
Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.
Timothy W. Yu, Maria H. Chahrour, Michael E. Coulter, Sarn Jiralerspong, Kazuko Okamura-Ikeda, Bulent Ataman, Klaus Schmitz-Abe, David A. Harmin, Mazhar Adli, Athar N. Malik, Alissa M. D'Gama, Elaine T. Lim, Stephan J. Sanders, Ganesh H. Mochida, Jennifer N. Partlow, Christine M. Sunu, Jillian M. Felie, Jacqueline Rodriguez, Ramzi H. Nasir, Janice Ware, Robert M. Joseph, R Sean Hill, Benjamin Y. Kwan, Muna Al-Saffar, Nahit M. Mukaddes, Asif Hashmi, Soher Balkhy, Generoso G. Gascon, Fuki M. Hisama, Elaine LeClair, Annapurna Poduri, Ozgur Oner, Samira Al-Saad, Sadika A. Al-Awadi, Laila Bastaki, Tawfeg Ben-Omran, Ahmad S. Teebi, Lihadh Al-Gazali, Valsamma Eapen, Christine R. Stevens, Leonard Rappaport, Stacey B. Gabriel, Kyriacos Markianos, Matthew W. State, Michael E. Greenberg, Hisaaki Taniguchi, Nancy E. Braverman, Eric M. Morrow and Christopher A. Walsh : Using whole-exome sequencing to identify inherited causes of autism., Neuron, Vol.77, No.2, 259-273, 2013.
(Summary)
Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole-exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1). At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.
(Keyword)
Adolescent / Animals / Autistic Disorder / Cells, Cultured / children / Child, Preschool / Cohort Studies / Exome / Female / Genome-Wide Association Study / Humans / Male / Pedigree / Rats / Sequence Analysis, DNA / Young Adult
Kiwamu Hyodo, Akira Mine, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Hisaaki Taniguchi and Tetsuro Okuno : ADP ribosylation factor 1 plays an essential role in the replication of a plant RNA virus., Journal of Virology, Vol.87, No.1, 163-176, 2013.
(Summary)
Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.
Akira Mine, Kiwamu Hyodo, Yuri Tajima, Kusumawaty Kusumanegara, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Hisaaki Taniguchi and Tetsuro Okuno : Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus., Journal of Virology, Vol.86, No.22, 12091-12104, 2012.
(Summary)
Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.
Kyoko Ishimoto, Takeo Iwata, Hisaaki Taniguchi, Noriko Mizusawa, Eiji Tanaka and Katsuhiko Yoshimoto : d-Dopachrome tautomerase promotes IL-6 expression and inhibits adipogenesis in preadipocytes., Cytokine, Vol.60, 772-777, 2012.
(Summary)
We previously identified d-dopachrome tautomerase (DDT) as a novel adipokine whose mRNA levels in adipocytes are negatively correlated with obesity-related clinical parameters, and which acts on adipocytes to regulate lipid metabolism. Here we investigated functions of DDT on preadipocytes. Recombinant DDT (rDDT) enhanced both the expression and secretion of interleukin-6 (IL-6) in SGBS cells, a human preadipocyte cell line. Treatment with rDDT increased levels of phosphorylated ERK1/2, but not p38, in SGBS cells, and rDDT-induced IL-6 mRNA expression was attenuated by pretreatment with an ERK inhibitor, U0126. Knockdown of CD74, but not CD44, inhibited rDDT-induced IL-6 mRNA expression in SGBS cells. These results suggested that the rDDT-induced IL-6 expression in preadipocytes occurred through the CD74-ERK pathway. Furthermore, in SGBS cells subjected to adipogenic induction, rDDT decreased the amount of triacylglycerol, number of cells with oil droplets, and levels of mRNA encoding adipocyte marker proteins. Increased expression of CCAAT/enhancer binding protein families and peroxisome proliferator-activated receptor 2 during adipogenesis was inhibited in the cells treated with rDDT. These results suggested DDT to inhibit adipogenesis by suppressing the expression of genes encoding adipogenic regulators in preadipocytes.
Dysfunction of PINK1, a mitochondrial Ser/Thr kinase, causes familial Parkinson's disease (PD). Recent studies have revealed that PINK1 is rapidly degraded in healthy mitochondria but accumulates on the membrane potential (ΔΨm)-deficient mitochondria, where it recruits another familial PD gene product, Parkin, to ubiquitylate the damaged mitochondria. Despite extensive study, the mechanism underlying the homeostatic control of PINK1 remains unknown. Here we report that PINK1 is autophosphorylated following a decrease in ΔΨm and that most disease-relevant mutations hinder this event. Mass spectrometric and mutational analyses demonstrate that PINK1 autophosphorylation occurs at Ser228 and Ser402, residues that are structurally clustered together. Importantly, Ala mutation of these sites abolishes autophosphorylation of PINK1 and inhibits Parkin recruitment onto depolarized mitochondria, whereas Asp (phosphorylation-mimic) mutation promotes mitochondrial localization of Parkin even though autophosphorylation was still compromised. We propose that autophosphorylation of Ser228 and Ser402 in PINK1 is essential for efficient mitochondrial localization of Parkin.
(Keyword)
Amino Acid Sequence / Animals / HeLa Cells / Humans / Membrane Potentials / Mice / Mitochondria / Molecular Sequence Data / Parkinson Disease / Phosphorylation / Protein Kinases / Protein Transport / Sequence Alignment / Ubiquitin-Protein Ligases
Hiro-Oki Iwakawa, Yuri Tajima, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Yukihide Tomari, Hisaaki Taniguchi and Tetsuro Okuno : Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region., Journal of Virology, Vol.86, No.15, 7836-7849, 2012.
(Summary)
Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.
Takeo Iwata, Hisaaki Taniguchi, Masamichi Kuwajima, Takako Taniguchi, Okuda Yuko, Akiko Sukeno, Kyoko Ishimoto, Noriko Mizusawa and Katsuhiko Yoshimoto : The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism, PLoS ONE, Vol.7, No.3, e33402, 2012.
(Summary)
Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.
(Keyword)
AMP-Activated Protein Kinases / Adipocytes / Adipokines / Animals / Blotting, Western / Body Mass Index / Cell Differentiation / DNA Primers / Gene Knockdown Techniques / Humans / Intramolecular Oxidoreductases / Lipid Metabolism / Mice / Microscopy, Fluorescence / Phosphorylation / Proteomics / Reverse Transcriptase Polymerase Chain Reaction / Signal Transduction
Michitaka Suzuki, Toshihiko Otsuka, Yuki Ohsaki, Jinglei Cheng, Takako Taniguchi, Hisashi Hashimoto, Hisaaki Taniguchi and Toyoshi Fujimoto : Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets., Molecular Biology of the Cell, Vol.23, No.5, 800-810, 2012.
(Summary)
Apolipoprotein B-100 (ApoB) is the principal component of very low density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the endoplasmic reticulum (ER) lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remain unknown. In the present study, we demonstrate that ApoB after lipidation is dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulates as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which is almost confined to LDs in this cell type, decreases recruitment of p97 to LDs and causes an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induces an accumulation of lipidated ApoB in the ER lumen but does not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bind with each other and with lipidated ApoB and show colocalization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the predislocation and postdislocation steps, respectively.
Akira Yamaguchi, Takako Saito, Lixy Yamada, Hisaaki Taniguchi, Yoshito Harada and Hitoshi Sawada : Identification and localization of the sperm CRISP family protein CiUrabin involved in gamete interaction in the ascidian Ciona intestinalis., Molecular Reproduction and Development, Vol.78, No.7, 488-497, 2011.
(Summary)
Ascidians are hermaphrodites, and most release sperm and eggs nearly simultaneously. Many species, including Halocynthia roretzi and Ciona intestinalis, are self-sterile. We previously reported that the interaction between a 12 EGF-like repeat-containing vitelline-coat (VC) protein, HrVC70, and a sperm GPI-anchored CRISP, HrUrabin, in lipid rafts plays a key role in self-/nonself-recognizable gamete interaction in H. roretzi. On the other hand, we recently identified two pairs of polymorphic genes responsible for self-incompatibility in C. intestinalis by positional cloning: The sperm polycystin 1-like receptors s-Themis-A/B and its fibrinogen-like ligand v-Themis-A/B on the VC. However, it is not known if the orthologs of HrVC70 and HrUrabin also participate in gamete interaction in C. intestinalis since they are from different orders. Here, we tested for a C. intestinalis ortholog (CiUrabin) of HrUrabin by searching the genome database and proteomes of sperm lipid rafts. The identified CiUrabin belongs to the CRISP family, with a PR domain and a GPI-anchor-attachment site. CiUrabin appears to be specifically expressed in the testis and localized at the surface of the sperm head, as revealed by Northern blotting and immunocytochemistry, respectively. The specific interaction between CiVC57, a C. intestinalis ortholog of HrVC70, and CiUrabin was confirmed by Far Western analysis, similarly to the interaction between HrVC70 and HrUrabin. The molecular interaction between CiVC57 and CiUrabin may be involved in the primary binding of sperm to the VC prior to the allorecognition process, mediated by v-Themis-A/B and s-Themis-A/B, during fertilization of C. intestinalis.
Toshinori Endo, Keisuke Ueno, Kouki Yonezawa, Katsuhiko Mineta, Kohji Hotta, Yutaka Satou, Lixy Yamada, Michio Ogasawara, Hiroki Takahashi, Ayako Nakajima, Mia Nakachi, Mamoru Nomura, Junko Yaguchi, Yasunori Sasakura, Chisato Yamasaki, Miho Sera, Akiyasu C. Yoshizawa, Tadashi Imanishi, Hisaaki Taniguchi and Kazuo Inaba : CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality., Nucleic Acids Research, Vol.39, No.Database issue, D807-14, 2010.
