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: 8716782
• 负责人: PETER S BRZOVIC
• 金额: $ 28.25万
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716782
• 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; Human Ubiquitin; 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讯影响真核细胞生物学的各个方面 调节对人类发展和生存至关重要的途径。 Ub 信号的畸变或缺陷可以 导致许多使人衰弱的疾病，包括神经退行性疾病、感染和癌症。尽管 几十年来取得了显着的进展，但我们对分子因素仍然只有初步的了解 以及控制调节 Ub 转移所需蛋白质组装的相互作用网络 发信号。干预与 Ub 信号转导相关的疾病的能力需要透彻了解 这些途径在分子水平上。 Ub修改的基本方案涉及协同活动 以及几种不同蛋白质的相互作用。该提案的重点是扩大我们对 泛素转移反应的核心参与者，这一类酶被称为泛素结合酶或 E2。一次 简单地认为 E2 可以将 Ub 运送到修饰位点，但新出现的证据表明 E2 可以发挥关键作用 在底物识别中的作用，确定靶蛋白 Ub 修饰的性质，甚至相互作用 与并影响传统 Ub 转移途径之外的蛋白质的活性。大约有 40 个 E2 人类基因组中的大多数被认为直接参与 Ub 转移。功能很少 大多数 E2 的信息均可用。本提案中概述的研究计划旨在 大大扩展了我们对E2功能的理解。使用各种生化方法、NMR 光谱学、晶体学和质谱学，该项目旨在开发一种分子 了解控制因素 1) E2~Ub 结合物对特定的内在活性 残基，2) E2~Ub 底物识别，以及 3) E2~Ub 缀合物的调节相互作用 调节主要 Ub 转移途径之外的蛋白质。我们已经适应并开发了新工具 用于识别被特定 E2~Ub 缀合物选择性修饰的蛋白质。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。该系统允许我们生成 特定的 E2~Ub 缀合物并研究它们在细胞中的活性，而不会因竞争 Ub-而出现并发症 转移反应。我们寻求获得的新知识将对我们的理解产生重大影响 人类 E2 结构和功能，大大加速涉及泛素化和 泛素信号传导。