Unconventional Interactions of Human Ubiquitin Conjugating Enzymes

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所有方面， 调节对人类发育和生存能力至关重要的途径。Ub信号传导的异常或缺陷可以 导致许多使人衰弱的疾病，包括神经变性疾病、感染和癌症。尽管 尽管在过去的几十年里取得了巨大的进步，但我们仍然对分子因素只有初步的了解。 和相互作用的网络，控制调节Ub转移所需的蛋白质的组装， 信号干预与Ub信号相关的疾病的能力需要彻底了解 在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的相互作用。这一建议的重点是扩大我们对一个 在Ub转移反应中的核心球员，被称为Ub结合酶或E2的酶类。一旦 虽然E2被认为只是将Ub运送到修改的位置，但新出现的证据表明，E2可以发挥关键作用。 在底物识别中起作用，决定靶蛋白的Ub修饰的性质，甚至相互作用 具有并影响传统Ub-转移途径外的蛋白质的活性。有~40个E2， 人类基因组和大多数被认为是直接参与Ub转移。功能非常少 对于大多数这些E2，信息是可用的。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。使用各种生物化学方法，NMR 光谱学，晶体学和质谱学，该项目旨在开发一种分子 理解支配1）E2-Ub缀合物对特定的细胞的固有活性的因素， 残基，2）底物的E2~Ub识别，和3）E2~Ub缀合物的调节相互作用， 调节主要Ub转移途径外的蛋白质。我们已经适应并开发了新的工具， 其用于鉴定被特定E2~Ub缀合物选择性修饰的蛋白质。我们利用一种新的“大肠杆菌” 构建了重组大肠杆菌Ub转运途径的表达系统。杆菌这个系统可以让我们产生 特别是E2-Ub缀合物，并研究它们在细胞中的活性，而不存在来自竞争性Ub- 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E2结构和功能以及大大加速了涉及泛素化和 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.