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Proteomic analysis of ubiquitin modification

泛素修饰的蛋白质组学分析

基本信息

批准号：
16370047
负责人：
YOKOSAWA Hideyoshi
金额：
$ 8.64万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

1.We established a simple method for determination of the ubiquitin chain linkage by peptide mass fingerprinting using MALDI-TOF mass spectrometry : Polyubiquitin chains tagged to target proteins are subjected to trypsin digestion and the chain type-specific linkages are determined by MALDI-TOF mass spectrometry. 2.We reconstituted the ubiquitin fusion degradation (UFD) pathway using E1(Uba1), E2(Ubc4), E3(Ufd4), and E4(Ufd2) enzymes purified from yeast : UFD substrates are first ubiquitinated by actions of Uba1, Ubc4, and Ufd4 and elongation of the ubiquitin chains is then catalyzed by Ufd2. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by peptide mass fingerprinting revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage in the UFD pathway. 3.We found that Lsb2, an SH3 domain-containing protein, is polyubiquitinated. Direct determination of the ubiquitin chain linkage type in polyubiquitin chains tagged to Lsb2 by peptide mass fingerprinting revealed that Lsb2 is polyubiquitinated through Lys63 linkage. In addition, we found that this polyubiquitination is catalyzed by Rsp5, a HECT type ubiquitin ligase and that the Lys80 residue is the ubiquitination site in Lsb2. 3.We determined novel target proteins for modification with a ubiquitin-like protein ISG15 (an interferon-stimulated gene product with a molecular mass of 15 kDa) by a proteomnic analysis. We then studied the consequence of ISG15 modification of protein phosphatase 2Cβ, one of the target proteins identified, and found that ISG15 modification of protein phosphatase 2Cβ suppresses the activity of protein phosphatase 2Cβ against TAK1/TAB1-induced NF-κB activation. 4.With respect to the ubiquitin-dependent proteolytic pathway, we characterized subunits of the 26S proteasome, a proteolytic machine in this proteolytic pathway. In addition, we succeeded in isolating novel compounds inhibiting this proteolytic pathway.

1.建立了一种用MALDI-TOF质谱法测定泛素链连接的简便方法：将标记在目的蛋白上的多聚泛素链用胰酶消化，用MALDI-TOF质谱仪确定链类型特异性连接。2.我们利用从酵母中纯化的E1(Uba1)、E2(Ubc4)、E3(Ufd4)和E4(Ufd2)酶重组了泛素融合降解(UFD)途径：Ufd底物首先在Uba1、Ubc4和Ufd4的作用下泛素化，然后Ufd2催化泛素链的伸长。用肽质量指纹图谱直接测定多泛素化UFD底物中泛素链的连接类型，发现Ufd2通过UFD途径中的Lys48连接催化泛素链的延长。3.我们发现含有SH3结构域的Lsb2是一种多泛素化的蛋白质。用肽质量指纹图谱直接测定Lsb2标记的多泛素链中的泛素链类型，发现Lsb2是通过Lys63连接的多泛素化。此外，我们发现这种多泛素化是由Hect类型泛素连接酶Rsp5催化的，Lys80残基是Lsb2中的泛素化位点。3.通过蛋白质组学分析确定了泛素样蛋白ISG15(一种干扰素刺激的基因产物，相对分子质量为15 kDa)修饰的新靶蛋白。然后我们研究了ISG15修饰蛋白磷酸酶2Cβ的结果，发现ISG15修饰蛋白磷酸酶2Cβ抑制了蛋白磷酸酶2Cβ的活性，对抗Tak1/Tab1诱导的NF-κB激活。4.关于泛素依赖的蛋白分解途径，我们确定了26S蛋白酶体的亚基，它是该蛋白分解途径中的蛋白分解机制。此外，我们还成功地分离出了抑制这一蛋白分解途径的新化合物。

项目成果

期刊论文数量（30）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Rpn7 is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae

DOI：
10.1074/jbc.m314231200
发表时间：
2004-06-25
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Isono, E;Saeki, Y;Toh-e, A
通讯作者：
Toh-e, A

Himeic acid: A new ubiquitin-activating enzyme inhibitor isolated from a mrine-derived fungus, Aspergillus sp.

姫酸：一种从鼠源真菌曲霉属中分离出来的新型泛素激活酶抑制剂。

DOI：
发表时间：
2005
期刊：
Bioorganic and Medicinal Chemistry Letters 15
影响因子：
0
作者：
水口峰之;他;Masahiro Fujimuro;Masahiro Fujimuro;Yasushi Saeki;Yuhsuke Kikukawa;Tomoharu Takeuchi;Tomoharu Takeuchi;Masahiro Fujimuro;Masahiro Fujimuro;Yasushi Saeki;Yuhsuke Kikukawa;Tomoharu Takeuchi;Tomoharu Takeuchi;Sachiko Tsukamoto;Masahiro Fujimuro;Masahiro Fujimuro;Tomoharu Takeuchi;Tomoharu Takeuchi;Sachiko Tsukamoto
通讯作者：
Sachiko Tsukamoto

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae