The role of the SUMO pathway in regulation of the nuclear function.

SUMO 途径在核功能调节中的作用。

基本信息

批准号：
15510164
负责人：
SAITOH Hisato
金额：
$ 2.5万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

1)We developed a binary vector system that introduces a synthetic SUMO-1 conjugation pathway into Escherichia coli and demonstrated that large amounts of sumoylated Ran GTPase activating protein 1 C-terminal region(RanGAP1-C2), Ran binding protein 2 internal repeat domain, p53 and promyelocytic leukemia were efficiently produced. The sumoylated recombinant RanGAP1-C2 appeared to retain the in vivo properties, since it was specifically sumoylated at lysine 517 as expected from in vivo studies. Our findings indicate the establishment of a biosynthetic route for producing large amounts of sumoylated recombinant proteins that will open up new avenues for studying the biochemical and structural aspects of the SUMO-1 modification pathway.(see FEBS Lett.564,85-90,2004 and Anal.Biochem.331,204-206,2004)2)We show that RanBP2's 30-kDa catalytic fragment is a largely unstructured protein. Despite two distinct but partially overlapping 79-residue catalytic domains, one of which is sufficient for maximal activity, RanBP2 binds to Ubc9 in a 1:1 stoichiometry. The identification of nine RanBP2 and three Ubc9 side chains that are important for RanBP2-dependent SUMOylation indicates largely hydrophobic interactions. These properties distinguish RanBP2 from all other known E3 ligases, and we speculate that RanBP2 exerts its catalytic effect by altering Ubc9's properties rather than by mediating target interactions.(see Nat.Struct.Mol.Biol.11,984-989,2004)

1）我们开发了一个二进制矢量系统，该系统将合成的SUMO-1结合途径引入大肠杆菌中，并证明大量的sumoyated Ran ran ran gtpase激活蛋白1 c末端区域（rangap1-c2），ran ran ran ran ran ran结合蛋白2内部重复结构域，p53，p53和promyElocycytic prodignity wayeremia waysemia waysemia。 sumoy的重组rangap1-c2似乎保留了体内特性，因为它是在体内研究中所预期的，在赖氨酸517处得到了特异性sumoyly。我们的发现表明，建立了一种生物合成途径，用于生产大量的Sumoymet重组蛋白，该蛋白将为SUMO-1修改途径的生化和结构方面开放新的途径。 RANBP2的30 kDa催化片段是一种非结构化的蛋白质。尽管有两个不同但部分重叠的79个残留催化域，其中一个足以达到最大活性，但RANBP2在1：1的化学计量学中与UBC9结合。鉴定9个RANBP2和三个UBC9侧链对RANBP2依赖性Sumoylation很重要，这在很大程度上表明了疏水相互作用。这些特性将RANBP2与所有其他已知的E3连接酶区分开来，我们推测RANBP2通过改变UBC9的性质而不是通过介导目标相互作用来发挥其催化作用（请参阅Nat.Struct.Mol.BIOL.11,11,984-989,2004））