CAREER: Identifying the Substrate Proteins of Nedd4 Family of E3 Ubiquitin Ligase by Orthogonal Ubiquitin Transfer (OUT)

来自生命过程化学项目（CLP）的CAREER奖将支持芝加哥大学的Jun Yin教授的工作，以阐明细胞中泛素信号蛋白降解的机制。尹教授将开发新的化学方法来设计蛋白质泛素化酶，并使用工程酶作为工具来绘制细胞中调节蛋白质寿命的信号转导途径。泛素是76个残基的小蛋白质。它通过一类称为E3泛素连接酶的酶附着在细胞中老化，未使用或受损的蛋白质上。与泛素连接的细胞蛋白质被26 S蛋白酶体降解。除了其管家作用外，泛素还作为信号来控制其所连接的蛋白质的生物活性和亚细胞位置。蛋白质泛素化的缺陷常常与癌症、病毒感染和神经退行性疾病有关。在细胞生物学中研究蛋白质泛素化的一个主要瓶颈是没有一个很好的方法来鉴定细胞中E3酶的底物蛋白。正因为如此，E3酶的生物学功能尚不清楚，并且很难研究E3缺陷的疾病原因。在这个CAREER奖的支持下，尹教授将设计E3酶，使每个工程E3将附加独特的化学标签到其底物蛋白。通过这种方式，E3酶可以与细胞中的泛素化靶点配对，并且可以阐明调节蛋白质降解的信号转导网络。在这项工作中，E3酶Nedd 4 -1，一种关键的癌症诱导剂，将被工程化以描述其底物特异性。通过将E3酶及其底物蛋白联系起来，该项目将提供一个通用平台来定义E3酶的生物学功能，阐明E3与各种疾病的病理联系，并鉴定特定的E3酶作为药物开发的治疗靶点。CAREER奖还将支持尹教授培训本科生和研究生在生物研究中使用蛋白质工程。它将赞助一个免费访问的在线数据库，其中全面收集了蛋白质工程的方法和应用。此外，还将为芝加哥南部当地高中的师生提供一个参观殷教授实验室的机会，并亲身体验化学和分子生物学的现代研究。总的来说，尹教授将建立一个综合的研究和教育计划，以培养未来的科学家在化学和生物学的接口，并培养K-12学生在他们的学习生涯的早期阶段对科学的兴趣。

The CAREER award from the Chemistry of Life Processes Program (CLP) will support work by Professor Jun Yin at the University of Chicago to elucidate the mechanism of ubiquitin signaled protein degradation in the cell. Professor Yin will develop new chemical methods to engineer protein ubiquitination enzymes and use the engineered enzymes as tools to map the signal transduction pathways in the cell that regulate the life span of proteins. Ubiquitin is a small protein of 76 residues. It is attached to aged, unused or damaged proteins in the cell by a class of enzymes known as E3 ubiquitin ligase. Cellular proteins attached with ubiquitin are degraded by the 26S proteasome. Besides its housekeeping role, ubiquitin also serves as a signal to control the biological activities and subcellular locations of the proteins it is attached to. Quite often defects of protein ubiquitination are implicated in cancer, virus infection and neurodegenerative disorders. A major bottleneck in investigating protein ubiquitination in cell biology is that there is not a good method to identify the substrate proteins of E3 enzymes in the cell. Because of this, the biological functions of E3 enzymes are unclear and the cause of disease by defective E3s is hard to study. With the support of this CAREER award, Professor Yin will engineer the E3 enzymes so that each engineered E3 will attach unique chemical labels to their substrate proteins. In this way a E3 enzyme can be paired with its ubiquitination targets in the cell and the signal transduction networks that regulate protein degradation can be elucidated. In this work, E3 enzyme Nedd4-1, a key inducer of cancer, will be engineered in ordered to profile its substrate specificity. By associating E3 enzymes and their substrate proteins, this project will provide a general platform to define the biological function of the E3 enzymes, to elucidate the pathological connection of E3 with various diseases and to identify specific E3 enzymes as therapeutic targets for drug development. The CAREER award will also support Professor Yin to train undergraduate and graduate students to use protein engineering in biological research. It will sponsor a freely accessible online database with a comprehensive collection of methods and applications of protein engineering. Furthermore it will provide an opportunity for the teachers and students from the local high schools in south side Chicago to visit Professor Yin's lab and get hands-on experience of modern research in chemistry and molecular biology. Overall Professor Yin will establish an integrated research and educational program to train future scientists at the interface of chemistry and biology and to nurture the interests of K-12 students in science at the early stage of their learning career.