(Summary)
The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36,034 unique sequences, but for higher usability it covers 89,683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10,000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.
Takako Taniguchi, Shinsuke Kido, Emiko Yamauchi, Masahiro Abe, Toshio Matsumoto and Hisaaki Taniguchi : Induction of endosomal/lysosomal pathways in differentiating osteoblasts as revealed by combined proteomic and transcriptomic analyses., FEBS Letters, Vol.584, No.18, 3969-3974, 2010.
(Summary)
We have analyzed proteome changes associated with bone-forming osteoblast differentiation by quantitative differential proteomic and transcriptomic analyses using in vitro differentiation model. Sixty nine proteins were found up-regulated (>2-fold) and 18 were down-regulated (<0.5-fold) at protein level. The mRNA levels of these proteins were then analyzed by quantitative real-time PCR combined with clustering analysis. The most prominent cluster with increased protein and mRNA levels contains endosomal and lysosomal proteins, demonstrating the drastic induction of degradative endosomal/lysosomal pathways in osteoblasts. Osteoblasts, therefore, are involved not only in the synthesis but also in the turnover of the extracellular matrix proteins such as collagens.
Kazuko Okamura-Ikeda, Harumi Hosaka, Nobuo Maita, Kazuko Fujiwara, Akiyasu Yoshizawa, Atsushi Nakagawa and Hisaaki Taniguchi : Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system, --- IMPLICATIONS FOR RECOGNITION OF LIPOYL PROTEIN SUBSTRATE, DISEASE-RELATED MUTATIONS, AND REACTION MECHANISM ---, The Journal of Biological Chemistry, Vol.285, No.24, 18684-18692, 2010.
(Summary)
Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate. Several mutations in the human T-protein gene are known to cause nonketotic hyperglycinemia. Here, we report the crystal structure of Escherichia coli T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction. The structure of the complex shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Invariant Arg(292) of the T-protein is essential for complex assembly. The structure also provides novel insights in understanding the disease-causing mutations, in addition to the disease-related impairment in the cofactor-enzyme interactions reported previously. Furthermore, structural and mutational analyses suggest that the reversible transfer of the methylene group between the lipoate and tetrahydrofolate should proceed through the electron relay-assisted iminium intermediate formation.
Yoko Ishino and Hisaaki Taniguchi : Dead time loss correction of mass errors occurring in high-throughput proteomics based on electrospray ionization time-of-flight tandem mass spectrometry., Rapid Communications in Mass Spectrometry: RCM, Vol.24, No.10, 1490-1495, 2010.
Akira Mine, Atsushi Takeda, Takako Taniguchi, Hisaaki Taniguchi, Masanori Kaido, Kazuyuki Mise and Tetsuro Okuno : Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus., Journal of Virology, Vol.84, No.12, 6070-6081, 2010.
(Summary)
Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.
Kazuko Fujiwara, Nobuo Maita, Harumi Hosaka, Kazuko Okamura-Ikeda, Atsushi Nakagawa and Hisaaki Taniguchi : Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A, The Journal of Biological Chemistry, Vol.285, No.13, 9971-9980, 2010.
(Summary)
Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645-33651). Here, we report two new LplA structures, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5'-AMP reaction intermediate and rotation of the C-terminal domain by about 180 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
R. Ban, H. Matsuzaki, T Akashi, G. Sakashita, Hisaaki Taniguchi, Y S Park, H. Tanaka, K Furukawa and T Urano : Mitotic regulation of the stability of microtubule plus-end tracking protein EB3 by ubiquitin ligase SIAH-1 and Aurora mitotic kinases, The Journal of Biological Chemistry, Vol.284, No.41, 28367-28381, 2009.
(Summary)
Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G(1) phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.
(Keyword)
Amino Acid Sequence / Animals / COS Cells / Cercopithecus aethiops / HeLa Cells / Humans / Isoenzymes / Microtubule-Associated Proteins / Microtubules / Mitosis / Molecular Sequence Data / Nuclear Proteins / Protein-Serine-Threonine Kinases / RNA, Small Interfering / Recombinant Fusion Proteins / Two-Hybrid System Techniques / Ubiquitin-Protein Ligases
K Tashiro, T Tsunematsu, H Okubo, T Ohta, E Sano, Emiko Yamauchi, Hisaaki Taniguchi and Hiroaki Konishi : GAREM, a novel adaptor protein for growth factor receptor-bound protein 2, contributes to cellular transformation through the activation of extracellular signal-regulated kinase signaling., The Journal of Biological Chemistry, Vol.284, No.30, 20206-20214, 2009.
(Summary)
Adaptor proteins for the various growth factor receptors play a crucial role in signal transduction through tyrosine phosphorylation. Several candidates for adaptor proteins with potential effects on the epidermal growth factor (EGF) receptor-mediated signaling pathway have been identified by recent phosphoproteomic studies. Here, we focus on a novel protein, GAREM (Grb2-associated and regulator of Erk/MAPK) as a downstream molecule of the EGF receptor. GAREM is phosphorylated at tyrosine 105 and 453 after EGF stimulation. Grb2 was identified as its binding partner, and the proline-rich motifs of GAREM are recognized by the N- and C-terminal SH3 domains of Grb2. In addition, the tyrosine phosphorylations of GAREM are necessary for its binding to Grb2. Because the amino acid sequence surrounding tyrosine 453 is similar to the immunoreceptor tyrosine-based inhibitory motif, Shp2, a positive regulator of Erk, binds to GAREM in this phosphorylation-dependent manner. Consequently, Erk activation in response to EGF stimulation is regulated by the expression of GAREM in COS-7 and HeLa cells, which occurs independent of the presence of other binding proteins, such as Gab1 and SOS, to the activated EGF receptor. Furthermore, the expression of GAREM has an effect on the transformation activity of cultured cells. Together, these findings suggest that GAREM plays a key role in the ligand-mediated signaling pathway of the EGF receptor and the tumorigenesis of cells.
(Keyword)
Amino Acid Motifs / Animals / Binding Sites / COS Cells / Cercopithecus aethiops / Extracellular Signal-Regulated MAP Kinases / GRB2 Adaptor Protein / Gene Expression / HeLa Cells / Humans / Molecular Sequence Data / Phosphorylation / Proline / Protein Binding / Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Receptor, Epidermal Growth Factor / Tyrosine / src Homology Domains
L Yamada, T Saito, Hisaaki Taniguchi, H Sawada and Y Harada : Comprehensive egg-coat proteome of an ascidian Ciona intestinalis reveals gamete recognition molecules involved in self-sterility., The Journal of Biological Chemistry, Vol.284, No.14, 9402-9410, 2009.
(Summary)
Despite central roles of egg coat proteins in gamete recognition, their functions and composition are poorly understood. Here, we report that the proteome of the egg coat in the solitary ascidian Ciona intestinalis, called vitelline coat (VC) fraction, contains more than 800 proteins identified by mass spectrometry-based analyses. Over 100 proteins were enriched in the VC fraction compared with the VC-free egg proteome. The most abundant component in the VC was an apolipoprotein-like protein. The VC contained multiple homologs of mammalian zona pellucida (ZP) proteins, the number of which was unexpectedly large and most of which possessed epidermal growth factor-like repeats. Furthermore, the present study revealed that two fibrinogen-like proteins, v-Themis-A and -B, both of which are expressed in the VC, are the molecules responsible for the two self-sterility loci that were identified by our previous genetic study in this species.
Etsuko Sano, Shigeichi Shono, Kyoko Tashiro, Hiroaki Konishi, Emiko Yamauchi and Hisaaki Taniguchi : Novel tyrosine phosphorylated and cardiolipin-binding protein CLPABP functions as mitochondrial RNA granule., Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1783, No.6, 1036-1047, 2008.
(Summary)
We identified a new protein containing the pleckstrin homology (PH) domain through tyrosine phosphoproteomics using epidermal growth factor-stimulated cells. The tandem PH domains of this protein can bind to mitochondria-specific phospholipid, cardiolipin or its dehydro product, phosphatidic acid; therefore, we have designated this protein as cardiolipin and phosphatidic acid-binding protein (CLPABP). In this study, we show that CLPABP is localized on the tubulin network and the mitochondrial surface in the granular form along with other proteins and RNA. The affinity of CLPABP to mitochondria is elevated depending on the extent of tyrosine phosphorylation. The CLPABP complex contains various proteins related to cytoplasmic mRNA metabolism. The unique subcellular localization of CLPABP requires its PH domains and a multifunctional protein, SF2p32, as its binding protein. The CLPABP granule also contains the cytochrome c transcript, which may be mediated by the RNA-binding protein HuR. Immunofluorescence staining reveals that the CLPABP granule is colocalized with cytochrome c and various ribosomal proteins that are present in the CLPABP complex. Therefore, the CLPABP RNA-protein complex may play a role in transporting cytochrome c mRNA and its translated product to the mitochondria.
Shizue Omi, Rie Nakata, Kazuko Okamura-Ikeda, Hiroaki Konishi and Hisaaki Taniguchi : Contribution of Peroxisome-specific Isoform of Lon Protease in Sorting PTS1 Proteins to Peroxisomes., The Journal of Biochemistry, Vol.143, No.5, 649-660, 2008.
(Summary)
Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in beta-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the beta-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.
(Keyword)
Acyl-CoA Oxidase / Catalase / Cell Line / Enzymes / Humans / Peroxisomes / Protease La / Protein Isoforms / Protein Sorting Signals / Protein Transport / Proteins
Yoshito Harada, Yuhei Takagaki, Masahiko Sunagawa, Takako Saito, Lixy Yamada, Hisaaki Taniguchi, Eiichi Shoguchi and Hitoshi Sawada : Mechanism of self-sterility in a hermaphroditic chordate., Science, Vol.320, No.5875, 548-550, 2008.
