The molecular mechanisms underlying the aging process are poorly understood, thereby hindering the development of therapeutics to delay the onset of aging and age-related degenerative diseases. Genetic studies in the widely used model organism, Caenorhabditis elegans, have generated daf-2 mutants defective in insulin/IGF-1 signaling (IIS), which have significantly extended lifespans. The daf-2 mutation activates DAF-16, a transcription factor, which initiates downstream gene expression changes that mediate the life-extension phenotype. We have identified protein-activity changes that are downstream effects of DAF-16 activation using the tools of chemical proteomics. These studies complement conventional genomic and proteomic approaches by providing insight into low-abundance proteins and posttranslational modifications (PTMs) implicated in IIS. From our preliminary studies, we identified a lipid-binding protein, LBP-3, which upon RNAi-mediated knockdown increases both lifespan and dauer formation in C. elegans. Given the established dysregulation of lipid metabolism in IIS, and the confirmed role of other lipid-binding proteins in controlling lifespan and stress resistance, we hypothesize that LBP-3 is a novel mediator of IIS. To test this hypothesis, we will determine the mechanism by which LBP-3 acts within known nodes of the IIS pathway to regulate lifespan. Furthermore, since mammalian homologs of LBP-3 are known to be redox regulated, and dysregulation of reactive oxygen species (ROS) levels is a characteristic feature of IIS, we will investigate the in vivo oxidation state of LBP-3. In additio to revealing a novel mode of regulation for C. elegans LBP-3, these studies will also serve to more globally evaluate protein oxidation events accompanying IIS. Lastly, since oxidation of mammalian LBPs is known to affect protein stability and lipid binding, we will evaluate the effect of oxidation on C. elegans LBP-3 function. LBP-3 contains a highly reactive cysteine residue, Cys154, which is the predicted site of oxidation. We will exploit this reactive cysteine to develop covalent small-molecule probes and inhibitors to pharmacologically modulate LBP-3 function in C. elegans. Together, these studies will: (1) characterize a novel downstream mediator of C. elegans IIS; (2) reveal the role of protein oxidation in governing the function of LBP-3 and other C. elegans proteins during IIS; and (3) demonstrate that LBP-3 can be targeted by small molecules to pharmacologically modulate C. elegans lifespan.
We propose to apply chemical proteomic technologies to discover changes in cysteine- mediated protein activities during insulin/IGF-1 signaling and aging in C. elegans. Our studies are geared toward discovering novel protein activities and posttranslational modes of regulation implicated in aging.
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