Elucidating the Roles of E3 Ligases and Deubiquitinating Enzymes in Mitophagy by Orthogonal Ubiquitin Transfer and Linkage-Specific Ubiquitin Probes

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The Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Jun Yin from Georgia State University and Dr. Ashton Cropp of Virginia Commonwealth University to investigate the roles of protein modification by ubiquitin (UB) as a fundamental mechanism for the cell to cope with oxidative damage. UB is a small protein mediating protein interaction for the regulation of various cellular processes, including those carried out by mitochondria. Mitochondria are “powerhouses” in the cell for energy production, but at the same time, they are “heavy polluters” that release reactive oxygen species (ROS) into their local environments, which inflicts damage on cells. During the process of aging, cells are less capable of managing ROS produced by mitochondria, and the accumulated damage could result in brain damage and heart failure at the end of the human life span. For self-protection, cells initiate a process called mitophagy with enzymes such as E3 UB ligases, where flagging the damaged mitochondria with UB takes place so damaged mitochondria can be isolated and removed before they release a substantial amount of damaging ROS in the cell. The joint teams of Yin and Cropp will assemble an integrated technology platform with a combined use of protein engineering, reactivity-based chemical probe design, and protein analysis methods to elucidate the mechanisms of mitophagy regulation that result from protein modification by UB. Their work will define the cellular steps underpinning ROS response and reveal how the breakdown of such processes may contribute to human aging. The integrated outreach program of the project will provide substantial training opportunities to K-12 students from local communities in Atlanta, GA and Richmond, VA. The highly diverse student body in the urban areas of the two cities will benefit from early exposure to the development of science and technology and the rewarding career opportunities in chemically driven biological research. Protein modification by UB plays an essential role in sensing damaged mitochondria and signaling their removal by mitophagy. Various E3 UB ligases have been found to be recruited to damaged mitochondria and decorate them with UB chains to induce mitophagy. Currently, the ubiquitination targets of the E3s are not well characterized, so the E3-regulaued mitophagy pathways are undefined. Furthermore, E3s can assemble a network of UB chains of diverse linkages around damaged mitochondria, but little is known about how the UB chains are trimmed and edited by deubiquitinating enzymes (DUBs) to counteract the UB chain extension reaction catalyzed by the E3s. The joint team from the Yin and Cropp laboratories plans to fill these knowledge gaps by profiling the substrates of various E3s at the initiation phase of mitophagy with an innovative proteomic platform known as orthogonal UB transfer (OUT). OUT will enable the identification of the direct substrates of the E3s to map the associated cell signaling pathways regulating mitophagy. By comparing the substrate profiles of various E3s, the team will differentiate the roles of E3s in mitophagy regulation. Furthermore, the team has developed an innovative method to synthesize linkage-specific UB chains and UB-substrate conjugates based on unnatural amino acid (UAA) incorporation and plans to use the UB conjugates as probes to capture DUBs that regulate mitophagy. By accomplishing the research plan, the team expects to map the protein ubiquitination pathways regulating mitophagy and identify DUBs interacting with the E3s in editing diverse linkages of UB chains for proper encoding of the mitophagy signals. The significance of the work is to develop powerful technologies to map cell signaling pathways mediated by UB transfer, elucidate the actions of E3s and DUBs in mitophagy signaling, and decipher the cellular mechanisms underlying mitochondria quality control to neutralize the damage by ROS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.