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

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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.