Grant: Discovery Grants Program - Individual

Lysine methylation is a prevalent post-translational modification (PTM) that plays an important role in normal physiology and disease pathogenesis. While Lys methylation occurring on histones constitutes an essential part of the epigenetic program that controls chromatin structure and gene expression, non-histone protein Lys methylation is associated with numerous cellular functions ranging from signal transduction to cell differentiation. The human proteome is predicted to harbor more than 30,000 Lys methylation (Kme) sites of which fewer than 5,000 has been identified to date. This discrepancy highlights an unmet need in the study of Lys methylation and calls for the development of an effective strategy for systematic Kme identification. This NSERC DG program is focused on developing novel reagents for the effective capturing of Lys methylated peptides or proteins and accompanying mass spectrometry-based proteomics strategies that enable large-scale identification and functional annotation of Lys methylation. Our Objectives are: 1.We will engineer methyllysine-superbinders by in vitro evolution of naturally occuring Kme-binding domains (KMBD) to acquire markedly enhanced affinities for methyllysine. This will be accomplished by creating phage-displayed libraries of KMBDs followed by selection with Kme-containing peptide probes. The resulting KMBD mutants with increased affinities, dubbed Kme-superbinders, will be used to enrich Lys methylated proteins or peptides prior to their identification by mass spectrometry (MS). 2.We will develop MS-based proteomic methods to systematically identify protein Lys methylation. To this end, we will employ KMBD mutants developed to date and the Kme-superbinders developed in Objective 1 as affinity purification (AP) reagents for Kme-containing peptides or proteins (and the associated protein complexes) followed by identification by tandem MS (MS/MS) or targeted MRM (Multiple Reaction Monitoring) analysis. 3.We will characterize the dynamic changes and function of Lys methylation in cellular processes. Using stem cell neuronal differentiation and T cell activation/inhibition as examples, we will employ quantitative MS strategy enabled by the Kme- and pTyr-superbinders to elucidate how dynamic changes in Lys methylation and its crosstalk with Tyr phosphorylation regulate cell differentiation and immune function. Our long-term goal is to use the innovative proteomic strategies developed herein to map the methyllysine proteome at the organismal level and in key cellular processes. The decoding of the methyllysine proteome would lay the foundation for its functional annotation in health and disease at unprecedented resolution and scale.