Structural Studies of SUMO Protein Modification

描述（由申请方提供）：小泛素样修饰物SUMO对细胞蛋白质的修饰对于酵母中的真核细胞核过程和细胞周期进程至关重要。与泛素缀合不同，泛素缀合可以靶向蛋白质以被蛋白质体降解，SUMO修饰靶向蛋白质以改变活性和定位。SUMO被翻译为前体蛋白，由蛋白酶加工产生成熟形式，然后通过C-末端腺苷酸化活化，并通过SUMO活化酶E1转移到分子内半胱氨酸，在SUMO和E1之间形成硫酯键。然后将E1-SUMO硫酯转移至E2缀合蛋白内的半胱氨酸残基，以在E2和SUMO之间形成硫酯键。虽然E2-SUMO复合物能够将SUMO转移到蛋白质靶标上的赖氨酸残基，但E3连接酶通过结合底物和E2-SUMO或通过激活E2-SUMO复合物进行缀合来促进该过程。SUMO修饰可以被蛋白酶逆转，所述蛋白酶释放底物和SUMO用于额外的缀合轮次。SUMO的既定功能正在增长，现在包括调节核质转运，核体代谢，转录，细胞定位，染色体分离，DNA修复和复制，以及调节细胞周期进程，因为酿酒酵母的SUMO基因，蛋白酶，E1和E2都是细胞周期通过G2-M所需的。综上所述，这些观察结果表明，SUMO结合在真核细胞核代谢和细胞周期控制的调控过程中起着核心作用，这些过程与人类健康，癌症和NIH的使命直接相关。该提案旨在解决SUMO缀合装置的组件的功能和结构意义。我们将利用结构、生物化学和遗传学技术来建立1）SUMO酶识别SUMO异构体的基础，2）E1、SUMO和E2之间的相互作用，3）SUMO E3和通过不同的SUMO结合结构域识别SUMO的因子催化的活性，4）RING型E3 SUMO连接酶的动力学和结构基础。除了有助于我们理解SUMO结合，SUMO途径的彻底结构-功能分析将产生对其他泛素和泛素样结合途径中使用的保守机制的见解。

DESCRIPTION (provided by applicant): Modification of cellular proteins by the Small Ubiquitin-like Modifier SUMO is essential for eukaryotic nuclear processes and cell cycle progression in yeast. Unlike ubiquitin conjugation, which can target proteins for degradation by the proteosome, SUMO modification targets proteins for changes in activity and localization. SUMO is translated as a precursor protein that is processed by proteases to generate a mature form which is then activated via C-terminal adenylation and transferred to an intramolecular cysteine by the SUMO activating enzyme E1 to form a thioester bond between SUMO and E1. The E1-SUMO thioester is then transferred to a cysteine residue within the E2 conjugating protein to form a thioester bond between E2 and SUMO. While the E2-SUMO complex is competent to transfer SUMO to lysine residues on the protein target, E3 ligases facilitate this process by binding substrates and E2-SUMO or by activating the E2-SUMO complex for conjugation. SUMO modification can be reversed by proteases that liberate both substrate and SUMO for additional rounds of conjugation. Established functions for SUMO are growing and now include regulating nucleocytoplasmic transport, nuclear body metabolism, transcription, cellular localization, chromosome segregation, DNA repair and replication, and regulation of cell cycle progression in so much as Saccharomyces cerevisiae genes for SUMO, a protease, E1, and E2 are all required for cell cycle progression through G2-M. Taken together, these observations suggest that SUMO conjugation plays a central role in regulatory processes involved in eukaryotic nuclear metabolism and cell cycle control, processes that are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional and structural significance for components of the SUMO conjugation apparatus. We will utilize structural, biochemical and genetic techniques to establish the basis for 1) SUMO isoform recognition by SUMO enzymes, 2) interactions between E1, SUMO, and E2 3) activities catalyzed by SUMO E3s and factors that recognize SUMO via distinct SUMO binding domains, 4) the kinetic and structural basis for RING-type E3 SUMO ligases. In addition to contributing to our understanding of SUMO conjugation, a thorough structure-function analysis of the SUMO pathway will yield insights into conserved mechanisms utilized in other ubiquitin and ubiquitin-like conjugation pathways.