Chemical Approaches to Study Protein Post-translational Modifications

Summary Most biological events in the cell are mediated at some level by protein post-translational modifications. For example, aberrant protein phosphorylation catalyzed by kinase and phosphatase enzymes is linked to a wide variety of cancers. Similarly, the unregulated acetylation state of histone proteins, controlled by histone deacetylase (HDAC) proteins, can lead to epigenetic changes in transcription and ultimately disease. Key to characterizing both healthy and disease states is a detailed molecular understanding of the role played by protein post-translational modifications, such as phosphorylation and acetylation, on protein function and interactions. Importantly, enzymes regulating protein post-translational modifications, including kinase, phosphatase, and HDAC proteins, are targets of therapeutics. Yet, tools linking specific protein modifications to downstream biological activities are often limited or unavailable, which has stalled progress in disease characterization and drug development. The NIGMS-funded projects in the Pflum lab address the critical need to develop innovative chemical approaches to discover unanticipated roles of protein post-translational modifications and their modifying enzymes in cell biology. In our work with protein phosphorylation, we pioneered use of g-phosphoryl modified ATP analogs in kinase-catalyzed labeling reactions. Building on this prior work, we propose in the next 5 years to 1) develop a new suite of methods with unique abilities to probe kinase- and phosphatase- substrate pairs and multi-protein complexes in cells, and 2) apply our innovative tools to a variety of biological problems in collaboration with biologists. In our work with protein acetylation, we have demonstrated the power of using inactive mutants as traps to discover non-histone substrates of HDAC1 and HDAC6, which has revealed unexpected roles in cell biology. In the next 5 years, we will apply this powerful trapping strategy to additional HDAC protein isoforms, which will establish the role of HDAC proteins in activities beyond epigenetics and transcriptional regulation. In a new direction for the program, trapping will be expanded to demethylase enzymes, which regulate protein methylation. Given the critical role of kinase, phosphatase, demethylases, HDAC enzymes in disease and drug treatment, yet the inadequate tools available to study these enzymes in complex cellular systems, the enabling chemical strategies proposed in this application will strengthen biomedical research in cell biology and drug design.