PROJECT SUMMARY Protein citrullination is increased in inflammatory disease and cancer, and the evidence indicates that this post-translational modification is a major contributor to disease pathogenesis. In fact, inhibition of protein citrullination decreases disease severity in animal models of rheumatoid arthritis, ulcerative colitis, nerve damage, and cancer. Thus, the Protein Arginine Deiminases (PADs), which catalyze this PTM, represent novel therapeutic targets. The overall goal of this renewal application is to investigate how protein citrullination impacts human cell signaling. Specific questions to be addressed include: (1) What proteins are citrullinated?; (2) How does citrullination affect activity?; (3) Does citrullination impact proteolytic events? and (4) Can we generate isozyme specific inhibitors? Answers to these questions will be obtained by (1) developing chemoproteomic platforms to identify citrullination events; (2) developing and using a genetic code expansion platform to evaluate the functional consequences of individual citrullination events; and (3) developing chemical probes and isozyme selective PAD inhibitors to modulate cellular citrullination levels. By addressing each of these questions, the PI expects to provide a holistic understanding for how the PADs contribute to human biology. Given the outstanding track record of the PI and his leadership position in the PAD field, combined with his strong network of collaborators, this project will provide new insights and sorely needed research tools to the scientific community. Key outcomes include the discovery of new PAD biology, which is predicted to have major translational impacts on autoimmunity and cancer.