Elucidating Cell Signaling by Orthogonal Ubiquitin Transfer and Linkage-Specific di-Ubiquitin Probes

美国国家科学基金会化学部的生命过程化学项目为佐治亚州立大学(GSU)的尹军教授和弗吉尼亚联邦大学(VCU)的托马斯·A·克罗普教授提供资金。这项研究试图了解泛素(UB)进行的细胞信号转导机制。UB是一种蛋白质，它与细胞中的其他蛋白质相连，调节细胞过程。几种不同的酶形成一个继电器，将UB转移到细胞蛋白质上。这些传输响应于单元内发生的更改而发生。这些转移酶是如何选择UB转移的目标的，人们对此知之甚少。因此，由UB辅助的小区信令网络被模糊地定义。殷博士和克罗普博士组成的团队正在准备和实施探测器，以找出关键酶的底物分布及其在细胞生物学中的作用。研究人员还在生成确定连接的UB链，以研究转移酶的催化机制。该项目联合培养本科生和研究生，研究细胞生物学的化学基础。通过对这个项目的贡献，GSU和VCU的不同背景的学生获得了化学、分子生物学和细胞生物学方面的关键研究技能。综合外展计划为亚特兰大地区欠发达社区的初中生和高中生提供研究机会。这个联合项目利用YIN实验室开发的UB正交转移(OUT)技术和Cropp实验室开发的非天然氨基酸(UAA)掺入技术来研究UB介导的细胞信号。殷实验室使用噬菌体和酵母细胞表面展示技术来设计出用于细胞调节的关键E3S酶的级联，如c-Cbl、Parkin和E6AP。外级联在细胞中表达，蛋白质组学用于分析特定E3的底物，以揭示它们的生物学功能。Yen和Cropp实验室通过在UB的不同靶位传递UAA来合成已定义连接的di-UB偶联物。测定了具有di-UB共轭的E2s的共晶结构，以阐明E2s如何合成不同键的UB链。总体而言，该项目为UB信令网络和“编写”UB代码的化学机制提供了亟需的见解。

The Chemistry of Life Processes Program in the Chemistry Division of NSF is funding Professor Jun Yin of the Georgia State University (GSU) and Professor Thomas A. Cropp of the Virginia Commonwealth University (VCU). The research seeks to understand the cell signaling mechanism carried out by by ubiquitin (UB). UB is a protein that is connected to other proteins in the cell to regulate cellular processes. Several different enzymes form a relay to transfer UB to cellular proteins. These transfers occur in response to the changes that occur within a cell. How these transfer enzymes select the targets of UB transfer is poorly understood. Consequently, the cell signaling networks that are aided by UB are vaguely defined. The team of Dr. Yin and Dr. Cropp is preparing and implementing probes to figure out the substrate profiles of key enzymes and their roles in cell biology. The investigators are also generating UB chains of defined linkages to investigate the catalytic mechanisms of the transfer enzymes. The project jointly trains undergraduate and graduate students to investigate the chemical basis of cell biology. By contributing to this project, the GSU and VCU students of diverse backgrounds obtain key research skills in chemistry, molecular biology, and cell biology. Integrated outreach programs provide research opportunities for middle school and high school students in underdeveloped communities in the Atlanta area. This joint project takes advantage of the orthogonal UB transfer (OUT) technology developed in the Yin laboratory and the unnatural amino acid (UAA) incorporation technology developed in the Cropp laboratory to study UB-mediated cell signaling. The Yin lab uses phage and yeast cell surface display to engineer OUT cascades of key E3s enzymes for cell regulation such as c-Cbl, Parkin, and E6AP. The OUT cascades is expressed in the cell and proteomics is used to profile the substrates of specific E3s to reveal their biological functions. The Yin and Cropp labs synthesize di-UB conjugates of defined linkages by delivering UAA in various targeted positions in UB. The co-crystal structures of E2s with di-UB conjugates are determined in order to elucidate how E2s synthesize UB chains of diverse linkages. Overall the project provides much-needed insights in UB signaling networks and the chemical mechanism for "writing" the UB code.