出版者・発行元：Cold Spring Harbor Laboratory  

<title>Abstract</title>Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

DOI： 10.1101/2021.12.13.472378

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.14889/jpdm.2021.0002

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.14889/jpdm.2020.0002

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

N6-Methyladenosine (m6A) modification is the major chemical modification in mRNA that controls fundamental biological processes, including cell proliferation. Herein, we demonstrate that fat mass and obesity-associated (FTO) demethylates m6A modification of cyclin D1, the key regulator for G1 phase progression and controls cell proliferation in vitro and in vivo. FTO depletion upregulates cyclin D1 m6A modification, which in turn accelerates the degradation of cyclin D1 mRNA, leading to the impairment of G1 progression. m6A modification of cyclin D1 oscillates in a cell-cycle-dependent manner; m6A levels are suppressed during the G1 phase and enhanced during other phases. Low m6A levels during G1 are associated with the nuclear translocation of FTO from the cytosol. Furthermore, nucleocytoplasmic shuttling of FTO is regulated by casein kinase II-mediated phosphorylation of FTO. Our results highlight the role of m6A in regulating cyclin D1 mRNA stability and add another layer of complexity to cell-cycle regulation.

DOI： 10.1016/j.celrep.2020.03.028

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担当区分：筆頭著者   記述言語：英語  

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumorigenesis or the curatives have not been established. Using unique trascriptome and proteome integration method, iPEACH (1), we previously identified translationally controlled tumor protein (TCTP) as a novel biological target for NF1-associated tumors (2). Here, we identified specific TCTP-interacting proteins by sequential affinity purification and data-independent mass spectrometry acquisition (AP-DIA/SWATH) to investigate the role of TCTP in NF1-associated malignant tumors. TCTP mainly interacts with proteins related to protein synthesis and especially to elongation factor complex components, including EF1A2, EF1B, EF1D, EF1G, and valyl-tRNA synthetase (VARS), in NF1-deficient malignant tumor cells. Interestingly, TCTP preferentially binds to EF1A2 (normally found only in neural and skeletal-muscle cells and several cancer cells), rather than EF1A1 despite the high homologies (98%) in their sequences. The docking simulation and further validations to study the interaction between TCTP and EF1A2 revealed that TCTP directly binds with EF1A2 via the contact areas of EF1A2 dimerization. Using unique and common sequences between EF1A2 and EF1A1 in AP-DIA/SWATH, we quantitatively validated the interaction of EF1A2 and TCTP/other elongation factors and found that TCTP coordinates the translational machinery of elongation factors via the association with EF1A2. These data suggest that TCTP activates EF1A2-dependent translation by mediating complex formation with other elongation factors. Inhibiting the TCTP-EF1A2 interaction with EF1A2 siRNAs or a TCTP inhibitor, artesunate, significantly down-regulated the factors related to protein translation and caused dramatic suppression of growth/translation in NF1-associated tumors. Our findings demonstrate that a specific protein translation machinery related to the TCTP-EF1A2 interaction is functionally implicated in the tumorigenesis and progression of NF1-associated tumors and could represent a therapeutic target.

DOI： 10.1074/mcp.RA118.001014

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1093/nar/gky899

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1007/s12032-018-1229-x

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その他リンク： http://link.springer.com/content/pdf/10.1007/s12032-018-1229-x.pdf

記述言語：英語   掲載種別：研究論文（学術雑誌）  

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.

DOI： 10.1038/s41467-018-05187-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Recently, we reported that bacterial incorporation induces cellular transdifferentiation of human fibroblasts. However, the bacterium-intrinsic cellular-transdifferentiation factor remained unknown. Here, we found that cellular transdifferentiation is caused by ribosomes. Ribosomes, isolated from both prokaryotic and eukaryotic cells, induce the formation of embryoid body-like cell clusters. Numerous ribosomes are incorporated into both the cytoplasm and nucleus through trypsin-activated endocytosis, which leads to cell-cluster formation. Although ribosome-induced cell clusters (RICs) express several stemness markers and differentiate into derivatives of all three germ layers in heterogeneous cell populations, RICs fail to proliferate, alter the methylation states of pluripotent genes, or contribute to teratoma or chimera formation. However, RICs express markers of epithelial-mesenchymal transition without altering the cell cycle, despite their proliferation obstruction. These findings demonstrate that incorporation of ribosomes into host cells induces cell transdifferentiation and alters cellular plasticity.

