Unconventional Interactions of Human Ubiquitin Conjugating Enzymes

通过泛素(Ub)信号的细胞内通讯影响真核细胞生物学的方方面面 调节对人类发展和生存至关重要的途径。Ub信号中的异常或缺陷可能 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 尽管几十年来取得了显著的进步，但我们对分子因素仍然只有一个初步的了解 以及管理受调控的Ub转移所需的蛋白质组装的相互作用网络和 发信号。干预与Ub信号相关的疾病的能力需要彻底了解 分子水平上的这些途径。Ub修改的基本方案涉及协同活动 以及几种不同蛋白质之间的相互作用。这项建议的重点是扩大我们对 Ub转移反应的中心参与者，这类酶被称为Ub结合酶或E2。一次 人们认为只是将Ub送到修饰的地方，越来越多的证据表明，E2可以发挥关键作用 在底物识别中的作用，确定目标蛋白的Ub修饰的性质，甚至相互作用 与传统的Ub转移途径以外的蛋白质的活性有关并对其产生影响。有大约40个E2在 人类基因组和大多数被认为直接参与了Ub的转移。功能很少 这些E2中的大多数都有相关信息。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，核磁共振 光谱学、结晶学和质谱学，这个项目试图开发一种分子 对1)E_2~Ub结合物的内在活性的理解 残基，2)E_2~Ub底物的识别，以及3)E_2~Ub结合物的调节相互作用 调节主要Ub转运途径之外的蛋白质。我们已经调整和开发了新的工具， 用于鉴定被特定的E2~Ub偶联物选择性修饰的蛋白质。我们利用了一种新的“在大肠杆菌中” 可在大肠杆菌中重建Ub转移途径的表达系统。这个系统允许我们生成 特异性的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-Ub的并发症 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的结构和功能，极大地促进了涉及泛素化和 泛素信号。

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.