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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 泛素化调节了各种过程，包括DNA修复，信号转导，细胞周期进展，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是由E3蛋白连接酶进行的，该蛋白属于两个主要家族：环E3连接酶和Hect E3连接酶。 Hect E3连接酶每个都包含大约350个氨基酸C末端催化结构域，具有保守的活性位点半胱氨酸，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。高度可变的E3S的N末端区域与特定蛋白底物相互作用，而催化域与其同源E2相互作用并进行催化反应。已经在HECT结构蛋白的N末端观察到了多种域（WW结构域，C2结构域，类似重复，BH3，UBA等），并且已用于进一步对这些蛋白质进行分类。与HECT E3连接酶不同，环E3连接酶不会直接进行泛素转移至底物，而是将活化的E2及其底物汇总在一起，从而允许进行催化。 我们建议确定人E3连接酶的高分辨率结构及其与同源E2，底物和调节因素的复合物，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，结晶并表征这些晶体。但是，我们需要AP上的出色X射线，以便从弱衍射的晶体中收集数据并从重原子衍生物中收集异常数据。