DOI： 10.1038/s41598-018-20057-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：C M B ASSOC  

Chemotherapy for cholangiocarcinoma (CCA) is not quite successful. In this study, we revisited the possibility of artesunate (ART) and chloroquine (CQ), the antimalarial drugs, as therapeutic agents against CCA. The possible mechanisms of these drugs to exert cytotoxicity on CCA cells were also explored. The effects of ART and CQ on proliferation and death patterns of two CCA cell lines, KKU-214 and its highly metastatic subtype KKU-214L5, were examined using water soluble tetrazolium (WST) assay and time-lapse photometry, respectively. To differentiate and verify the death patterns between necrosis and apoptosis, lactate dehydrogenase (LDH) release, and caspase 3 activity were measured. CellROX (TM) green reagent staining method was used to assess reactive oxygen species (ROS) production in ART-and CQ-treated cells. ART and CQ significantly inhibited proliferation of CCA cells. Both drugs kill malarial parasites via similar mechanism depending on ROS formation, however, ART induced necrotic cell death and CQ induced apoptotic cell death in CCA cells. ART induced LDH release, whereas CQ activated caspase 3, confirming induction of necrotic and apoptotic cell deaths by ART and CQ, respectively. ART treatment induced higher ROS production than CQ. ART and CQ induce CCA cells death via different death pathways. ART should be suitable for necrosis-sensitive CCA, whereas CQ is more suitable for apoptosis-sensitive CCA.

DOI： 10.14715/cmb/2018.64.10.18

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press  

Major advancements have recently been made in mass spectrometry-based proteomics, yielding an increasing number of datasets from various proteomics projects worldwide. In order to facilitate the sharing and reuse of promising datasets, it is important to construct appropriate, high-quality public data repositories. jPOSTrepo (https://repository. jpostdb.org/) has successfully implemented several unique features, including high-speed file uploading, flexible file management and easy-to-use interfaces. This repository has been launched as a public repository containing various proteomic datasets and is available for researchers worldwide. In addition, our repository has joined the ProteomeXchange consortium, which includes the most popular public repositories such as PRIDE in Europe for MS/MS datasets and PASSEL for SRM datasets in the USA. Later MassIVE was introduced in the USA and accepted into the ProteomeXchange, as was our repository in July 2016, providing important datasets from Asia/Oceania. Accordingly, this repository thus contributes to a global alliance to share and store all datasets from a wide variety of proteomics experiments. Thus, the repository is expected to become a major repository, particularly for data collected in the Asia/Oceania region.

DOI： 10.1093/nar/gkw1080

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担当区分：筆頭著者,　責任著者   記述言語：日本語   出版者・発行元：日本プロテオーム学会  

<p>プロテオミクスデータに基づいて抽出した生物学的に意義のある新規候補分子群に対して，どのような生物学的役割を果たすのか検証することが最終的に求められる．siRNA/shRNAや低分子化合物などによる分子機能阻害，および発現プラスミドによる分子の過剰発現によって生じる細胞の表現型を，顕微鏡観察や各種アッセイ系によって評価していくことが，一般的な細胞生物学的検証法として挙げられる．しかしながら，細胞生物学的実験手法は候補分子によって様々であることから，実はプロテオーム解析よりも時間がかかるステップとなっている．したがって，プロテオミクスデータに即して細胞生物学的な検証を円滑に進めていくには，プロテオーム解析に用いたサンプルの調製と前処理，質量分析などのプロテオーム解析手法は勿論のこと，in-silicoのGene Ontology解析や分子ネットワーク解析によって新規分子群を抽出する過程が大変重要となってくる．本内容では神経系腫瘍の解析を通じて，我々がいかにしてプロテオミクスデータで同定された新規候補分子群を細胞生物学的に検証したのか報告する．</p>

