Chemical Approaches to Characterizing Protein Post-translational Modifications by Mass Spectrometry

总结

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 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 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 application of ATP analogs in kinase- catalyzed labeling reactions to develop a suite of methods to probe kinase-substrate pairs and multi-protein complexes in cells. This supplemental funding application will support this project by applying our innovative tools to complex biological problems in collaboration with biologists. In our work with protein acetylation, we have demonstrated in the last several 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 this supplemental funding application, we will apply this powerful trapping strategy to discover the substrates of HDAC1 in sickle cell anemia model systems, which will reveal unanticipated molecular mechanisms in sickle cell and possible new therapeutics. Importantly, these studies will provide an ideal training ground to expose the pre-doctoral candidate to proteomics-based mass spectrometry, biochemistry, and cell biology to prepare for entry into the biomedical workforce.