(Summary)
Hermaphroditic organisms avoid inbreeding by a system of self-incompatibility (SI). A primitive chordate (ascidian) Ciona intestinalis is an example of such an organism, but the molecular mechanism underlying its SI system is not known. Here, we show that the SI system is governed by two gene loci that act cooperatively. Each locus contains a tightly linked pair of polycystin 1-related receptor (s-Themis) and fibrinogen-like ligand (v-Themis) genes, the latter of which is located in the first intron of s-Themis but transcribed in the opposite direction. These genes may encode male- and female-side self-recognition molecules. The SI system of C. intestinalis has a similar framework to that of flowering plants but utilizing different molecules.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Ciona intestinalis / Disorders of Sex Development / Female / Fertility / Fertilization / Genes / Male / Molecular Sequence Data / Ovum / Spermatozoa / TRPP Cation Channels
Yoko Ishino and Hisaaki Taniguchi : An a posteriori calibration method for improving protein identification accuracy in proteomics using electrospray ionization time-of-flight tandem mass spectrometry., Rapid Communications in Mass Spectrometry: RCM, Vol.22, No.8, 1335-1338, 2008.
(Keyword)
Bacterial Proteins / Calibration / Chromatography, High Pressure Liquid / Proteomics / Reproducibility of Results / Spectrometry, Mass, Electrospray Ionization / Synechocystis / Tandem Mass Spectrometry
Yoko Ishino, Hitomi Okada, Masahiko Ikeuchi and Hisaaki Taniguchi : Mass spectrometry-based prokaryote gene annotation., Proteomics, Vol.7, No.22, 4053-4065, 2007.
(Summary)
MS combined with database searching has become the preferred method for identifying proteins present in cell or tissue samples. The technique enables us to execute large-scale proteome analyses of species whose genomes have already been sequenced. Searching mass spectrometric data against protein databases composed of annotated genes has been widely conducted. However, there are some issues with this technique; wrong annotations in protein databases cause deterioration in the accuracy of protein identification, and only proteins that have already been annotated can be identified. We propose a new framework that can detect correct ORFs by integrating an MS/MS proteomic data mapping and a knowledge-based system regarding the translation initiation sites. This technique can provide correction of predicted coding sequences, together with the possibility of identifying novel genes. We have developed a computational system; it should first conduct the probabilistic peptide-matching against all possible translational frames using MS/MS data, then search for discriminative DNA patterns around the detected peptides, and lastly integrate the facts using empirical knowledge stored in knowledge bases to obtain correct ORFs. We used photosynthetic bacteria Synechocystis sp. PCC6803 as a sample prokaryote, resulting in the finding of 14 N-terminus annotation errors and several new candidate genes.
Kazuko Fujiwara, Hosaka Harumi, Matsuda Makoto, Kazuko Okamura-Ikeda, Motokawa Yutaro, Suzuki Mamoru, Nakagawa Atsushi and Hisaaki Taniguchi : Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP, Journal of Molecular Biology, Vol.371, No.1, 222-234, 2007.
(Summary)
Lipoic acid is an essential cofactor of the alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. It is covalently attached to a specific lysine residue of the subunit of the complexes. The bovine lipoyltransferase (bLT) catalyzes the lipoic acid attachment reaction using lipoyl-AMP as a substrate, forming a lipoylated protein and AMP. To gain insights into the reaction mechanism at the atomic level, we have determined the crystal structure of bLT at 2.10 A resolution. Unexpectedly, the purified recombinant bLT contains endogenous lipoyl-AMP. The structure of bLT consists of N-terminal and C-terminal domains, and lipoyl-AMP is bound to the active site in the N-terminal domain, adopting a U-shaped conformation. The lipoyl moiety is buried in the hydrophobic pocket, forming van der Waals interactions, and the AMP moiety forms numerous hydrogen bonds with bLT in another tunnel-like cavity. These interactions work together to expose the C10 atom of lipoyl-AMP to the surface of the bLT molecule. The carbonyl oxygen atom of lipoyl-AMP interacts with the invariant Lys135. The interaction might stimulate the positive charge of the C10 atom of lipoyl-AMP, and consequently facilitate the nucleophilic attack by the lysine residue of the lipoate-acceptor protein, accompanying the bond cleavage between the carbonyl group and the phosphate group. We discuss the structural differences between bLT and the lipoate-protein ligase A from Escherichia coli and Thermoplasma acidophilum. We further demonstrate that bLT in mitochondria also contains endogenous lipoylmononucleotide, being ready for the lipoylation of apoproteins.
Toshinobu Nakamura, Yoshikazu Arai, Hiroki Umehara, Masaaki Masuhara, Tohru Kimura, Hisaaki Taniguchi, Toshihiro Sekimoto, Masahito Ikawa, Yoshihiro Yoneda, Masaru Okabe, Satoshi Tanaka, Kunio Shiota and Toru Nakano : PGC7/Stella protects against DNA demethylation in early embryogenesis., Nature Cell Biology, Vol.9, No.1, 64-71, 2007.
(Summary)
DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.
(Keyword)
Animals / COS Cells / Cell Line / Cell Nucleus / Cercopithecus aethiops / DNA Methylation / Embryonic Development / Epigenesis, Genetic / Female / Fertilization / Gene Expression Regulation, Developmental / Humans / Male / Mice / Proteins / Transfection
S Ohashi, G Sakashita, R Ban, M Nagasawa, H Matsuzaki, Y Murata, Hisaaki Taniguchi, H Shima, K Furukawa and T Urano : Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies., Oncogene, Vol.25, No.59, 7691-7702, 2006.
(Summary)
Mammalian Aurora-A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae Ipl1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and define its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase(s) of Aurora-A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora-A forms complexes with the negative regulator protein serine/threonine phosphatase type 1 (PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho-specific Aurora-A monoclonal antibodies restrain Aurora-A kinase activity in vitro, providing further therapeutic avenues to explore.
Toru Tobe, Scott A. Beatson, Hisaaki Taniguchi, Hiroyuki Abe, Christopher M. Bailey, Amanda Fivian, Rasha Younis, Sophie Matthews, Olivier Marches, Gad Frankel, Tetsuya Hayashi and Mark J. Pallen : An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination., Proceedings of the National Academy of Sciences of the United States of America, Vol.103, No.40, 14941-14946, 2006.
(Summary)
Several pathogenic strains of Escherichia coli exploit type III secretion to inject "effector proteins" into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage "metagenome," acting as a crucible for the evolution of pathogenicity.
Hiroaki Konishi, Kyoko Tashiro, Yasunobu Murata, Hiromi Nabeshi, Emiko Yamauchi and Hisaaki Taniguchi : CFBP is a novel tyrosine-phosphorylated protein that might function as a regulator of CIN85/CD2AP., The Journal of Biological Chemistry, Vol.281, No.39, 28919-28931, 2006.
(Summary)
To decipher the global network of the epidermal growth factor (EGF) receptor-mediated signaling pathway, a large scale proteomic analysis of tyrosine-phosphorylated proteins was conducted. Here, we focus on characterizing a novel protein, CFBP (CIN85/CD2AP family binding protein), identified in the study. CFBP was found to be phosphorylated at tyrosine 204 upon EGF stimulation, and the CIN85/CD2AP family was identified as a binding partner. A proline-rich motif of CFBP is recognized by one of the three Src-homology 3 domains of CIN85/CD2AP, and the affinity of the interaction is regulated by the tyrosine phosphorylation of CFBP. They co-localize in actinenriched structures, and overexpression of CFBP induced morphological changes with actin reorganization. Furthermore, CFBP accelerated the EGF receptor's down-regulation by facilitating the recruitment of Cbl to the CD2AP/CIN85 complex. Two spliced variants of CFBP lacking either exon 5 or 8 are also expressed, and the variant lacking exon 5 without the proline-rich motif lacks the ability to bind to the CIN85/CD2AP family. The CFBP protein seems to play a key role in the ligand-mediated internalization and down-regulation of the EGF receptor.
(Keyword)
Adaptor Proteins, Signal Transducing / Amino Acid Sequence / Animals / Base Sequence / COS Cells / Carrier Proteins / Cell Line, Tumor / Cercopithecus aethiops / Cytoskeletal Proteins / HeLa Cells / Humans / Molecular Sequence Data / Phosphoproteins / Phosphorylation / Receptor, Epidermal Growth Factor / Tyrosine
Kazuki Maezawa, Shuji Shigenobu, Hisaaki Taniguchi, Takeo Kubo, Shin-ich Aizawa and Mizue Morioka : Hundreds of flagellar basal bodies cover the cell surface of the endosymbiotic bacterium Buchnera aphidicola sp. strain APS., Journal of Bacteriology, Vol.188, No.18, 6539-6543, 2006.
(Summary)
Buchnera aphidicola is the endosymbiotic bacterium of the pea aphid. Due to its small genome size, Buchnera lacks many essential genes for autogenous life but obtains nutrients from the host. Although the Buchnera cell is nonmotile, it retains clusters of flagellar genes that lack the late genes necessary for motility, including the flagellin gene. In this study, we show that the flagellar genes are actually transcribed and translated and that the Buchnera cell surface is covered with hundreds of hook-basal-body (HBB) complexes. The abundance of HBB complexes suggests a role other than motility. We discuss the possibility that the HBB complex may serve as a protein transporter not only for the flagellar proteins but also for other proteins to maintain the symbiotic system.
