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. Such modification may lead to loss of physiological functions, protein-protein disulfide crosslinks, and eventual cataract formation. We also propose that the reversible protein thiolation observed in early stage of oxidative stress is mediated by two redox regulating systems, the GSH-dependent thioltransferase (TTase) and the NADPH-dependent thioredoxin/thioredoxin reductase (TRx/TR), to repair the oxidative damage and to restore thiol/disulfide homeostasis in the lens. Thus, cataract will form if the repair systems fail.
The specific aims are 1) To determine the distributions of free and bound-cysteine and glutathione, the irreversible thiol oxidation end products, and the activities of TTase and TRx in relationship to age and the site of opacity in human lenses. 2) To study if transulfuration pathway is present in the lens and contribute to the observed elevations of cysteine and PSSC in the fiber cells of oxidant-induced cataracts. 3) To study the physiological functions of TTase in maintaining the redox homeostasis and cell viability. 4) To study/TR system in the lens for its contribution to oxidation damage repair of lens proteins/enzymes. We will use LOCSIII to classify the ECCE lenses (nuclear portion) into cataract subtypes and the conventional method to classify the ICCE (intact) human cataractous lenses. Both will be used for various studies. Immunohistochemical and immunocytochemical techniques will be used to study the distributions of PSSG/PSSC and TTase/TRx in the human tissues. The significance of TRx in the lens will be verified by using TRx overexpressed and suppressed cells. The importance of TTase in maintaining lens clarify will be clarified using TTase transgenic and knockout mice. Non-invasive dynamic light scattering probe will be used to detect large protein aggregates possibly formed in the TTase knockout mice. Human lens epithelial cells (B3) and human donor lenses will be used throughout the studies. The results of these experiments will further our understanding on the mechanism of cataractogenesis and the importance of repair systems to protect the lens from cataract formation.
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