DOI： 10.14889/jpros.1.1_37

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2015.05.102

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記述言語：日本語   出版者・発行元：医歯薬出版  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Due to the lack of information on the molecular mechanism of NF1-associated tumor pathogenesis or biomarkers/therapeutic targets, an effective treatment for NF1 tumors has not been established. In this study, the novel NF1-associated protein, translationally controlled tumor protein (TCTP), was identified by integrated proteomics and found to be up-regulated via activated MAPK/PI3K-AKT signaling in response to growth factors in NF1-deficient Schwann cells. Immunohistochemical analysis of NF1-associated tumors revealed that the TCTP expression level correlated with tumorigenicity. In NF1-deficient MPNST cells, TCTP protein but not mRNA was down-regulated by NF1 GTPase-activating protein-related domain or MAPK/PI3K inhibitors, and this correlated with suppression of mammalian target of rapamycin (mTOR) signaling. mTOR inhibition by rapamycin also down-regulated TCTP protein expression, whereas knockdown or overexpression of TCTP suppressed or activated mTOR signaling, respectively, and affected cell viability. These results suggest that a positive feedback loop between TCTP and mTOR contributes to NF1-associated tumor formation. Last, the anti-tumor effect of artesunate, which binds to and degrades TCTP, was evaluated. Artesunate significantly suppressed the viability of MPNST cells but not normal Schwann cells, and the TCTP level inversely correlated with artesunate sensitivity. Moreover, combinational use of artesunate and rapamycin enhanced the cytotoxic effect on MPNST cells. These findings suggest that TCTP is functionally implicated in the progression of NF1-associated tumors and could serve as a biological target for their therapy.

DOI： 10.1074/jbc.M114.568253

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担当区分：最終著者   記述言語：日本語   出版者・発行元：日本電気泳動学会  

DOI： 10.2198/sbk.58.39

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.6.0

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up-or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

DOI： 10.1074/mcp.M112.024802

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：5  

DOI： 10.1371/journal.pone.0059558

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up-and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin alpha, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied. Molecular & Cellular Proteomics 8: 2350-2367, 2009.

DOI： 10.1074/mcp.M900179-MCP200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

DOI： 10.1074/jbc.M708206200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TNIV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5'-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at - 32 and - 99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The beta-glucuronidase (GUS) reporter gene analysis with serial 5'-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues - 509 to - 288 in gene ngr1 and a TMV-responsive region at the residues - 401 to - 174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TNIV responsive element: GT1, and the WUN-motif at positions between - 401 and - 174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3.

DOI： 10.1271/bbb.67.2574

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) m and 1.75 x 10(-7) m), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) m and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.

DOI： 10.1271/bbb.65.2259

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記述言語：日本語   出版者・発行元：生命科学系学会合同年次大会運営事務局  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2016.0.77.0

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2015.0.187.0

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記述言語：日本語   出版者・発行元：日本臨床プロテオーム研究会  

DOI： 10.14905/jscp.2015.0_19

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2015.0.107.0

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.123.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.55.0

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記述言語：英語   出版者・発行元：日本癌学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2012.0.172.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2011.0.104.0

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER ASSOC CANCER RESEARCH  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2008.0.97.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

