Mechanisms of Cell Regulation and Manipulation by the Ubiquitin System

Yale University, New Haven CT

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Abstract

Eukaryotic cells have highly conserved enzymatic systems for covalently linking ubiquitin (Ub) and related proteins such as SUMO to proteins. The modifications may lead to degradation of the targeted protein, usually by the proteasome. Ub and SUMO modifications are highly dynamic due to specialized proteases that efficiently remove them. Both modifiers have many crucial roles, including important contributions to human biology. Many human disorders, including neurodegenerative diseases and different cancers, are associated with abnormalities in the Ub system. Multiple drugs that target the Ub system are already in the clinic. In this renewal, the PI proposes to undertake studies on several recently discovered regulatory mechanisms. One focus is on the assembly and intracellular trafficking of proteasomes, which normally concentrate in the nucleus but undergo dramatic relocalization and changes in assembly when grown in specific nutrient-limited conditions. A key mechanism may be the regulated Ub- independent degradation of a transport adaptor that controls proteasome translocation into the nucleus. Another research direction addresses Ub-system enzymes secreted by endosymbiotic bacteria that infect humans and other species. One target is a deubiquitylating enzyme (DUB) from Orientia tsutsugamushi, the causative agent of scrub typhus, a highly lethal disease. Small- molecule inhibitors are being developed to antagonize the Orientia DUB. Another important DUB is from Wolbachia, bacteria that infect millions of arthropod species and exploit this unusual DUB to alter host reproduction to promote their own inheritance. A related system utilizes a nuclease to similar effect, which will also be investigated. Wolbachia are being deployed as agents for fighting disease vectors such as the mosquitoes that transmit dengue fever and malaria. Regarding SUMO protease functions, several new directions are being followed. One derives from a discovery that, following in vitro evolution, a mutation in yeast SUMO greatly mitigates the loss of a crucial SUMO protease. Another mutation at the same position blocks all SUMO cleavage. Initial data indicate that the inability to tolerate loss of SUMO cleavage is linked to thiamine (vitamin B1) deficiency. A final area of focus is on the question of why Ub, SUMO, and almost all other Ub-like proteins (Ubls) are synthesized with C-terminal extensions that must be removed. Potentially, tail cleavage serves as a proofreading step to prevent aberrant forms of the Ubl from getting attached to targets. Overall, the combination of genetic, biochemical, cell biological, and genomic studies proposed should give a much deeper understanding of the mechanisms of SUMO and Ub pathway regulation with important implications for human health.