Development of spatiotemporal reactivity mapping in live C. elegans and mechanistic investigations into reactive lipid electrophile sensing & signaling in stress response

Lipid peroxidation products, particularly the reactive aldehyde, 4-hydroxynonenal (4-HNE), is an important marker for oxidative stress and a potential contributor to the pathogenesis of chronic diseases, including neurodegenerative disorders. This thesis discusses the novel methodologies to (i) quantitatively map the 4-HNE responsive proteins in an organ-specific manner in live C. elegans, (ii) uncover the animal's innate detoxification mechanisms at a molecular level, altered in response to electrophilic stress induced by 4-HNE and its derivatives at a single protein-site specific context, and (iii) identify the new mode of lipid homeostasis regulation in the context of exogenous and endogenous stress in C. elegans neuronal disease models. Chapter 1 introduces lipid droplet (LD) assembly, regulation, degradation mechanisms of conserved importance from worms to man, and human disease relevance, and the current strategies, including state-of-the-art chemical biology tools, for studying LD metabolism-associated proteins. Chapter 2 explains the principles and the developments of OS-Localis-REX and OS-UltraID, two proteomics platforms enabling, respectively, electrophile function-guided or local proteome abundance-guided proximity mapping, their applications in C. elegans, and advantages and limitations of the two approaches based on our mapping outcomes. The functional relevance of 15 outstanding targets mapped by OS-Localis-REX was further studied by RNA interference and phenotypic screening in the context of 4-HNE stress. Chapter 3 focuses on one such hit that we found to manifest prominent 4-HNE stress-responsive roles, including in C. elegans Huntington's disease models: cyp-33e1 and its human ortholog, CYP2A6. Further studies in mammalian cells and worms revealed a partitioning between enzyme inhibition and localized production of a metabolite that we found critical for lipid storage regulation. This metabolite, 4-hydroxynonenoic acid (4-HNA), is produced through 4-HNE labeling at the conserved catalytic cysteine. Interestingly, an additional off-catalytic site cysteine functionally participates in 4-HNE stress sensing that we found to govern intestinal homeostasis. Chapter 4 discusses a parallel collaborative investigation into another electrophile-responsive protein we discovered, SAHH, whose loss of HNE responsivity alters the amino-acid metabolism.