Kyoko Tashiro, Hiroaki Konishi, Etsuko Sano, Hiromi Nabeshi, Emiko Yamauchi and Hisaaki Taniguchi : Suppression of the ligand-mediated downregulation of epidermal growth factor receptor by Ymer, a novel tyrosine phosphorylated and ubiquitinated protein., The Journal of Biological Chemistry, Vol.281, No.34, 24612-24622, 2006.
(Summary)
The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.
(Keyword)
Cell Line / Down-Regulation / Humans / Intracellular Signaling Peptides and Proteins / Ligands / Phosphorylation / Proteins / Receptor, Epidermal Growth Factor / Signal Transduction / Tyrosine / Ubiquitin
Fumiaki Imamura, Hiroshi Nagao, Hiromi Naritsuka, Yasunobu Murata, Hisaaki Taniguchi and Kensaku Mori : A leucine-rich repeat membrane protein, 5T4, is expressed by a subtype of granule cells with dendritic arbors in specific strata of the mouse olfactory bulb., The Journal of Comparative Neurology, Vol.495, No.6, 754-768, 2006.
(Summary)
Segregation of neuron-type-specific synaptic connections in different strata is a characteristic feature shared by the olfactory bulb (OB) and retina. In the mammalian OB, mitral cells form dendrodendritic synapses with granule cells (GCs) in the deep stratum of the external plexiform layer (EPL), whereas tufted cells form dendrodendritic synapses in the superficial stratum. In the search for membrane proteins with strata-specific expression patterns, we found that a leucine-rich repeat membrane protein (5T4 oncofetal trophoblast glycoprotein) was expressed selectively by a subset of superficial GCs. The somata of 5T4-positive GCs were localized in or near the mitral cell layer, and their apical dendrites ramified preferentially in the superficial stratum of the EPL, where tufted cell dendrites ramified. Strata-specific expression of 5T4 was found also in the retina: 5T4 was expressed selectively by rod-bipolar cells and a subset of amacrine cells whose dendrites ramified in a specific sublamina of the inner plexiform layer. During the perinatal and postnatal development of the OB, 5T4 expression paralleled in time the formation of dendrodendritic synapses in the EPL. Odor deprivation during the first postnatal month selectively reduced the thickness of the superficial stratum of the EPL and the number of 5T4-positive GCs. Because 5T4 is known to interact with actin cytoskeleton, these observations suggest that 5T4 is involved in the formation or maintenance of strata-specific dendritic ramification or synaptic connection of subsets of local interneurons.
Kazuko Fujiwara, Sachiko Toma, Kazuko Okamura-Ikeda, Yutaro Motokawa, Atsushi Nakagawa and Hisaaki Taniguchi : Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli: determination of the lipoic acid-binding site., The Journal of Biological Chemistry, Vol.280, No.39, 33645-33651, 2006.
(Summary)
Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.
Takayuki Kawakami, Yujin Hoshida, Fumihiko Kanai, Yasuo Tanaka, Keisuke Tateishi, Tsuneo Ikenoue, Shuntaro Obi, Shinpei Sato, Takuma Teratani, Shuichiro Shiina, Takao Kawabe, Takamasa Suzuki, Naoya Hatano, Hisaaki Taniguchi and Masao Omata : Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment., Proteomics, Vol.5, No.16, 4287-4295, 2005.
(Summary)
Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.
M Watanabe, K Nomura, A Ohyama, R Ishikawa, Y Komiya, K Hosaka, Emiko Yamauchi, Hisaaki Taniguchi, N Sasakawa, K Kumakura, T Ushiki, O Sato, M Ikebe and M. Igarashi : Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A., Molecular Biology of the Cell, Vol.16, No.10, 4519-4530, 2005.
(Summary)
Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.
(Keyword)
Amino Acid Sequence / Animals / Brain / Calcium / Cells, Cultured / Chromaffin Cells / Exocytosis / Microscopy, Atomic Force / Molecular Sequence Data / Myosin Heavy Chains / Myosin Type V / Protein Binding / Rats / Synaptic Vesicles / Syntaxin 1
Kazuko Okamura-Ikeda, Harumi Hosaka, Masato Yoshimura, Eiki Yamashita, Sachiko Toma, Atsushi Nakagawa, Kazuko Fujiwara, Yutaro Motokawa and Hisaaki Taniguchi : Crystal Structure of Human T-protein of Glycine Cleavage System at 2.0 Å Resolution and its Implication for Understanding Non-ketotic Hyperglycinemia, Journal of Molecular Biology, Vol.351, No.5, 1146-1159, 2005.
(Summary)
T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.
(Keyword)
Amino Acid Sequence / Aminomethyltransferase / Animals / Arginine / Asparagine / Binding Sites / Catalytic Domain / Cluster Analysis / Crystallography, X-Ray / DNA Mutational Analysis / Formaldehyde / Glycine / Humans / Hydrogen Bonding / Hydroxymethyl and Formyl Transferases / Hyperglycinemia, Nonketotic / Kinetics / Models, Chemical / Models, Molecular / Molecular Conformation / Molecular Sequence Data / Mutation / Protein Binding / Protein Conformation / Protein Structure, Secondary / Protein Structure, Tertiary / Sequence Homology, Amino Acid
Shintaro Ozeki, Jinglei Cheng, Kumi Tauchi-Sato, Naoya Hatano, Hisaaki Taniguchi and Toyoshi Fujimoto : Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane., Journal of Cell Science, Vol.118, No.Pt 12, 2601-2611, 2005.
(Summary)
Lipid droplets (LDs) are organelles that store neutral lipids, but their regulatory mechanism is not well understood. In the present study, we identified Rab18 as an LD component of HepG2 cells by proteomic analysis, and confirmed its localization by immunohistochemistry and western blotting. Wild-type and dominant-active Rab18 localized to LDs but the dominant-negative form did not. Endogenous Rab18 coexisted with adipocyte differentiation-related protein (ADRP) in LDs, but the labeling intensity of the two proteins showed clear reciprocity. Consistent with this observation, overexpression of Rab18 induced a decrease in the amounts of ADRP in LDs in HepG2 and BALB/c 3T3 cells. Furthermore, Rab18 overexpression caused close apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.
Yasunobu Murata, Tomoko Doi, Hisaaki Taniguchi and Yoshinori Fujiyoshi : Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor., Biochemical and Biophysical Research Communications, Vol.327, No.1, 183-191, 2005.
(Summary)
Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.
Xiangyu Li, Gyosuke Sakashita, Hideki Matsuzaki, Kenji Sugimoto, Keiji Kimura, Fumio Hanaoka, Hisaaki Taniguchi, Koichi Furukawa and Takeshi Urano : Direct association with inner centromere protein (INCENP) activates the novel chromosomal passenger protein, Aurora-C, The Journal of Biological Chemistry, Vol.279, No.45, 47201-47211, 2004.
(Summary)
A family of serine/threonine kinase Aurora constitutes a key regulator in the orchestration of mitotic events. The human Aurora paralogues Aurora-A, Aurora-B, and Aurora-C have a highly conserved catalytic domain. Extensive studies on the role of Aurora-A and Aurora-B have revealed distinct localizations and functions in regulating mitotic processes, whereas little is known about Aurora-C. The present study shows that human Aurora-C is a chromosomal passenger protein that forms complexes with Aurora-B and inner centromere protein (INCENP), which are known passenger proteins. We show that INCENP binds and activates Aurora-C in vivo and in vitro. Furthermore, Aurora-C co-expressed with INCENP elicits the phosphorylation of endogenous histone H3 in mammalian cells, even though this phosphorylation is not sufficient to establish chromosome condensation in interphase cells. We therefore suggest that Aurora-C is a novel chromosomal passenger protein that cooperates with Aurora-B to regulate mitotic chromosome dynamics in mammalian cells.
(Keyword)
Animals / Antibodies, Monoclonal / COS Cells / Catalytic Domain / Cell Cycle / Cell Line / Centromere / Chromatin / Chromosomal Proteins, Non-Histone / Chromosomes / Fluorescent Antibody Technique, Indirect / Gene Expression Regulation / HeLa Cells / Histones / Humans / Immunoprecipitation / Interphase / Microscopy, Confocal / Mitosis / Models, Chemical / Molecular Sequence Data / Phosphorylation / Plasmids / Protein Binding / Protein Biosynthesis / Protein Structure, Tertiary / Protein-Serine-Threonine Kinases / Subcellular Fractions / Time Factors / Transfection
Mamoru Matsubara, Toru Nakatsu, Hiroaki Kato and Hisaaki Taniguchi : Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca2+/calmodulin., The EMBO Journal, Vol.23, No.4, 712-718, 2004.
(Summary)
A variety of viral and signal transduction proteins are known to be myristoylated. Although the role of myristoylation in protein-lipid interaction is well established, the involvement of myristoylation in protein-protein interactions is less well understood. CAP-23/NAP-22 is a brain-specific protein kinase C substrate protein that is involved in axon regeneration. Although the protein lacks any canonical calmodulin (CaM)-binding domain, it binds CaM with high affinity. The binding of CAP-23/NAP-22 to CaM is myristoylation dependent and the N-terminal myristoyl group is directly involved in the protein-protein interaction. Here we show the crystal structure of Ca2+-CaM bound to a myristoylated peptide corresponding to the N-terminal domain of CAP-23/NAP-22. The myristoyl moiety of the peptide goes through a hydrophobic tunnel created by the hydrophobic pockets in the N- and C-terminal domains of CaM. In addition to the myristoyl group, several amino-acid residues in the peptide are important for CaM binding. This is a novel mode of binding and is very different from the mechanism of binding in other CaM-target complexes.
