Development and Application of Chemical Biology Approaches for Understanding Protein Arginylation

Project summary Posttranslational arginylation on proteins installed by arginyltransferase ATE1 is a critical modification for cardiovascular and heart development. The absence of this modification in ATE1 knockout mice was embryonically lethal with various signs of cardiovascular defects, emphasizing the importance of arginylation in physiological processes. Existing methods have proposed plausible arginylation sites on a handful of proteins, most of which have not been further validated or functionally investigated. Therefore, protein arginylation remains a vastly understudied field. It is still largely unclear how many proteins are arginylation substrates in cardiac tissue/cell proteomes, and what the effects of arginylation are on cardiovascular development? In this proposal, we will first establish a chemical proteomics platform for enriching the arginylation sites to facilitate the discovery of this challenging PTM. We will apply this technology to cardiac cells and clinical heart tissues to comprehensively profile the arginylation sites hidden in cardiovascular networks. In addition, our preliminary data discover that another cardiovascular-related molecule, homoarginine, is a proteinogenic amino acid, we thus will use isotopic homoarginine to unbiasedly discover the occurrence and distribution of the “homoarginylome” and homoarginine-associated metabolites in cardiovascular samples as well. These investigations will provide insights into the biological effects of homoarginine as an amino acid building block and a metabolite. We next focus on the elucidation of protein arginylation function in selected systems. A combination of mass spectrometry, biochemistry, and next-generation sequencing techniques will be used to study the conformational alterations of nucleosome after arginylation of histones to understand how epigenetic mechanisms can be regulated by arginylation during cardiomyoblast differentiation. By controlling the calreticulin arginylation levels, we will also study the impacts of arginylation on cardiac differentiation and development using stem cell and cardiology techniques. To further explore the therapeutic potential of protein arginylation, we aim to establish the arginylation targeting chimera (ArgTAC) technology for targeted PCSK9 arginylation and degradation. Successful degradation of PCSK9 by this arginylation system will potentially provide a novel therapeutic strategy in lowering LDL particles leading to improved cardiovascular conditions. Since protein degradation is the only known mechanism to remove protein arginylation, we will further explore the novel possibility that enzymes exist for de-arginylation of proteins (reversing arginylation). Towards this, we will apply CRISPR whole genome screening to identify putative de-arginylation enzymes before validation using enzymatic assays to characterize their de-arginylation activity. The proposed studies will unveil the prevalence and noncanonical biological roles of this essential PTM, and the novel technologies from this proposal will greatly benefit the research fields of protein arginylation, epigenetics, and cardiovascular disease.