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)我们开发了一种二元载体系统，将合成的SUMO-1缀合途径引入大肠杆菌，并证明大量的SUMO化Ran GTPase激活蛋白1 C端区域（RanGAP1-C2）、Ran结合蛋白2内部重复结构域、 p53和早幼粒细胞白血病被有效产生。苏酰化的重组 RanGAP1-C2 似乎保留了体内特性，因为正如体内研究所预期的那样，它在赖氨酸 517 处被特异性苏酰化。我们的研究结果表明建立了一条生产大量苏酰化重组蛋白的生物合成途径，这将为研究 SUMO-1 修饰途径的生化和结构方面开辟新途径。（参见 FEBS Lett.564,85-90,2004和Anal.Biochem.331,204-206,2004)2)我们表明RanBP2的30-kDa催化片段很大程度上是一个非结构蛋白。尽管有两个不同但部分重叠的 79 个残基催化结构域（其中之一足以实现最大活性），RanBP2 仍以 1:1 的化学计量与 Ubc9 结合。对 RanBP2 依赖性 SUMOylation 很重要的 9 个 RanBP2 和 3 个 Ubc9 侧链的鉴定表明很大程度上是疏水相互作用。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 的特性而不是通过介导靶标相互作用来发挥其催化作用。（参见 Nat.Struct.Mol.Biol.11,984-989,2004）