| Project/Area Number |
10680615
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
TSUJI Akihiko The University of Tokushima, Faculty of Engineering, Associate Professor, 工学部, 助教授 (20155360)
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| Co-Investigator(Kenkyū-buntansha) |
KURODA Rieko The University of Tokushima, Faculty of Engineering, Assistant Professor, 工学部(平成11年2月着任のため分担者として長浜氏と入れ替え), 助手 (30314850)
NAGAHAMA Masami The University of Tokushima, Faculty of Engineering, Assistant Professor, 工学部(平成10年10月東京薬科大へ転出), 助手 (60281169)
MATSUDA Yoshiko The University of Tokushima, Faculty of Engineering, Professor, 工学部, 教授 (40035449)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | protease / subtilisin-like tease / serine tease / tide hormone / alubumin / SPC4 / PACE4 / processing / ササチライシン様プロテアーゼ / セリンプロテアーゼ / ペプチドホルモン |
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| Research Abstract |
PACE4 (SPC4) is a member of the mammalian subtilisin-like proprotein convertase (SPC) family which participate in maturation of precursor proteins. In this project, we have analyzed the mechanisms for activation and transcriptional regulation of PACE4 (SPC4), effects of its antisense RNA expression on the processing of plasma protein and its enzymatic properties in order to clarify the function of PACE4 in vivo.
1. In transfected fibroblast cells, the 110-kDa proSPC4 undergoes slow cleavage to generate a 103-kDa matule enzyme in the endoplasmic reticulum (ER). Site-directed mutagenesis studiesdemonstrate that the proteolytic activation of SPC4 occurs mainly through a unimolecular autocatalytic process and the propeptide cleavage is prerequisite to its export from the ER.
2. The expression of PACE4 antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing in HepG2 cells like furin and PC8, indicating that PACE4 and PC7/8, as well as furin, are involved in
… More
the processing of proalbumin in HepG2 cells.
3. The 5'- flanking region of PACE4 gene contains twelve E-boxes (E1 to E12) within 1kb upstream of the transcription initiation site. Site-directed mutagenesis of E-boxes in those regulatory elements showed that the E10 E-box act as positive regulator whereas an E-box cluster (E4-E9) acts as a negative regulator in both cells. E2 E-box acts as positive regnlatoronly in HepG2 cells.
4. We characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific assay. α1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in the conditioned medium by PACE4 overexpression. PACE4 is a CaィイD12+ィエD1-dependent protease having an optimal CaィイD12+ィエD1 requirement of 2 mM, and highest activity at weakly basic pH. PACE4 activity can be inhibited by αl-PDX as wellas furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by α1-PDX causes abnormal embryogenic development. Less
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