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 of protein post-translational modifications, such as phosphorylation and acetylation, on protein function and interactions. Importantly, enzymes regulating post-translational modifications, including kinase, phosphatase, and HDAC proteins, are targets of drug treatment. Yet, tools linking 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 functions of protein modifying enzymes in cell biology. In our work with protein phosphorylation, we have pioneered in the last 10 years use of ATP analogs for 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 multiple biologists. In our work with protein acetylation, we have demonstrated in the last two years the power of using trapping mutants to discover non-histone substrates of HDAC1, which has revealed unexpected roles of HDAC1 proteins 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. Given the critical role of kinase, phosphatase, and HDAC enzymes in disease and drug treatment, yet the inadequate tools available to study these enzymes in cellular systems, the enabling chemical strategies proposed in this application will strengthen biomedical research in cell signaling and drug design.
Protein post-translational modifications mediate myriad of cellular events, and unregulated protein modifications are widely associated with disease states. The application outlines new chemical approaches to characterizing the enzymes governing two protein modifications, phosphorylation and acetylation, which will lead to a greater understanding of disease formation and development of new treatments.
