Enzyme-engineering Approaches to Dissect Protein Methylation Profiles

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DESCRIPTION (Provided by the applicant) Abstract: Epigenetic regulations are involved in numerous biological processes and errors in these processes have been implicated in many diseases such as cancer. Among the key enzymes that orchestrate epigenetics are over 60 human protein methyltransferases. Accumulated evidence showed that epigenetic diversity requires protein methyltransferases to act on histone and nonhistone targets. Nonetheless, elucidating the physiological and pathological roles of these processes has been significantly hindered by our inability to unambiguously profile the targets of the over 60 human protein methyltransferases in vivo. To address this formidable challenge and thus vertically advance the epigenetic research, we propose to develop a highly innovative technology, which we termed Bioorthogonal Profiling of Protein Methylation (BPPM) and, as a paradigm, apply the approach for target profiling of cancer-relevant protein methyltransferases. In conjunction with our supportive preliminary results, we envisioned that protein methyltransferases can be rationally engineered to exploit S-adenosyl-L-methionine analogues as alternate cofactors and thus label their targets with distinct chemical groups. The distinct modifications can be selectively enriched and unambiguously characterized with respective reporters for BPPM. We further plan to leverage the approach for in vivo BPPM by incorporating a creative approach for in situ production of SAM analogues. With regard to expected outcomes, the proposed work is aimed at providing transformative technological advancement and generally applicable reagents for target profiling of protein methyltransferases. Compared with conventional approaches, our BPPM is featured as the high integrity by using intact methyltransferases in context of cellular proteome, the high specificity by linking cell-type-specific methylation profiles to designated methyltransferases, and the high sensitivity by selectively enriching modified targets from proteome. The results are expected to reveal how the methylation profiles differ between diverse protein methyltransferases, in normal versus pathological stages or in non-aggressive versus aggressive diseases. Public Health Relevance: Deregulated protein methylation can cause developmental abnormalities, neurological disorders and cancer. The successful completion of this proposal is expected to provide the valuable tools to understand the molecular mechanisms involved in these disease processes. The resultant knowledge is expected to enhance our understanding of aberrant methylation pathways in these diseases and likely lead to the identification of novel biomarkers for disease diagnosis or targets for pharmacological intervention. Additionally, this research will contribute to a broader understanding of stem cell differentiation, for which protein-methylationinvolved epigenetic regulations have not been well understood.