SORTASE AND CHEMICAL BIOLOGY AS TOOLS TO STUDY UBIQUITINATION

Ubiquitin, a small protein added post-translationally to proteins, is implicated in a plethora of essential cellular processes including but not limited to protein homeostasis, protein trafficking, cell signaling cascades, and gene expression. Despite nearly 50 years’ worth of scientific research, many questions still surround the impacts and roles of protein ubiquitination due to limitations in the current technologies available to study the role of this common post-translational modification. To address limitations in this field, we leverage the power of sortase, a bacterial transpeptidase enzyme crucial to cell wall synthesis, to develop new strategies for studying protein ubiquitination. As one new technology, we introduce point mutations to the ubiquitin sequence in combination with click chemistry and genetic code expansion to develop a strategy for site-specific modification of proteins of interest with user-defined polyubiquitin chains. We exemplify the broad utility of this strategy for multiple different proteins of interest, including DNA replication protein PCNA. As an additional technology, we leverage this sortase-mediated ubiquitination system in combination with dehydroalanine chemistry to begin developing context-specific deubiquitinating enzyme (DUB) probes. We demonstrate early prospects of this strategy to capture DUB enzymes such as USP2 and highlight some key optimization points for future development of this strategy. Finally, we leverage the complementarity of sortase and ubiquitination to develop a strategy for enrichment of ubiquitinated protein sites with applications in mass spectrometry proteomics workflows. We demonstrate a versatile enrichment strategy using biotin-containing peptides on several ubiquitinated proteins following tryptic digest and begin to highlight future directions towards applying this strategy to proteomics studies in whole cell lysates. Taken together, these studies highlight complementarity between ubiquitination and sortase-mediated ligation and aim to increase accessibility to new aspects of ubiquitin biology that were previously inaccessible.