Miki Kikuchi, Naoya Hatano, Sadaki Yokota, Nobuyuki Shimozawa, Tsuneo Imanaka and Hisaaki Taniguchi : Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease., The Journal of Biological Chemistry, Vol.279, No.1, 421-429, 2004.
(Summary)
Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperone-like activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis.
Mamoru Matsubara, Koichi Titani, Hisaaki Taniguchi and Nobuhiro Hayashi : Direct involvement of protein myristoylation in myristoylated alanine-rich C kinase substrate (MARCKS)-calmodulin interaction., The Journal of Biological Chemistry, Vol.278, No.49, 48898-488902, 2003.
(Summary)
MARCKS, a major in vivo substrate of protein kinase C, interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner. Although we have previously observed that myristoylated and non-myristoylated MARCKS proteins behave differently during calmodulin-agarose chromatography, the role of protein myristoylation in the MARCKS-calmodulin interaction remained to be elucidated. Here we demonstrate that the myristoyl moiety together with the N-terminal protein domain is directly involved in the MARCKS-calmodulin interaction. Both myristoylated and non-myristoylated recombinant MARCKS bound to calmodulin-agarose at low ionic strengths, but only the former retained the affinity at high ionic strengths. A quantitative analysis obtained with dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-calmodulin showed that myristoylated MARCKS has an affinity higher than the non-myristoylated protein. Furthermore, a synthetic peptide based on the N-terminal sequence was found to bind calmodulin only when it was myristoylated. Only the N-terminal peptide but not the canonical calmodulin-binding domain showed the ionic strength-independent calmodulin binding. A mutation study suggested that the importance of the positive charge in the N-terminal protein domain in the binding.
Emiko Yamauchi, Toru Nakatsu, Mamoru Matsubra, Hiroaki Kato and Hisaaki Taniguchi : Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca(2+)-calmodulin., Nature Structural Biology, Vol.10, No.3, 226-231, 2003.
(Summary)
The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.
(Keyword)
Amino Acid Sequence / Binding Sites / Calcium / Calmodulin / Crystallography, X-Ray / Hydrophobic and Hydrophilic Interactions / Intracellular Signaling Peptides and Proteins / Macromolecular Substances / Membrane Proteins / Models, Molecular / Molecular Sequence Data / Peptides / Phosphoproteins / Protein Conformation / Protein Structure, Tertiary
T Yamamoto, Emiko Yamauchi, Hisaaki Taniguchi, H Matsuzaki and Ushio Kikkawa : Tyrosine phosphorylation of protein kinase C., Methods in Molecular Biology, Vol.233, 207-216, 2003.
(Keyword)
Amino Acid Sequence / Animals / COS Cells / Isoenzymes / Mass Spectrometry / Phosphorylation / Protein Kinase C / Tyrosine
Hideyuki Yamamoto, Emiko Yamauchi, Hisaaki Taniguchi, Tsuneyuki Ono and Eishichi Miyamoto : Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites., Archives of Biochemistry and Biophysics, Vol.408, No.2, 255-262, 2002.
Akihiro Ohyama, Kohei Hosaka, Yoshiaki komiya, Kimio Akagawa, Emiko Yamauchi, Hisaaki Taniguchi, Nobuyuki Sasagawa, Konosuke Kumakura, Sumiko Mochida, Takashi Yamauchi and Michihiro Igarashi : Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A., The Journal of Neuroscience, Vol.22, No.9, 3342-3351, 2002.
(Summary)
Syntaxin 1A/HPC-1 is a key component of the exocytotic molecular machinery, namely, the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor mechanism. Although >10 syntaxin-binding proteins have been identified, they cannot completely explain the regulation of exocytosis. Thus, novel proteins may interact with syntaxin. Because exocytosis requires both Ca2+ and ATP, we searched for Ca2+/ATP-dependent syntaxin-binding proteins from the rat brain and discovered Ca2+/calmodulin-activated protein kinase II (CaMKII)-alpha. At Ca2+ concentrations of >10(-6) m, only autophosphorylated CaMKII bound to syntaxin. Bound CaMKII was released from syntaxin by EGTA or by phosphatase, indicating that the binding is reversible. CaMKII bound to the linker domain of syntaxin, unlike any other known syntaxin-binding proteins. CaMKII-syntaxin complexes were also detected in synaptosomes by immunoprecipitation, and when reconstituted in vitro, they recruited larger amounts of synaptotagmin and SNAP-25 than syntaxin alone. The microinjected CaMKII-binding domain of syntaxin specifically affected exocytosis in chromaffin cells and in neurons. These results indicate that the Ca2+/ATP-dependent binding of CaMKII to syntaxin is an important process in the regulation of exocytosis.
Hiroaki Konishi, Emiko Yamauchi, Hisaaki Taniguchi, Toshiyoshi Yamamoto, Hidenori Matsuzaki, Yukitoshi Takemura, Kyoko Ohmae, Ushio Kikkawa and Yasutomi Nishizuka : Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro., Proceedings of the National Academy of Sciences of the United States of America, Vol.98, No.12, 6587-6592, 2001.
(Summary)
Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H(2)O(2), the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H(2)O(2)-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H(2)O(2).
(Keyword)
Amino Acid Sequence / Animals / COS Cells / Enzyme Activation / Hydrogen Peroxide / Immunoblotting / Isoenzymes / Molecular Sequence Data / Phosphorylation / Protein Kinase C / Protein Kinase C-delta / Protein-Tyrosine Kinases / Tyrosine
Ken-Ichi Sano, Kayo Maeda, Hisaaki Taniguchi and Yoichiro Maeda : Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule., European Journal of Biochemistry, Vol.267, No.15, 4870-4877, 2000.
(Summary)
Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.
(Keyword)
Actins / Adenosine Triphosphatases / Amino Acid Sequence / Amino Acids / Animals / Chymotrypsin / Circular Dichroism / DNA, Complementary / Dose-Response Relationship, Drug / Kinetics / Mass Spectrometry / Molecular Sequence Data / Muscle Fibers, Fast-Twitch / Muscle Fibers, Slow-Twitch / Mutagenesis / Myosins / Nephropidae / Potassium Chloride / Protein Binding / Protein Conformation / Protein Isoforms / Sequence Homology, Amino Acid / Temperature / Time Factors / Tropomyosin / Trypsin
Miki Morimatsu, Akio Nakamura, Hiroki Sumiyoshi, Nana Sakaba, Hisaaki Taniguchi, Kazuhiro Kohama and Sugie Higashi-Fujime : The molecular structure of the fastest myosin from green algae, Chara., Biochemical and Biophysical Research Communications, Vol.270, No.1, 147-152, 2000.
(Summary)
Chara myosin in green algae, Chara corallina, is the fastest myosin of all those observed so far. To shed light on the molecular mechanism of this fast sliding, we determined the primary structure of Chara myosin heavy chain (hc). It has a motor domain, six IQ motifs for calmodulin binding, a coiled-coil structure to dimerize, and a globular tail. Chara myosin hc is very similar to some plant myosins and has been predicted to belong to the class XI. Short loop 1 and loop 2 may account for the characteristics of mechanochemical properties of Chara myosin.
(Keyword)
Amino Acid Sequence / Cell Compartmentation / Chlorophyta / Cytoplasmic Streaming / Fluorescent Antibody Technique / Gene Library / Molecular Motor Proteins / Molecular Sequence Data / Myosin Heavy Chains / Peptide Fragments / Protein Structure, Tertiary / Sequence Analysis, DNA / Sequence Analysis, Protein / Sequence Homology, Amino Acid / Time Factors
Hisaaki Taniguchi : Protein myristoylation in protein-lipid and protein-protein interactions., Biophysical Chemistry, Vol.82, No.2-3, 129-137, 1999.
59.
Stephane Manenti, Hisaaki Taniguchi, Odile Sorokine, Emiko Yamauchi, Alain Dorsselaer Van, Hisaaki Taniguchi and Bernard Ducommun : Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro., The Biochemical Journal, Vol.340, 775-782, 1999.
60.
Akihiko Takasaki, Nobuhiro Hayashi, Mamoru Matsubara, Emiko Yamauchi and Hisaaki Taniguchi : Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction., The Journal of Biological Chemistry, Vol.274, No.17, 11848-11853, 1999.
61.
Hatsuho Uedaira, Hisayuki Morii, Miyuki Ishimura, Hisaaki Taniguchi and Keiichi Namba : Domain organization of flagellar hook protein from Salmonella typhimurium., FEBS Letters, Vol.445, No.1, 126-130, 1999.
62.
Ferenc Vonderviszt, Katsumi Imada, Yukio Furukawa, Hatsuho Uedaira and Hisaaki Taniguchi : Mechanism of self-association and filament capping by flagellar HAP2., Journal of Molecular Biology, Vol.284, No.5, 1399-1416, 1998.
63.
Emiko Yamauchi, Reiko Kiyonami, Michiko Kanai and Hisaaki Taniguchi : Presence of conserved domains in the C-terminus of MARCKS, a major in vivo substrate of protein kinase C: application of ion trap mass spectrometry to the elucidation of protein structures., The Journal of Biochemistry, Vol.123, No.4, 760-765, 1998.
64.
Hisaaki Taniguchi and Nobuhiro Hayashi : A liquid chromatography/electrospray mass spectrometric study on the post-transcriptional modification of tRNA., Nucleic Acids Research, Vol.26, No.6, 1481-1486, 1998.
