Chemical Strategies to Reveal Cellular Glycation and Ubiquitination - Protein post-translational modification (PTM) networks are tremendously promising, but vast, frontiers for drug discovery. It is therefore essential to learn how specific PTMs are integrated into signaling networks in living cells. To address this need, the Scheck lab pioneers new chemical biology tools that will provide critical insight about PTM networks in living cells. Our focus is on PTMs like glycation and ubiquitination, which have been particularly difficult to study using traditional tools that inhibit or profile specific enzyme activities through genetic knockout or pharmacologic inhibition. Our novel methods will unlock previously unattainable information that will be used to address longstanding questions about the role of glycation, ubiquitination—and their interplay—in diabetes, cancer, inflammation, neurodegenerative disease, and other age-related disorders. Learning how these signals are integrated into cellular signaling processes will provide access to new targets for preventing or treating numerous diseases. This MIRA project describes chemical strategies that rely on our knowledge of PTM chemistry and mechanism, making us uniquely suited to develop needed methods to track specific glycation or ubiquitination events in living cells. One series of projects builds on our significant published and unpublished work that has uncovered the chemical and molecular features that underpin selective glycation, enabling us to create a novel tool (called dialAGE) that predictably modulates site-specific glycation events in living cells. The proposed studies include the use of deuterated methylglyoxal probes to differentiate previously indistinguishable AGE isomers using unbiased quantitative proteomics. We will also evaluate how differential ubiquitin glycation influences the global ubiquitinome and learn how it influences protein turnover through proteasomal or autophagic pathways. We will also create a new set of dialAGE tools to manipulate histone glycation, enabling studies that will reveal how glycation influences chromatin compaction, PTM crosstalk, and in vitro transcription. Another series of projects builds on a new tool we recently reported, called targeted Charging of Ubiquitin to E2 (tCUbE), that tracks the fate of ubiquitin through its sequential E1-E2-E3 cascade all the way to its ultimate target. The proposed studies will optimize tCUbE as an enabling technology that can be broadly used to profile each of the 38 known human E2s. We will also use tCUbE to interrogate specific E2s, such as UBE2L3, which we hypothesize to exhibit multiple activities that differentially engage disease-relevant signaling pathways. Uncovering new E2-substrate interactions or cascades will enable discovery of targeted degradation or stabilization therapies to disrupt or rewire specific steps within the UPS. Together, this work will uncover direct links between glycation, ubiquitination, and critical intracellular processes, including proteasomal degradation, mitophagy, and chromatin remodeling. These studies will dramatically improve our understanding of glycation and ubiquitination, and will have an immediate impact on our appreciation for how they influence human health, aging, and disease.
