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: 8293759
• 负责人: PETER S BRZOVIC
• 金额: $ 28.32万
• 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/8293759
• 关键词: Active Sites; Address; Amaze; Amino Acids; Attention; Bacterial Infections; Binding; Biochemical; Biological; Biological Assay; Cells; Cellular biology; Communication; Complex; Critical Pathways; Crystallography; Defect; Disease; Environment; Enzymes; Escherichia coli; Eukaryotic Cell; Event; Foundations; Human; Human Development; Human Genome; Human Ubiquitin; Infection; 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; Signal Transduction; Site; Solid; Structure; System; Testing; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Virus Diseases; Work; base; biological research; enzyme activity; functional group; human disease; insight; pathogen; protein complex; protein function; tool; ubiquitin isopeptidase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): 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 approximately 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 also 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. PUBLIC HEALTH RELEVANCE: Ubiquitin (Ub) signaling in cells is critical to pathways involved in human development and viability and defects or aberrations in Ub-signaling can result in numerous debilitating human diseases including neurodegenerative diseases, bacterial and viral infections, and cancer. This study seeks to understand key enzymes involved in Ub transfer, the class of enzymes known as E2 Ub-conjugating enzymes, as remarkably little is known about how most human E2s work, what proteins they might target, or the pathways in which they function. The new knowledge we obtain will greatly facilitate the ability of researchers to identify, test, manipulate, and target specific ubiquitylation events in a cell, hel us understand how bacterial pathogens can hijack Ub pathways, and will generate new ways for researchers to investigate the biological impacts of Ub transfer pathways.

描述（由申请人提供）：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各个方面，并调节对人类发展和生存能力至关重要的途径。 UB信号的畸变或缺陷会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。尽管在过去的几十年中取得了显着进展，但我们仍然对控制UB传输和信号传导所需的蛋白质组装的相互作用的分子因素和相互作用网络有基本的理解。干预与UB信号有关的疾病的能力需要在分子水平上透彻了解这些途径。 UB修饰的基本方案涉及几种不同蛋白质的协同活动和相互作用。该提案着重于扩展我们对UB转移反应中核心参与者的理解，即称为UB偶联酶或E2的酶类。一旦思想仅仅是为了将UB穿梭到修饰部位，新兴的证据表明，E2可以在底物识别中起关键作用，确定靶蛋白的UB修饰的性质，甚至与传统UB-Transfer途径之外的蛋白质相互作用并影响蛋白质的活性。人类基因组中约有40个E2，大多数被认为直接参与UB转移。这些E2中的大多数可用的功能信息很少。该提案中概述的研究计划旨在大大扩展我们对E2功能的理解。该项目使用各种生化方法，NMR光谱学，晶体学和质谱法，试图发展对控制因素的分子理解。我们已经适应和开发了新工具，可以通过这些工具来识别特定的E2〜UB偶联物选择性修饰的蛋白质。我们还利用了一个新的“大肠杆菌”表达系统，该系统可以重建大肠杆菌中的UB转移途径。该系统允许我们生成特定的E2〜UB偶联物并研究其在细胞中的活性，而不会引起竞争UB转移反应并发症。我们寻求获得的新知识将对我们对人类E2结构和功能的理解产生重大影响，并极大地加速了涉及泛素化和泛素信号传导的生物学研究。 公共卫生相关性：细胞中的泛素（UB）信号对于参与人类发育的途径以及UB信号中的缺陷或畸变至关重要，可能会导致许多使人衰弱的人类疾病，包括神经退行性疾病，细菌和病毒感染以及癌症。这项研究试图了解参与UB转移的关键酶，UB转移，称为E2 UB偶联酶的酶类别，对大多数人E2的工作方式，它们可能靶向的蛋白质或它们发挥作用的途径知之甚少。我们获得的新知识将极大地促进研究人员在细胞中识别，测试，操纵和针对特定的泛素化事件的能力，Hel我们了解细菌病原体如何劫持UB UB途径，并为研究人员研究UB转移途径的生物学影响。