This-proposal is-to clarify the mechanisms for human senile-cataract formation.-Emphasis will be to-provide"""""""" evidences for the hypothesis that under oxidative stress, the formation of protein-thiol mixed disulfide (protein thiolation) with glutathione (PSSG) and cysteine (PSSC) is one of the early oxidative damage to lens proteins/enzymes. Thiol modification may lead to loss of physiological functions, protein-protein disulfide crosslinks, and eventual cataract formation. We also hypothesize that the enzyme systems of glutathionedependent thioltransferase (TTase) and NADPH-dependent thioredoxin/ thioredoxin reductase (TRx/TR) can dethiolate and repair some of the damaged lens proteins/enzymes induced by oxidative stress, thus maintain the cellular redox status and lens clarity. We further propose that TTase and TRx may have physiological functions to mediate redox signaling for cell proliferation and other functions.
The specific aims are 1) To determine the distributions and activities of TTase and TRx oxidation repair systems in relationship to age and site of opacity in human lenses. 2). To study the antioxidation, anti-apoptotic properties of TTase1/2, and the physiological functions of TTase-1 in regulating redox signaling-mediated growth factor mitogenic action. The mitochondrial target substrates for TTase-2 will be isolated and identified 3) To study the cytokine-like function of TRx and the in situ bioavailability regulated by TRx specific binding protein. We will use normal human lenses and LOCSIII classified ECCE cataract subtypes for activity assay and immunolocalization of the enzymes. The importance of TTase-1/2 in maintaining lens clarify, cell growth and function will be clarified using TTase-1/2 knockout mice. The reversible S-thiolation of low molecular weight protein tyrosine phophatase (LMWPTP) and actin cytoskeleton protein will be used as the models for testing the function of TTase-1 using wild type, TTase-1 knockout or overexpressed lens epithelial cells. The redox substrate trapping technique for TTase-2 targets includes separating by affinity column made with chimeric TTase-2 (CxxS, SxxC) and sequencing by mass spectroscopy. Cytokine-like property of TRx will be examined for TRx receptor, MAPK signaling and gene response. The negative regulation of TRx will be examined under oxidative stress and osmotic stress conditions. The results of these studies will further our understanding on the mechanism of cataractogenesis and the importance of the thiol repair systems to maintain lens clarity.