神経線維腫症１型(NF1)は、多発性神経線維腫を始め多彩な病態を示す遺伝性疾患である。NF1遺伝子産物NeurofibrominはRas-GAP相同領域を有し、その機能の欠損による、Rasを介した細胞内シグナル伝達異常は、特に神経系細胞分化異常を誘起し、NF1の病態に関わると考えられる。Neurofibrominの神経系細胞内機能を明らかにするために、RNA干渉(SiRNA)法を用いてNeurofibromin発現を抑制することによって、神経系細胞に及ぼすNGF依存性神経分化への影響を解析し、生じた表現型の細胞内責任シグナル分子群をiTRAQ法用いて詳細に検討した。PC12細胞において、NF1ノックダウン細胞はNGF刺激によるneurite outgrowthが経時的に阻害され、細胞骨格系の制御異常、運動能の亢進が観察された。最も形態変化の顕著なNGF刺激48時間後、それぞれの細胞から8M Urea、4%CHAPSを含む溶液でタンパク質を抽出し、アセトン沈殿にて脱塩濃縮後、トリプシン分解により得られたペプチドをiTRAQ(114：NGF_-_siRNA_-_, 115:NGF_-_siRNA＋, 116:NGF＋siRNA_-_, 117:NGF＋siRNA＋)試薬で修飾した。それぞれ等量混合した後、陽イオン交換クロマトにより分画し、これらをnanoLC-QqTOF解析に供し、iTRAQ修飾ペプチドの定量と同定を行った。現在までに約800種類以上のタンパク質を同定し、その内NGF刺激により特異的な発現差異を示す約60種のタンパク質を同定した。これらには既知のNGF誘導分子とともに新規の神経系分化関連分子が含まれていた。さらにNF1ノックダウンにより、NGF刺激PC12細胞内で特異的に上昇を示す19種のタンパク質と減少を示す19種のタンパク質を確認した。これらにはRasの下流に位置するGタンパク質Rho、プロスタグランジン、酸化還元酵素群、腫瘍関連分子群の上昇、また神経疾患の原因分子NMDAレセプターのチロシンキナーゼやそのレセプター下流の細胞骨格制御因子およびcalmodulin、刺激因子伝達分子群、特異的キナーゼ群などの減少が見られ、これらが総合的に細胞の分化異常に関連すると考えられた。現在、その他NF1腫瘍抑制遺伝子関連タンパク質の更なる同定を試み、その役割について検証中である。

DOI： 10.14889/jhupo.2006.0.47.0

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記述言語：日本語   出版者・発行元：日本プロテオーム学会（日本ヒトプロテオーム機構）  

神経系腫瘍抑制遺伝子産物（NF1蛋白；neurofibromin, NF2蛋白；merlin）の細胞内機能を、プロテオミクスの手法を用いて生化学的、細胞生物学的に解析している。今回、NF1高変異部位であるC末端部位、及びNF2高変異部位であるN末端部位に対する結合タンパク質群をiTRAQ法およびTAP法を用いたProteomic affinity cellular mappingにより網羅的かつ定量的に同定し、NF両蛋白質を介する細胞内シグナルネットワークを解析した。また、NF1／NF2 SiRNAを用いた細胞内NF1・NF2蛋白質のノックダウンを行い、これらの蛋白質の発現抑制による細胞内シグナルの変化を生化学・形態学的に検討するとともに、ノックダウン細胞内特異的変動蛋白質群をProteomic differential displayによって解析した。NF1蛋白質に特異的に結合する蛋白質群として、Neuron Regulators、リン酸化・脱リン酸化酵素群、アダプター分子群、細胞骨格系・細胞接着系制御分子群、蛋白質合成関連分子群など28種類の蛋白質が同定された。NF1結合蛋白質群中でneuronのaxon形成に関わるcollapsin response mediator protein 2(CRMP2), tumbulin, coffilinとのinteractionに注目したところ、これらの分子はPC12細胞のNGF刺激後、NF1蛋白質を介してGSK3B及びRho kinase、LIMKinaseによるリン酸化が相互制御されることによってneurite の形成と伸展に関わっていることがNF1ノックダウン細胞にて証明された。また、NF2蛋白質ノックダウン細胞においても、細胞運動能、細胞接着能、骨格系の異常が認められ、DNA修復分子群、アポトーシス関連分子群、細胞骨格系・細胞接着系制御分子群、Neuron Regulators、細胞周期関連分子群を含む約25種類のNF2結合蛋白質群が同定された。NF1蛋白質とNF2蛋白質に共通するNeuron Regulatorや細胞骨格制御因子などの結合蛋白質群の存在や、両分子のノックダウンによる形態変化と、それに伴う変動蛋白質分子群に共通性があることから、これら共通分子を介したシグナルの異常がNF1及びNF2に類似する病態に関連する可能性が考えられた。

DOI： 10.14889/jhupo.2006.0.45.0

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記述言語：日本語  

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