The broad and long range goal of this proposal is to understand and clarify the underlining 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, which may lead to protein-protein disulfide crosslinks with eventual cataract formation, and that the reversible protein thiolation observed in early stge of oxidative stress is mediated by a novel enzyme, thioltransferase (Ttase), to repair the oxidative damage and to restore thiol/disulfide homeostasis in the lens.
The specific aims are 1). To immunochemically map thiolated proteins in the lens during cataract progression and to isolated the thiolated crystallins and localize the site and nature of thiolation. 3). To study the association of PSSG and PSSC to lens opacity and pigmentation in human lenses and the effect of aging on thiol/disulfide homeostasis). To investigate the source of PSSC and its role in cataract formation. 4). To clarify if the dethiolation process in situ is catalyzed enzymatically and to study the substrate specificity of PSSG and PSSC to Ttase. 5). To examine the oxidative defense function of Ttase in the lens. 6). To evaluate the effect of oxidation on key metabolic enzymes in glucose metabolism and to clarify if Ttase involves in modulating their catalytic activities. The methods to be used for this proposal are 1). The in situ thiolated proteins will be generated by two cataract models: H2O2 (cortical) in vitro and hyperbaric O2 (nuclear) in vivo and in vitro. 2). Mass spectrometry will be used to located the site of thiolation in major thiolated crystallins. 3). PSSG and PSSC specific monoclonal antibodies will be used to located the site of thiolation in major thiolated crystallins. 3). PSSG and PSSC specific monoclonal antibodies will be used to locate the cellular distribution of PSSG and PSSC by immunohistochemical and immunocytochemical techniques. 4). In situ hybridization will be used to quantitate Ttase mRNA in normal and cataractous lens epithelial cells in humans. 5). HPLC will be used to quantitate fluorescence labeled free cysteine. 6). Immunochemistry and radiolabeling will be employed to characterize the in situ thiolated proteins in the cells. 7) Human epithelial cell line (B3) will be used to study the oxidative defense function of Ttase in normal-expressed, over-expressed and under-express human lens Ttase gene. 8). Recombinant human lens Ttase will be used to modulate the function/activity of several model proteins and enzymes that are damaged by oxidation. 9). The effect of H2O2 on glucose metabolism will be studied by radiolabeled C1-glucose and C6-glucose for hexose monophosphate shunt and glycolysis activities.
Showing the most recent 10 out of 30 publications
