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

1）我们开发了一个双元载体系统，将合成的SUMO-1缀合途径引入大肠杆菌，并证明了大量sumoylated Ran GT3激活蛋白1C-末端区域（RanGAP 1-C2）、Ran结合蛋白2内部重复结构域、p53和早幼粒细胞白血病的有效产生。类小泛素化的重组RanGAP 1-C2似乎保留了体内性质，因为它在赖氨酸517处特异性地被类小泛素化，正如体内研究所预期的那样。我们的研究结果表明，建立了一个生物合成路线，生产大量的sumoylated重组蛋白，这将开辟新的途径，研究生化和结构方面的SUMO-1修饰途径。(see FEBS Lett.564，85 - 90，2004和Anal.Biochem.331，204 - 206，2004）2）我们表明RanBP 2的30-kDa催化片段是一种大部分未结构化的蛋白质。尽管有两个不同但部分重叠的79个残基的催化结构域，其中之一足以达到最大活性，RanBP 2以1：1的化学计量与Ubc 9结合。对RanBP 2依赖性SUMO化重要的9个RanBP 2和3个Ubc 9侧链的鉴定表明主要是疏水相互作用。这些特性将RanBP 2与所有其他已知的E3连接酶区分开来，我们推测RanBP 2通过改变Ubc 9的特性而不是通过介导靶相互作用来发挥其催化作用。(see Nat.Struct.Mol.Biol.11，984 - 989，2004）

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)