STRUCTURAL BIOLOGY OF HUMAN UBIQUITIN E3 LIGASES AND INTERACTING FACTORS

人类泛素 E3 连接酶和相互作用因子的结构生物学

• 批准号: 8169329
• 负责人: Alexei Bochkarev
• 金额: $ 0.7万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169329
• 关键词: Active Sites; Amino Acids; C-terminal; C2 Domain; Catalysis; Catalytic Domain; Cell Cycle Progression; Complex; Computer Retrieval of Information on Scientific Projects Database; Cysteine; DNA Repair; Data; Disease; Endocytosis; Family; Family Dasypodidae; Funding; Grant; Human; Human Ubiquitin; Institution; Ligase; N-terminal; Process; Proteins; Reaction; Regulation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Signal Transduction; Source; Specificity; Structure; Substrate Specificity; Sulfhydryl Compounds; System; Techniques; Tertiary Protein Structure; Ubiquitin; United States National Institutes of Health; protein degradation; structural biology; structural genomics; ubiquitin-protein ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ubiquitylation regulates a variety of processes, including DNA repair, signal transduction, cell cycle progression, endocytosis and protein degradation, amongst others, and has been implicated in disease. The substrate specificity of the ubiquitylation system is carried out by the E3 protein ligases, which belong to two major families: the RING E3 ligases and the HECT E3 ligases. The HECT E3 ligases each contain an approximately 350 amino acid C-terminal catalytic domain with a conserved active site cysteine that forms a thiol-intermediate with ubiquitin, transferring it from an E2 ligase to the protein substrate. The highly variable N-terminal regions of the HECT E3s interact with specific protein substrates while the catalytic domain interacts with its cognate E2 and carries out the catalytic reaction. A variety of domains (WW domain, C2 domain, armadillo like-repeats, BH3, UBA, amongst others) have been observed in the N-terminus of the HECT domain proteins and these have been used to further classify these proteins. Unlike the HECT E3 ligases, the RING E3 ligases do not directly carry out the transfer of ubiquitin to substrate, but instead bring together the activated E2 and its substrate thus allowing catalysis to occur. We propose to determine the high-resolution structures of human E3 ligases and their complexes with cognate E2s, substrates and regulating factors in order to further elucidate their catalytic mechanisms, specificity and regulation. As part of the Structural Genomics Consortium, we have the capability to use high-throughput techniques to express and purify proteins, crystallize and characterize these crystals. However we require the brilliant X-rays available at the APS in order to collect data from weakly diffracting crystals and to collect anomalous data from heavy-atom derivatives.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 泛素化调节多种过程，包括 DNA 修复、信号转导、细胞周期进程、内吞作用和蛋白质降解等，并且与疾病有关。泛素化系统的底物特异性由 E3 蛋白连接酶实现，E3 蛋白连接酶属于两个主要家族：RING E3 连接酶和 HECT E3 连接酶。每个 HECT E3 连接酶均包含约 350 个氨基酸的 C 端催化结构域，具有保守的活性位点半胱氨酸，可与泛素形成硫醇中间体，将其从 E2 连接酶转移到蛋白质底物。 HECT E3 的高度可变的 N 端区域与特定蛋白质底物相互作用，而催化结构域与其同源 E2 相互作用并进行催化反应。在 HECT 结构域蛋白的 N 末端观察到多种结构域（WW 结构域、C2 结构域、犰狳样重复序列、BH3、UBA 等），这些结构域已用于进一步对这些蛋白进行分类。与 HECT E3 连接酶不同，RING E3 连接酶不直接将泛素转移到底物上，而是将活化的 E2 及其底物结合在一起，从而发生催化作用。 我们建议确定人类 E3 连接酶及其与同源 E2、底物和调节因子的复合物的高分辨率结构，以进一步阐明其催化机制、特异性和调节。作为结构基因组学联盟的一部分，我们有能力使用高通量技术来表达和纯化蛋白质、结晶和表征这些晶体。然而，我们需要 APS 提供的明亮 X 射线，以便从弱衍射晶体收集数据，并从重原子衍生物收集异常数据。