(Summary)
Liquid chromatography/electrospray mass spectrometry is one of the rapidly developing techniques with which mass of large hydrophilic polymers such as proteins and nucleic acids can be determined precisely. The technique was applied to studies on the modifications of tRNAs. Various tRNA species purified from Escherichia coli were directly injected into a capillary reversed-phase column and the desalted and concentrated tRNAs were analyzed on-line with an electrospray mass spectrometer. In some cases, small but significant differences were noted between the theoretical and observed molecular masses, suggesting that there exist still unknown modifications. Under high resolution measurements, multiple peaks corresponding to species modified to a varying extent were resolved. To study the structures in detail, the isolated tRNA species were digested with ribonuclease T1, and the resulting mixture of fragments were analyzed by the same liquid chromatography/mass spectrometry. In this way, most of the fragments were easily identified solely from their masses, and the positions where the expected and real structures differ were revealed. The results obtained showed the presence of micro-heterogeneity among tRNAs and demonstrated at the same time the power of the hyphenated technique for the structural analysis on nucleic acids.
Emiko Yamauchi, Reiko Kiyonami, Michiko Kanai and Hisaaki Taniguchi : The C-terminal conserved domain of MARCKS is phosphorylated in vivo by proline-directed protein kinase. Application of ion trap mass spectrometry to the determination of protein phosphorylation sites., The Journal of Biological Chemistry, Vol.273, No.8, 4367-4371, 1998.
66.
Mykles L Donald, Julie L.S Cotton, Hisaaki Taniguchi, Ken-ichi Sano and Yuichiro Maeda : Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript., Journal of Muscle Research and Cell Motility, Vol.19, No.2, 105-115, 1998.
67.
Mamoru Matsubara, Emiko Yamauchi, Nobuhiro Hayashi and Hisaaki Taniguchi : MARCKS, a major protein kinase C substrate, assumes non-helical conformations both in solution and in complex with Ca2+-calmodulin., FEBS Letters, Vol.421, No.3, 203-207, 1998.
68.
Mamoru Matsubara, Nobuhiro Hayashi, Koiti Titani and Hisaaki Taniguchi : Circular dichroism and 1H NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. Nascent alpha-helical structures are stabilized by calmodulin both in the presence and absence of Ca2+., The Journal of Biological Chemistry, Vol.272, No.37, 23050-23056, 1997.
69.
Emiko Yamauchi, Koiti Tintani and Hisaaki Taniguchi : Specific binding of acidic phospholipids to microtubule-associated protein MAP1B regulates its interaction with tubulin., The Journal of Biological Chemistry, Vol.272, No.36, 22948-22953, 1997.
70.
Soichi Takeda, Tomoyoshi Kobayashi, Hisaaki Taniguchi, Hiroshi Hayashi and Yuichiro Maeda : Structural and functional domains of the troponin complex revealed by limited digestion., European Journal of Biochemistry, Vol.246, No.3, 611-617, 1997.
(Summary)
Troponin (Tn), consisting of three subunits, TnT, TnC, and TnI, plays a crucial role in the calcium-dependent regulation of vertebrate striated muscle contraction. In the present study, we have applied limited proteolysis to the Tn complex in order to study domain structures and to detect conformational differences of Tn under different conditions. We found that both TnT and TnI were susceptible to chymotryptic digestion: while TnT was cleaved into TnT-(1-158)-peptide and TnT-(159-259)-peptide irrespective of Ca2+ concentration, the cleavage sites of TnI were dependent on the Ca2+ occupancy of TnC. In addition, we characterized the effects of depletion of the C-terminal part of TnI on acto-S1 ATPase activity. The TnT-(159-259)-peptide-TnC-TnICa-frag complex [TnICa-frag = (TnI-(1-134 and 1-140)-peptide], which was produced in the presence of CaCl2 and MgCl2, retains both the activating and inhibitory capabilities of whole Tn on the acto-S1 ATPase activity, while TnT-(159-259)-peptide-TnC-TnIMg-frag complex [TnIMg-frag = (TnI-(1-116)-peptide], which was obtained in the presence of MgCl2 and EGTA, lost its ability to activate acto-S1 ATPase activity. Our results indicate that residues 117-134 or 117-140 of TnI undergo structural changes upon Ca(2+)-binding to the regulatory sites of TnC and are necessary for the Ca(2+)-dependent inhibitory action of the Tn complex on acto-S1 ATPase activity. We also showed that residues 135-181 or 141-181 of TnI are involved in the interaction of Tn with the tropomyosin-actin filament.
Stephane Manenti, Hisaaki Taniguchi, Odile Sorokine, Alain Dorsselaer Van and Jean-Marie Darbon : Specific proteolytic cleavage of the myristoylated alanine-rich C kinase substrate between Asn 147 and Glu 148 also occurs in brain., Journal of Neuroscience Research, Vol.48, No.3, 259-263, 1997.
72.
Yumiko Saijo, Soichi Takeda, Andrea Sherer, Tomoyoshi Kobayashi, Yuichiro Maeda, Hisaaki Taniguchi, Min Yao and Soichi Wakatsuki : Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I., Protein Science, Vol.6, No.4, 916-918, 1997.
73.
Nobuhiro Hayashi, Mamoru Matsubara, Koiti Titani and Hisaaki Taniguchi : Circular dichroism and 1H nuclear magnetic resonance studies on the solution and membrane structures of GAP-43 calmodulin-binding domain., The Journal of Biological Chemistry, Vol.272, No.12, 7639-7645, 1997.
74.
Mamoru Matsubara, Masashi Kusubata, Koichi Ishiguro, Tsuneko Uchida, Koiti Titani and Hisaaki Taniguchi : Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions., The Journal of Biological Chemistry, Vol.271, No.35, 21108-21113, 1996.
75.
Hisaaki Taniguchi, masami Suzuki, Stephane Manenti and Koiti Titani : A mass spectrometric study on the in vivo posttranslational modification of GAP-43., The Journal of Biological Chemistry, Vol.269, No.36, 22481-22484, 1994.
76.
Hisaaki Taniguchi, Stephane Manenti, Masami Suzuki and Koiti Titani : Myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, is an in vivo substrate of proline-directed protein kinase(s). A mass spectroscopic analysis of the post-translational modifications., The Journal of Biological Chemistry, Vol.269, No.28, 18299-18302, 1994.
77.
Stephane Manenti, Odile Sorokine, Alain Dorsselaer Van and Hisaaki Taniguchi : Demyristoylation of the major substrate of protein kinase C (MARCKS) by the cytoplasmic fraction of brain synaptosomes., The Journal of Biological Chemistry, Vol.269, No.11, 8309-8313, 1994.
78.
Hisaaki Taniguchi and Stephane Manenti : Interaction of myristoylated alanine-rich protein kinase C substrate (MARCKS) with membrane phospholipids., The Journal of Biological Chemistry, Vol.268, No.14, 9960-9963, 1993.
79.
Stephane Manenti, Odile Sorokine, Alain Dorsselaer Van and Hisaaki Taniguchi : Isolation of the non-myristoylated form of a major substrate of protein kinase C (MARCKS) from bovine brain., The Journal of Biological Chemistry, Vol.268, No.10, 6878-6881, 1993.
80.
Walter Pyerin and Hisaaki Taniguchi : Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450., The EMBO Journal, Vol.8, No.10, 3003-3010, 1989.
81.
Hisaaki Taniguchi, Yoshio Imai and Ryo Sato : Protein-protein and lipid-protein interactions in a reconstituted cytochrome P-450 dependent microsomal monooxygenase., Biochemistry, Vol.26, No.22, 7084-7090, 1987.
82.
Hisaaki Taniguchi, Yoshio Imai, Takashi Iyanagi and Ryo Sato : Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane of phosphatidylcholine vesicles., Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.550, No.2, 341-356, 1979.
(Summary)
Cytochrome P-450 and NADPH-cytochrome P-450 REDUctase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphoaditylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80--90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the sysTEM IN A SIMILAR WAY TO THE FAST-PHASE REDUCTION OF CYTOCHROME P-450, though the latter is not the rate-limiting step of the overall reaction.
Nobuo Maita, Hisaaki Taniguchi and Hitoshi Sakuraba : Crystallization, X-ray diffraction analysis and SIRAS phasing of human α-L-iduronidase, Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, Vol.68, No.11, 1363-1366, 2012.
(Summary)
Human lysosomal α-L-iduronidase, whose deficiency causes mucopolysaccharidosis type I, was crystallized using sodium/potassium tartrate and polyethylene glycol 3350 as a precipitant. Using synchrotron radiation, a native data set was collected from a single crystal at 100 K to 2.3 Å resolution. The crystal belonged to space group R3 with unit-cell dimensions of a = b = 259.22, c = 71.83 Å. To obtain the phase information, mercury-derivative crystals were prepared and a single-wavelength anomalous dispersion (SAD) data set was collected at the Hg peak wavelength. Phase calculation with the single isomorphous replacement with anomalous scattering (SIRAS) method successfully yielded an interpretable electron-density map.
Kyoko Tashiro, Hiroaki Konishi, Hiromi Nabeshi, Emiko Yamauchi and Hisaaki Taniguchi : [New functional proteins identified by proteomic analysis in the epidermal growth factor receptor-mediated signaling pathway and application for practical use]., Journal of the Pharmaceutical Society of Japan, Vol.130, No.4, 471-477, Apr. 2010.
(Summary)
To clarify the whole picture of epidermal growth factor (EGF) signaling pathway, we identified proteins from the EGF-stimulated A431 cells by anti-phospho-tyrosine antibody column chromatography. Over 150 proteins were detected including previously unidentified proteins as well as well-studied proteins. Among these proteins, we picked up four proteins that had not been known in EGF signaling pathway and analyzed their functions. We report the functions of these proteins in this article. 1) CFBP interacts with CD2AP family proteins and functions as a key component in downregulation of EGF receptor protein level following EGF stimulation. 2) Ymer is found to be phosphorylated and ubiquitinated upon EGF stimulation, and functions as a regulator for the downregulation and endocytosis of EGF receptor. 3) CLPABP binds to mitochondria-specific phospholipids, cardiolipin, through its PH domain, and its complex includes various proteins related to mRNA metabolism. 4) GAREM is associated with Grb2 and Shp2. Each association affects the ERK activity. Finally, we discuss the possibilities that these proteins can be used as a novel biomarker protein for cancer and other diseases.
