Structural Studies of SUMO Protein Modification

描述(申请人提供)：信号转导通路依靠可逆的化学修饰在细胞内和细胞间传递信息。泛素样蛋白SUMO(小泛素样修饰物)对蛋白质底物的共价修饰有助于调节核心细胞功能的途径，包括核运输、胞质分裂、染色体分离、G2-M细胞周期进展和转录调节等。泛素(Ub)和类泛素(Ubl)蛋白的翻译后修饰需要E1激活酶、E2结合酶和E3连接酶的顺序作用，而Ub/Ub1的加工和去结合是由Ub/Ubl特异性的酶催化的。由于相扑结合在真核细胞的核代谢和细胞周期控制中起着不可或缺的作用，我们的研究直接关系到人类的健康、癌症和美国国立卫生研究院的使命。这项建议旨在通过结构、生化和遗传学研究来解决相扑结合途径组件的功能意义，这些研究将为1)相扑和泛素激活，2)E2和E3酶的相扑接合，3)通过相扑结合结构域的表征和通过研究确定相扑表面在应对环境胁迫(如DNA损伤)中的重要性，3)相扑通路的调节，4)相扑修饰的增殖细胞核抗原相关的结构生物学及其被反重组解旋酶2识别的基础。由于构成相扑结合途径的酶、机制和辅助因子与其他Ub/Ub1途径中的酶、机制和辅助因子保守或在概念上相似，因此我们的研究将具有广泛的相关性，并将影响Ub和其他Ubl修饰剂对蛋白质结合的研究。

DESCRIPTION (provided by applicant): Signal transduction pathways rely on reversible chemical modifications to relay information within and across cells. Covalent modification of protein substrates by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) contributes to pathways that regulate core cellular functions including nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcriptional regulation among many others. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Because SUMO conjugation plays an integral role in eukaryotic nuclear metabolism and cell cycle control, our studies are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional significance for components of the SUMO conjugation pathway through structural, biochemical and genetic studies that will establish the basis for 1) SUMO and ubiquitin activation, 2) SUMO conjugation by E2 and E3 enzymes, 3) regulation of SUMO pathway through characterization of SUMO-binding domains and through studies that will determine the importance of SUMO surfaces in response to environmental stress such as DNA damage, 4) address the structural biology associated with SUMO modified PCNA and its recognition by the anti- recombinogenic helicase Srs2. Because the enzymes, mechanisms and co-factors that constitute the SUMO conjugation pathway are conserved or conceptually analogous to those in other Ub/Ubl pathways, our studies will be broadly relevant and will impact research on protein conjugation by Ub and other Ubl modifiers.