PROFILING OF LOW-OCCUPANCY, KINETICALLY PRIVILEGED ELECTROPHILE SENSORS IN THE C. ELEGANS CYSTEINOME: A WINDOW INTO ENDOGENOUS REDOX SIGNALING
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Urul, Daniel Ahmet
Recent advances in the pharmacokinetic understanding of irreversible inhibitors have led to renewed interest in covalent drug development. A major driving force behind this therapeutics renaissance is the emergence of a “redox-sensing proteome” that facilitates key intracellular signaling pathways and acute stress responses by utilizing reactive electrophilic species (RES) in lieu of canonical signaling biomolecules. The ability to detect RES at endogenous concentrations and convert RES flux into a downstream output is engendered by “kinetically privileged” cysteine residues able to facilitate rapid covalent modification. Thus, mining the cysteinome for such uniquely reactive targets can guide future pharmaceutical strategies. Indeed, several contemporary profiling techniques have been developed to capture these cysteines and their associated RES-sensor proteins. However, despite their continued usefulness, many of these systems require heavy exogenous dosing of the RES of interest. In treating the system with minimal temporal or spatial control, many RES-sensing proteins that undergo low endogenous modification of an otherwise “kinetically privileged” cysteine sensor may be missed, and the pathways facilitated by the ones that are captured may be obfuscated by the breadth of accompanying off-target effects. Furthermore, many of these methods are incompatible with live-animal models, further reducing the biological context within which these “low-occupancy” sensors operate. To better elucidate these RES sensors and their impact as signaling mediators, a novel profiling method must be able to facilitate controlled RES deployment within a whole-organism context. This work describes the development of one such method, adapting a comprehensive screening platform for the direct identification of novel, low-occupancy RES sensors for use in C. elegans, a renowned model organism. Following proof-of-concept targeted labeling of a known RES sensor in vivo, we uncovered novel redox-sensing functionality in S-adenosylhomocysteine hydrolase (SAHH) through pilot cysteinomic profiling efforts in C. elegans. This highly conserved SAHH also allowed us to investigate the nature of RES-sensing conservation across species, forming a basis through which sensing capability in seemingly disparate species can inform functionality in a human context. With the potential to further fine-tune future profiling efforts with tissue- and organelle-specificity, C. elegans offers a significant avenue for deepening our comprehension of RES-mediated signaling pathways with relevance to human biology.
C. elegans; Covalent inhibitors; Electrophile signaling; Reactive cysteines; Reactive electrophilic species; Redox signaling
Cerione, Richard A.
Aye, Yimon; Liu, Kelly Jun
Chemistry and Chemical Biology
Ph. D., Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis