Friday, May 10, 2024
5/10/2024
Project Summary Oxidative stress contributes to the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. One carbon metabolism influences the response to oxidative stress via antioxidant biosynthesis; however, this pathway is also perturbed upon oxidative stress in the Drosophila eye. One explanation for the observed dysregulation of one carbon metabolism under oxidative stress is because of oxidative modifications on the key enzyme, Adenosylhomocysteinase (Ahcy). Ahcy is a highly conserved enzyme and is solely responsible for the hydrolysis of S-adenosylhomocysteine (SAH) into adenosine and homocysteine in all higher eukaryotes. Upon inducing oxidative stress in Drosophila cells and eyes, Ahcy becomes oxidized at cysteine residue 195 (C195) correlating with increased SAH. This is important because increases in SAH can inhibit methylation reactions and alter the epigenome; however, the mechanism by which oxidative stress perturbs one carbon metabolism is unknown. Based on these data, I hypothesize that redox modifications at C195 inhibit Ahcy activity under oxidative stress leading to increased SAH and a decreased methylation capacity resulting in changes in gene expression. In the proposed studies, I will employ in vitro assays, biochemical, genome-wide sequencing, and genetic manipulation studies to elucidate if Ahcy is redox regulated and if it serves as a neuroprotective mechanism against retinal degeneration. In aim I, I will determine if C195 is the only (or major) oxidized cysteine, determine the type of oxidative modification, characterize the impact of C195 oxidation on enzymatic activity of Ahcy using a novel DESI-MS approach, and test if C195 mutations regulate Ahcy enzymatic function in vivo. In aim II, I will identify genes regulated by Ahcy, test if the genetic downregulation of Ahcy alleviates blue light induced retinal damage, and identify the genomic loci directly bound by Ahcy. Along with the experiments planned in these aims, I will obtain expertise in recombinant protein purification, enzymatic assay development and characterization, and proficiency in large- scale dataset bioinformatic analyses. These skillsets will be an asset for my future career goal of entering the pharmaceutical industry. Overall, this proposal will elucidate the regulation mechanism of Ahcy and may lay the foundation for future studies to develop targeted therapies to delay or prevent the onset ocular disease.
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