The aims of this proposal are to clarify the roles of glutathione-dependent thioltransferase (TTase) and NADPH-dependent thioredoxin (TRx) in redox mechanism which offer protection against oxidative damage and cataract formation and which stimulate cell proliferation. Emphasis will be to provide evidence for the hypothesis that under oxidative stress, the formation of protein-thiol mixed disulfide with glutathione and cysteine is one of the early events in oxidative damage to lens proteins/enzymes. Thiol modification may lead to loss of physiological functions, protein aggregation, and eventual lens opacity. We also hypothesize that TTase and TRx can dethiolate and repair some of the oxidation-damaged lens proteins/ enzymes, thus maintaining the cellular redox status and lens clarity. We further propose that TTase and TRx may regulate redox signaling for cell proliferation and other functions.
The specific aims are 1) To determine the activities and distributions of TTase and TRx oxidation repair systems in relationship to age and site of opacity in human lenses. 2) To study the effect of cytosolic TTase (TTase-1) deletion on lens opacity in aging and UV radiation, and to examine TTase-1 functions in antioxidation, anti-apoptotic, and control of redox mediated growth factor signaling. 3) The mitochondrial target substrates for TTase isozyme (TTase-2) will be isolated and identified. 4) 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 cataracts for activity assay and immunolocalization of the enzymes. The relationship of TTase-1 to lens clarify will be studied using TTase-1 knockout (KO), transgenic and wild type (WT) mice. Studies on cell growth and apoptosis will use human lens epithelial B3 cells with TTase 1or 2 overexpressed or deleted, or LEC isolated from KO and WT mice. The reversible S- thiolation of actin and low molecular weight protein tyrosine phosphatase will be used as the models for testing the function of TTase-1. The trapped TTase-2 targets will be separated on an affinity column made with chimeric TTase-2 (CxxS, SxxC) and sequenced by mass spectrometry. Cytokine-like property of TRx will be examined for TRx receptor and signaling cascade while TRx negative regulation will be studied by TRx binding protein-2. The results of these studies will further our understanding of the mechanism of cataractogenesis and the importance of the thiol repair systems in maintaining lens clarity.
Human age-related cataract is the major cause for blindness in the world, yet the reason for cataract formation is not well-understood. This proposed research is based on the hypothesis that cataract is induced by oxidative damage, thus the research will focus on the oxidative damage repair systems in the lens so that a better strategy can be developed for preserving lens clarity. ? ? ?
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