Unconventional Interactions of Human Ubiquitin Conjugating Enzymes

描述(由申请人提供)：通过泛素(Ub)信号的细胞内通信影响真核细胞生物学的各个方面，并调节对人类发育和生存至关重要的途径。Ub信号的异常或缺陷可导致许多衰弱的疾病，包括神经退行性疾病、感染和癌症。尽管几十年来取得了显著的进展，但我们仍然对调控Ub转移和信号转导所需的蛋白质组装的分子因素和相互作用网络只有一个初步的了解。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。这项提议的重点是扩大我们对Ub转移反应中的核心角色的理解，这是一类被称为Ub结合酶或E2s的酶。曾经被认为是简单地将Ub运送到修饰位置的新证据表明，E2s可以在底物识别、决定目标蛋白的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和影响活性方面发挥关键作用。人类基因组中大约有40个E2，其中大多数被认为直接参与了Ub的转移。对于这些E2中的大多数，可用的功能信息非常少。这项建议中概述的研究计划旨在大幅扩大我们对E2功能的理解。本项目利用各种生化手段、核磁共振光谱、结晶学和质谱学，试图从分子水平上理解以下因素：1)E_2~Ub结合物对特定残基的内在活性；2)E_2~Ub对底物的识别；3)E_2~Ub偶联物调节主要Ub转移途径外蛋白质的相互作用。我们已经适应和开发了新的工具，用来鉴定由特定的E2~Ub结合物选择性修饰的蛋白质。我们还利用了一种新的“在大肠杆菌中”的表达系统，可以在大肠杆菌中重建Ub转移途径。该系统使我们能够产生特定的E2~Ub结合物，并研究它们在细胞中的活性，而不会出现竞争Ub转移反应的并发症。我们寻求获得的新知识将对我们理解人类E2的结构和功能产生重大影响，并极大地促进涉及泛素化和泛素信号转导的生物学研究。 公共卫生相关性：细胞中的泛素(Ub)信号对参与人类发育和生存的途径至关重要，Ub信号的缺陷或异常可导致许多人类疾病，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Ub转移所涉及的关键酶，这类酶被称为E2 Ub结合酶，因为人们对大多数人类E2的工作原理、它们可能的靶向蛋白或它们的作用途径知之甚少。我们所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的泛素化事件的能力，帮助我们了解细菌病原体如何劫持Ub途径，并将为研究人员研究Ub转移途径的生物学影响提供新的途径。

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.