Unconventional Interactions of Human Ubiquitin Conjugating Enzymes

描述（由申请人提供）：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所有方面，并调节对人类发育和生存力至关重要的途径。Ub信号传导的异常或缺陷可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的进展，我们仍然只有一个初步的了解的分子因子和网络的相互作用，控制所需的调节Ub转移和信号传导的蛋白质的组装。干预与Ub信号相关的疾病的能力需要在分子水平上彻底了解这些途径。Ub修饰的基本方案涉及几种不同蛋白质的协同活性和相互作用。这项提案的重点是扩大我们对Ub转移反应中的核心参与者的理解，这类酶被称为Ub结合酶或E2。一旦被认为只是将Ub穿梭到修饰位点，新出现的证据表明E2可以在底物识别中发挥关键作用，决定靶蛋白的Ub修饰的性质，甚至与传统Ub转移途径之外的蛋白质相互作用并影响其活性。人类基因组中大约有40个E2，大多数被认为直接参与Ub转移。大多数E2的功能信息非常少。本提案中概述的研究计划旨在大幅扩展我们对E2功能的理解。利用各种生物化学方法，NMR光谱学，晶体学和质谱学，该项目旨在从分子上了解以下因素：1）E2~Ub缀合物对特定残基的内在活性，2）E2~Ub对底物的识别，3）E2~Ub缀合物调节主要Ub转移途径外蛋白质的调节相互作用。我们已经适应并开发了新的工具，用于鉴定被特定E2~Ub缀合物选择性修饰的蛋白质。我们还利用了一种新的“在大肠杆菌”表达系统，可以重建在大肠杆菌中的Ub转移途径。杆菌该系统使我们能够产生特定的E2~Ub缀合物，并研究它们在细胞中的活性，而不会因竞争性Ub转移反应而引起并发症。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，并大大加速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）信号传导对参与人类发育和生存力的途径至关重要，并且Ub信号传导中的缺陷或畸变可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。这项研究旨在了解参与Ub转移的关键酶，这类酶被称为E2 Ub结合酶，因为对大多数人类E2如何工作，它们可能靶向什么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识别，测试，操纵和靶向细胞中特定泛素化事件的能力，帮助我们了解细菌病原体如何劫持Ub途径，并将为研究人员提供新的方法来研究Ub转移途径的生物学影响。

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.