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

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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.