(Keyword)
Adaptor Proteins, Signal Transducing / Biological Markers / Carrier Proteins / chromatography / Down-Regulation / Drug Design / Endocytosis / GRB2 Adaptor Protein / Humans / Intracellular Signaling Peptides and Proteins / Phosphoproteins / proteomics / Receptor, Epidermal Growth Factor / signal transduction
Yuichiro Goda, Nori Sato, Takako Taniguchi, Yoichiro Takata, Hirofumi Kosaka, Toshinori Sakai, Kousaku Higashino, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Proteomic analysis of ligamentum flavum, 52nd International Spinal Cord Society (ISCoS), Oct. 2013.
2.
Yuichiro Goda, Nori Sato, Takako Taniguchi, Yoichiro Takata, Hirofumi Kosaka, Toshinori Sakai, Kousaku Higashino, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Proteomic analysis of ligamentum flavum from lumbar spinal canal stenosis, International Society for Study of the Lumbar Spine (ISSLS) 2013, May 2013.
3.
Yuichiro Goda, Nori Sato, Takako Taniguchi, Yoichiro Takata, Hirofumi Kosaka, Toshinori Sakai, Kousaku Higashino, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Proteomic Analysis of Ligamentum Flavum from Lumbar Spinal Canal Stenosis., 2013 Annual meeting of the Orthopaedic Research Society, January 26-29, 2013 (Poster Presentation ), San Antonio, TX, Jan. 2013.
4.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Hisaaki Taniguchi and Naoko Imamoto : Multisite phosphorylation of FG nucleoporins by MAP kinases is involved in the regulation of nucleocytoplasmic transport, Cold Spring Harbor Laboratory Meeting "Dynamic Organization of Nuclear Function", Cold Spring Harbor, USA, Sep. 2012.
5.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Multisite phosphorylation of nucleoporins by ERK and p38 MAP kinases is implicated in the regulation of nuclear transport, Gordon Research Conference "Phosphorylation & G-Protein Mediated Signaling Networks", Biddeford, USA, Jun. 2012.
6.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Phosphorylation of FG-repeat nucleoporins by MAP kinases is implicated in the control of nuclear transport, EMBO Conference "Cellular Signaling & Molecular Medicine", Dubrovnik, Croatia, May 2012.
7.
Nori Sato, Takako Taniguchi, Yuichiro Goda, Hirofumi Kosaka, Kousaku Higashino, Toshinori Sakai, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Quantitative proteomic analysis of human tendon and ligament, 2012 Annual Meeting of the Orthopaedic Research Society, Feb. 2012.
8.
Hidetaka Kosako, Naoki Tani, Shigehiro Yoshimura, Shingo Kose, Megumi Kawano, Hisaaki Taniguchi and Naoko Imamoto : Phosphorylation of FG nucleoporins by ERK and p38 MAP kinases is involved in the regulation of nucleocytoplasmic transport, 2011 American Society for Cell Biology Annual Meeting, Denver, USA, Dec. 2011.
9.
Masanori Kaido, Kazutomo Abe, Takako Taniguchi, Hisaaki Taniguchi, Kazuyuki Mise and Tetsuro Okuno : Chloroplastic Glyceraldehyde 3-Phophate Dehydrogenase of Nicotiana Benthamiana Plays a Positive Role in Cell-to-Cell Movement of Red Clover Necrotic Mosaic Virus., International Congress of Virology 2011, Sapporo, Sep. 2011.
10.
Hiro-Oki Iwakawa, Yuri Tajima, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Hisaaki Taniguchi and Tetsuro Okuno : Poly(A)-Binding Protein Stimulates Cap-Independent Translation of Uncapped/Nonpolyadenylated Viral RNA via Binding to the 3' Untranslated Region., International Congress of Virology 2011, Sapporo, Sep. 2011.
11.
Akira Mine, Takako Taniguchi, Masanori Kaido, Kazuyuki Mise, Hisaaki Taniguchi and Tetsuro Okuno : Host Heat Shock Protein 70 Regulates Proper Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus., International Congress of Virology 2011, Sapporo, Sep. 2011.
12.
Kazuko Okamura-Ikeda, Hosaka Harumi, Nobuo Maita, Kazuko Fujiwara, Yoshizawa C Akiyasu, Nakagawa Atsushi and Hisaaki Taniguchi : Crystal structure of T-H protein complex of the glycine cleavage system, Acta Crystallographica Section A, Vol.67, No.supplement, C434-C435, Madrid, Aug. 2011.
13.
Nobuo Maita, Hisaaki Taniguchi and Sakuraba Hitoshi : Crystal structure of human α-L-iduronidase, Acta Crystallographica Section A, Vol.67, No.supplement, C300, Madrid, Aug. 2011.
14.
Nori Sato, Takako Taniguchi, Yuichiro Goda, Hirofumi Kosaka, Kosaku Higashino, Toshinori Sakai, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Establishment of a Method for Proteomic Analysis of Human Achilles Tendon., 2011 Annual meeting of the Orthopaedic Research Society, Long Beach, California, Long Beach, California, Jan. 2011.
15.
Nori Sato, Taniguchi Takako, Goda Yuichiro, Kosaka Hirofumi, Higashino Kosaku, Toshinori Sakai, Koichi Sairyo, Shinsuke Katoh, Hisaaki Taniguchi and Natsuo Yasui : Development of a Method for Proteomic Analysis of Human Yellow Ligament., 2011 Annual meeting of the Orthopaedic Research Society, Long Beach, California, Long Beach, California, Jan. 2011.
16.
Toshinori Endo, Keisuke Ueno, Kouki Yonezawa, Katsuhiko Mineta, Kohji Hotta, Yutaka Satou, Lixy Yamada, Michio Ogasawara, Hiroki Takahashi, Ayako Nakajima, Mia Nakachi, Mamoru Nomura, Junko Yaguchi, Alu Konnno, Yoshinori Sasakura, Akiyasu Yoshizawa, Hisaaki Taniguchi, Chisato Yamasaki, Miho Sera, Tadashi Imanishi and Kazuo Inaba : CIPRO 2.5: Ciona intestinalis Protein Database, a unique integrated repository of large-scale omics data, bioinformatic analyses, and curated annotation, with ability for user rating and comments., 1st Tunicate Information System Meeting, Niece, Nov. 2010.
17.
Toshinori Endo, Keisuke Ueno, Kouki Yonezawa, Katsuhiko Mineta, Kohji Hotta, Yutaka Satou, Lixy Yamada, Michio Ogasawara, Hiroki Takahashi, Ayako Nakajima, Mia Nakachi, Mamoru Nomura, Junko Yaguchi, Alu Konnno, Yoshinori Sasakura, Akiyasu Yoshizawa, Hisaaki Taniguchi, Chisato Yamasaki, Miho Sera, Tadashi Imanishi and Kazuo Inaba : CIPRO 2.5: Ciona intestinalis Protein Database, a unique integrated repository of large-scale omics data, bioinformatic analyses, and curated annotation, with ability for user rating and comments., Beyond the Genome, Boston, Oct. 2010.
18.
Toshinori Endo, Keisuke Ueno, Kouki Yonezawa, Katsuhiko Mineta, Kohji Hotta, Yutaka Satou, Lixy Yamada, Michio Ogasawara, Hiroki Takahashi, Ayako Nakajima, Mia Nakachi, Mamoru Nomura, Junko Yaguchi, Alu Konnno, Yoshinori Sasakura, Akiyasu Yoshizawa, Hisaaki Taniguchi, Chisato Yamasaki, Miho Sera, Tadashi Imanishi and Kazuo Inaba : CIPRO 2.5: Ciona intestinalis Protein integrated database with large-scale omics data, bioinformatic analyses and curated annotation, with ability for user rating and comments, Biocuration 2010, Tokyo, Oct. 2010.
19.
Toshinori Endo, Keisuke Ueno, Kouki Yonezawa, Katsuhiko Mineta, Kohji Hotta, Yutaka Satou, Lixy Yamada, Michio Ogasawara, Hiroki Takahashi, Ayako Nakajima, Mia Nakachi, Mamoru Nomura, Junko Yaguchi, Alu Konnno, Yoshinori Sasakura, Akiyasu Yoshizawa, Hisaaki Taniguchi, Tadashi Imanishi and Kazuo Inaba : CIONA INTESTINALIS PROTEIN DATABASE CIPRO SHOWS A VARIETY OF PROTEOMES FOR A SINGLE SPECIES, Computational Biology Research Center Workshop 2010, Tokyo, Jul. 2010.
20.
Hisaaki Taniguchi : Annotation of prokaryote genomes using large-scale proteomic data., 10th RECOMB Satellite Meeting on Computational Proteomics,, San Diego, Mar. 2010.
21.
Takako Taniguchi and Hisaaki Taniguchi : ProteoFiT: a bundle of VBA macros for parsing, filtering and analyzing Mascot database search results for large-scale proteomics., 10th RECOMB Satellite Meeting on Computational Proteomics,, San Diego, Mar. 2010.
22.
A Yoshizawa, L Yamada and Hisaaki Taniguchi : Western Blot-like presentation of gel-enhanced LC/MS data and software-based detection of protein modifications., 10th RECOMB Satellite Meeting on Computational Proteomics,, San Diego, Mar. 2010.
