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

来自生命过程化学计划（CLP）的CAREER奖将支持芝加哥大学尹俊教授阐明细胞中泛素信号蛋白降解机制的工作。尹教授将开发新的化学方法来设计蛋白质泛素化酶，并利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。泛素是一种由76个残基组成的小蛋白。它通过一种被称为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。与泛素结合的细胞蛋白被26S蛋白酶体降解。除了它的管家作用，泛素还作为一个信号来控制它所附着的蛋白质的生物活性和亚细胞位置。通常，蛋白质泛素化缺陷与癌症、病毒感染和神经退行性疾病有关。细胞生物学中研究蛋白质泛素化的一个主要瓶颈是没有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，由缺陷的E3引起疾病的原因也很难研究。在CAREER奖的支持下，尹教授将设计E3酶，使每个设计的E3都能在其底物蛋白上附加独特的化学标签。通过这种方式，E3酶可以与其细胞中的泛素化靶点配对，从而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Nedd4-1，一个关键的癌症诱导剂，将被设计以分析其底物特异性。本项目将通过E3酶及其底物蛋白的关联，为明确E3酶的生物学功能、阐明E3与各种疾病的病理联系、确定特异性的E3酶作为药物开发的治疗靶点提供一个通用平台。该奖项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，全面收集蛋白质工程的方法和应用。此外，来自芝加哥南部当地高中的师生将有机会参观尹教授的实验室，并亲身体验化学和分子生物学的现代研究。尹教授将建立一个综合的研究和教育计划，以培养化学和生物学界面的未来科学家，并在他们学习生涯的早期培养K-12学生对科学的兴趣。

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.