Structural Studies of SUMO Protein Modification

描述（由申请人提供）：信号转导途径依赖于可逆的化学修饰，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，包括核转运、细胞质分裂、染色体分离、G2-M细胞周期进程和转录调节等。泛素（Ub）和泛素样（Ubl）蛋白如SUMO的翻译后修饰需要E1激活酶、E2偶联酶和E3连接酶的连续作用，而Ub/Ubl的加工和解偶联是由Ub/Ubl特异性蛋白酶催化的。由于SUMO偶联在真核生物核代谢和细胞周期控制中起着不可或缺的作用，我们的研究与人类健康、癌症和NIH的使命直接相关。该提案旨在通过结构、生化和遗传学研究来解决SUMO偶联途径组分的功能意义，这些研究将建立以下基础：1)SUMO和泛素激活；2)E2和E3酶的SUMO偶联；3)通过SUMO结合域的表征来调节SUMO途径，并通过研究确定SUMO表面在应对环境应激（如DNA损伤）中的重要性。4)研究SUMO修饰的PCNA及其被抗重组解旋酶Srs2识别的相关结构生物学。由于构成SUMO偶联途径的酶、机制和辅助因子是保守的，或者在概念上类似于其他Ub/Ubl途径，我们的研究将具有广泛的相关性，并将影响Ub和其他Ubl修饰剂的蛋白质偶联研究。

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. PUBLIC HEALTH RELEVANCE: Reversible post-translational modifications relay information within and across cells in signal transduction pathways that control the spatial and temporal distribution of protein substrates. Covalent modification of proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO (small ubiquitin-like modifier) and Nedd8 impact nearly all facets of cellular metabolism including cell cycle control, protein degradation, protein localization, nuclear transport, cytokinesis, chromosome segregation, and transcriptional regulation among many others. This proposal seeks to explore the mechanisms that underlie protein modification by SUMO through structural, biochemical and genetic studies that will address 1) Ub/Ubl activation by the E1 enzyme, 2) SUMO conjugation by E2 and E3 enzymes, 3) structural determinants required for recognition of SUMO by SUMO binding proteins, and 4) structural analysis of PCNA, its modification by SUMO, and its recognition by Srs2. The SUMO pathway regulates many pathways that are associated with human disease conditions including neurodegenerative disorders, cancer, and inflammation.