Unconventional Interactions of Human Ubiquitin Conjugating Enzymes

人类泛素结合酶的非常规相互作用

• 批准号: 9276349
• 负责人: PETER S BRZOVIC
• 金额: $ 4.87万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-10 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276349
• 关键词: Active Sites; Address; Amaze; Amino Acids; Attention; Bacterial Infections; Binding; Biochemical; Biological Assay; Cells; Cellular biology; Collaborations; Communication; Complex; Critical Pathways; Crystallography; Defect; Disease; Environment; Enzymes; Escherichia coli; Eukaryotic Cell; Event; Foundations; Human; Human Development; Human Genome; In Vitro; Infection; Intervention; Investigation; Knowledge; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Mining; Modification; Molecular; Monitor; Mono-S; NMR Spectroscopy; Nature; Neurodegenerative Disorders; Pathogenicity; Pathway interactions; Phosphotransferases; Play; Polyubiquitin; Post-Translational Protein Processing; Predictive Value; Proteins; Reaction; Research; Research Personnel; Role; Scheme; Shigella Infections; Signal Pathway; Signal Transduction; Site; Solid; Structure; System; Testing; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Universities; Virus Diseases; Work; base; biological research; enzyme activity; functional group; in vivo; innovation; insight; mouse model; pathogen; protein complex; protein function; tool; ubiquitin isopeptidase; ubiquitin-protein ligase

Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of eukaryotic cell biology and regulates pathways critical to human development and viability. Aberrations or defects in Ub-signaling can result in numerous debilitating diseases including neurodegenerative diseases, infections, and cancer. Despite remarkable progress over the decades, we still have only a rudimentary understanding of the molecular factors and networks of interactions that govern the assembly of the proteins required for regulated Ub transfer and signaling. The ability to intervene in diseases related to Ub-signaling requires a thorough understanding of these pathways at the molecular level. The basic scheme for Ub modification involves the concerted activities and interactions of several different proteins. This proposal focuses on expanding our understanding of a central player in Ub-transfer reactions, the class of enzymes known as Ub-Conjugating Enzymes or E2s. Once thought simply to shuttle Ub to the site of modification, emerging evidence suggests that E2s can play a pivotal role in substrate recognition, determining the nature of Ub modification of a target protein, and even interacting with and influencing the activities of proteins outside the traditional Ub-transfer pathway. There are ~40 E2s in the human genome and most are thought to be directly involved in Ub transfer. Very little functional information is available for the majority of these E2s. The Research Plan outlined in this proposal seeks to substantially expand our understanding of E2 function. Using various biochemical approaches, NMR spectroscopy, crystallography, and mass spectrometry, this project seeks to develop a molecular understanding of the factors that govern 1) the intrinsic activity of E2~Ub conjugates toward particular residues, 2) E2~Ub recognition of substrates, and 3) and regulatory interactions of E2~Ub conjugates that regulate proteins outside the main Ub-transfer pathway. We have adapted and developed new tools with which to identify proteins selectively modified by particular E2~Ub conjugates. We make use of a new "in coli" expression system that can reconstruct Ub-transfer pathways in E. coli. This system allows us to generate particular E2~Ub conjugates and investigate their activity in cells without complications from competing Ub- transfer reactions. The new knowledge we seek to obtain will have a significant impact our understanding of human E2 structure and function and greatly accelerate biological research involving ubiquitylation and ubiquitin signaling.

通过泛素（UB）信号传导的细胞内通信影响真核细胞生物学的所有方面 调节对人类发展至关重要的途径。 UB信号中的畸变或缺陷可以 导致许多令人衰弱的疾病，包括神经退行性疾病，感染和癌症。尽管 在过去的几十年中，我们仍然对分子因素有基本的了解 和互动网络，这些互动的组装组装的蛋白质是受规范的UB转移所需的蛋白质和 信号。干预与UB信号有关的疾病的能力需要彻底了解 这些途径在分子水平。 UB修改的基本方案涉及协同活动 和几种不同蛋白质的相互作用。该提议着重于扩大我们对 UB转移反应的中心参与者，称为UB偶联酶或E2的酶类。一次 思想仅仅是为了将UB穿梭到修改地点，新兴的证据表明E2可以发挥关键作用 在底物识别中的作用，确定靶蛋白的UB修饰的性质，甚至相互作用 并影响并影响传统UB转移途径之外的蛋白质活性。有〜40 e2 人类基因组和大多数人被认为直接参与UB转移。功能很小 这些E2大多数都可以使用。该提案中概述的研究计划试图 实质上扩展了我们对E2功能的理解。使用各种生化方法，NMR 光谱，晶体学和质谱法，该项目旨在发展分子 理解控制因素1）E2〜ub偶联的固有活性 残基，2）e2〜ub识别底物，3）E2〜ub结合的调节相互作用 调节主要UB转移途径外的蛋白质。我们已经适应并开发了新工具 可以通过特定的E2〜UB偶联物选择性地识别蛋白质。我们利用新的“大肠杆菌” 可以在大肠杆菌中重建UB转移途径的表达系统。该系统使我们能够生成 特定的e2〜ub结合并研究它们在细胞中的活性，而没有竞争UB的并发症 转移反应。我们寻求获得的新知识将产生我们对的理解 人E2结构和功能，并大大加速了涉及泛素化和的生物学研究 泛素信号传导。