23.
Hidetaka Kosako, Chizuru Aranami, Nozomi Yamaguchi, Hitomi Suzuki, Shingo Kose, Naoko Imamoto, Hisaaki Taniguchi, Eisuke Nishida and Seisuke Hattori : Phosphorylation of nuclear pore complex proteins by ERK MAP kinase regulates interaction with transport receptors, 49th Annual Meeting of American Society for Cell Biology, San Diego, USA, Dec. 2009.
24.
t Saito, L Yamada, Hisaaki Taniguchi, Y Harada and H Sawada : dentification of the ascidian egg-coat proteins involved in gamete interaction and self-incompatibility., Gordon conference on Fertilization & Activation Of Development, Holderness, NH, USA, Jul. 2009.
25.
T Saito, L Yamada, Hisaaki Taniguchi, H Sawada and Y Harada : Proteomic analysis of egg-coat proteins in Ciona intestinalis: Identification of allorecognition proteins, v-Themis-A and -BT, The 5th International Tunicate Meeting, Naha, Jun. 2009.
26.
A Yoshizawa, L Yamada and Hisaaki Taniguchi : Ascidian Adult Body Map: A proteomic view of adult ascidian tissues., The 5th International Tunicate Meeting, Naha, Jun. 2009.
27.
L Yamada and Hisaaki Taniguchi : Global protein profiling of ascidian C. intestinalis: toward the comprehensive understanding at the protein level,, The 5th International Tunicate Meeting, Naha, Jun. 2009.
28.
Yuko Okuda, Takako Tniguchi, Yukiko Unemi, Akiko Sukeno, Masamichi Kuwajima and Hisaaki Taniguchi : Secretome Analysis of Human Adipocytes Obtained from Visceral and Subcutaneous Adipose Tissues, HUPO 6th Annual World Congress, Seoul, Oct. 2007.
29.
Takako Miyamoto, Shinsuke Kido, Masahiro Abe, Toshio Matsumoto and Hisaaki Taniguchi : Expression Profiling by Quantitative and Large-scale Proteomics in Differentiating Osteoblasts, HUPO 5th Annual World Congress, Los Angeles, Nov. 2006.
30.
Rie Nakata, Shizue Omi, Kazuko Okamura-Ikeda and Hisaaki Taniguchi : Purification and Characterization of Human Peroxisomal Lon Protease Expressed in E. coli, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jul. 2006.
31.
Shizue Omi, Rie Nakata, Kazuko Okamura-Ikeda and Hisaaki Taniguchi : Effects of Overexpression of Human Peroxisomal Lon Protease on the Peroxisomal Organization, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jul. 2006.
32.
Takako Miyamoto, Shinsuke Kido, Toshio Matsumoto and Hisaaki Taniguchi : Proteomic Analysis of Osteoblast Differentiation, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jul. 2006.
33.
Kyoko Tashiro, Hiroaki Konishi, Emiko Yamauchi, Hiromi Nabeshi and Hisaaki Taniguchi : A novel tyrosine phosphorylated protein, Ymer, has the inhibitory effect on downregulation of EGF receptor, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jul. 2006.
34.
Hiroaki Konishi, Kyoko Tashiro, Yasunobu Murata, Hiromi Nabeshi, Emiko Yamauchi and Hisaaki Taniguchi : CFBP is a novel tyrosine phsphorylated protein which functions as aregulator of CIN85/CD2AP, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jul. 2006.
35.
Takako Miyamoto and Hisaaki Taniguchi : Analysis of Extracellular Matrix Proteins Expressed during Osteoblast Differential Expression Profiling, Conference on Mass Spectrometry, Seattle, May 2006.
36.
Hisaaki Taniguchi, M Kikuchi, Rie Nakata, Shizue Omi and Kazuko Okamura-Ikeda : Proteomic Analysis of Rat Liver Peroxisome:Immunoisolation and Functional Analysis, Molecular & Cellular Proteomics, Vol.4, No.8, S489, San Francisco, Aug. 2005.
37.
Hisaaki Taniguchi, Emiko Yamauchi and Y Murata : Toward the global analysis of cellular signaling pathways, Uehara Memorial Foundation Symposium, Tokyo, Jun. 2002.
38.
Hisaaki Taniguchi, Y Murata and Emiko Yamauchi : Global Analysis of Protein Phosphorylation in Signaling Complex, Sixth International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 2001.
39.
Hisaaki Taniguchi : Proteomic Analysis of Posttranslational Modifications, International Proteome Proteomics Conference, Kisarazu, Aug. 1999.
40.
Hisaaki Taniguchi : Mass Spectrometry of Proteins and Nucleic Acids, Electrophoresis'99, Ohmiya, May 1999.
Yoshiyuki Kamiya, Takuji Nakashima, Takako Taniguchi, Masato Iwatsuki, Yoko Takahashi, Hisaaki Taniguchi and Satoshi Omura : Mass spectrometric analysis of mangromicins and detection of new analogs, 日本農芸化学会大会2014, Mar. 2014.
Takako Taniguchi and Hisaaki Taniguchi : 定量プロテオミクス解析への新たなアプローチ, 第54回日本生化学会中国·四国支部例会, May 2013.
8.
Seongpil Chung, Takako Taniguchi, Tani Naoki and Hisaaki Taniguchi : Dansyl-labeling reversed-phase liquid chromatography- and mass spectrometry-based metabolomic investigation of human urine sample, 第54回日本生化学会中国・四国支部例会, May 2013.
(Summary)
Metabolomics is the systematic study of endogenous small-molecule metabolites, the chemical entities that are transformed during metabolism, within a biological system. Analysis of these key metabolites is one of the powerful tools for the detection of biomarkers for diagnosis of diseases. The reversed-phase (RP) liquid chromatography (LC)/mass spectrometry (MS) is clearly the method of choice to analyze complex mixtures: the application of the method to proteomic analyses became the golden standard in the field. However, the presence of charged metabolites, especially, basic molecules containing amines in the metabolites makes it difficult to get good separation during the reversed-phase chromatography. In this study, we introduced a method using RPLC/MS. The dansyl chloride has been used in the N-terminal labeling of proteins and peptides, and its chemistry has been well studied. Dansylation of metabolites containing primary amine, secondary amine, or phenolic hydroxyl group, the presence of which deteriorate the chromatographic separation due to their interaction with the residual silanol group of the reversed-phase column material, not only improves the chromatographic separation but also increases the ionization efficiency during the electrospray ionization process. To increase the number of metabolite to be detected, the biological extracts were first separated using an ion-paring reversed-phase liquid chromatography with heptafluorobutyric acid as ion-paring reagent, and the individual fractions were subjected to the LC/MS analysis. This method was applied for acquiring metabolite profile from human urine samples. Analysis of the LC-MS data were performed by XCMS program (http://metlinscripps.edu/xcms), which automatically eliminated isotopic peaks, common adduct ions, multiply charged ions. Ion spectra differentially altered between sample groups were analyzed based on retention time and accurate mass matches. As the results, 416 ion spectra or metabolites were detected from nine ion-pair RPLC fractions. In addition, using a library of 220 amine- and phenol-containing metabolite standards, we were able to identify 58 metabolites containing amine and 28 amino acids. These studies can provide useful metabolomic information that has important implications for understanding the biology of disease state and identifying potential biomarkers.
9.
Nobuo Maita, 月村 孝宏, 大野 一樹, Hisaaki Taniguchi and 櫻庭 均 : Crystal Structure of Human -L-Iduronidase, 第35回日本分子生物学会年会, Dec. 2012.
10.
合田 有一郎, 谷口 貴子, Nori Sato, Natsuo Yasui and Hisaaki Taniguchi : 腰部脊柱管狭窄症における肥厚黄色靱帯のプロテオーム解析, 第37回日本医用マススペクトル学会年会, Oct. 2012.
Hisaaki Taniguchi : 質量分析を基盤とするプロテオミクスの医学・生物学研究への応用, 第56回質量分析総合討論会, May 2008.
24.
Hisaaki Taniguchi : Application of mass spectrometry-based proteomics to medical research, IGM-COE Symposium Uniderstanding Health and Disease Through Functional Genomics, Jan. 2008.
Molecular basis of multi-layered nonhost resistance against virus and a novel cultivar-specific virus resistance in plants (Project/Area Number: 16H04881 )
Analyzing glomerular renin angiotensin system with peptidomics for development of therapeutic and diagnostic methods inhibiting disease progression of chronic kidney disease (Project/Area Number: 26461614 )
Studies on a plant virus replication complexes as the transportation hub (Project/Area Number: 25450058 )
Proteomic analysis of extracellular matrix proteins (Project/Area Number: 23659727 )
The promotion of hepatic regeneration by lipid droplet's proteins operation (Project/Area Number: 23659094 )
Studies on the relevance of cell-to-cell movement and replication mechanisms of Dianthovirus (Project/Area Number: 22580047 )
Global analysis of post-translational modifications in super-molecular protein complexes by targeted proteomics (Project/Area Number: 22370042 )
Molecular mechanisms underlying viral strategies for circumvention and utilization of host defense systems (Project/Area Number: 22248002 )
Frontier of genome biology toward understanding molecular systems supporting cellular life (Project/Area Number: 12205001 )
PROTEOMIC ANALYSIS OF SYNAPTIC MOLECULAR STRUCTURES AND FUNCTIONAL REGULATION BY PROTEIN PHOSPHORYLATION (Project/Area Number: 11680768 )
Development of high-throughput mass-spec based sequencing and its application to global analysis of protein phosphorylation (Project/Area Number: 11558082 )