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

1)我们建立了一个二元载体系统，将合成的SUMO-1偶联途径引入大肠杆菌，并证明了大量sumo化的Ran GTPase激活蛋白1 c末端区域（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的有效产生。重组RanGAP1-C2似乎保留了体内特性，因为它在体内研究中被特异性地在赖氨酸517处进行了sumylated。我们的研究结果表明，建立了一种生物合成途径来生产大量的SUMO-1修饰蛋白，这将为研究SUMO-1修饰途径的生化和结构方面开辟新的途径。(见《生物工程学报》，2004年第4期，第85-90期；2)我们发现RanBP2的30 kda催化片段是一个很大程度上非结构化的蛋白。尽管有两个不同但部分重叠的79-残基催化结构域，其中一个足以达到最大活性，RanBP2以1:1的化学计量与Ubc9结合。9个RanBP2侧链和3个Ubc9侧链的鉴定对RanBP2依赖的SUMOylation很重要，表明主要是疏水相互作用。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。(见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)