Role of post-translational modifications to lens proteins in cataract formation Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/2f75rb50f

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  • Cataract is a leading cause of blindness throughout the world, yet the fundamental biochemical causes are unknown. A rodent model of the biochemical processes is selenite cataract. This cataract shows some of the features of human cataracts such as increased lens calcium, proteolysis of proteins, and insolubilization leading to lens opacity. The goals of the current experiments were: (1) To measure changes in transcript levels for calpains and caspase 3 and oxidation of epithelial proteins in selenite cataract. (2) To elucidate changes in calpain 10 and its interaction with other calpains in selenite cataract. (3) To investigate changes in stability of βB1-crystallin caused by deamidation and truncation. These data would provide roles for apoptosis, protein insolubilization, proteolysis and deamidation observed in cataract. To induce cataract, 12-day old rats were injected with an overdose of Na₂SeO₃. Epithelium was analyzed by competitive RT-PCR, zymography, and thiol-blotting. Calpains were detected by western-blotting. For βB1-crystallin stability studies, recombinant βB1-crystallins were denatured by urea or heat. Urea stability was measured by circular dichroism and fluorescence spectrometry, and heat stability was measured by light scattering at 405 nm. During selenite cataract formation, calpains in epithelium were activated resulting in increased proteolysis of crystallins, but mRNA levels for calpains did not show appreciable changes. Oxidation of sulthydryls in epithelial proteins was minimal during cataract formation. These results suggested that calpain-induced proteolysis in the epithelium contribute to selenite cataract. In selenite cataract, calpain 10 proteins disappeared, which appeared to be due to degradation by calpain 2 and Lp82 calpain. Deamidated βB1-crystallin was less stable in urea and heat, compared to wildtype. When the terminal extensions were removed, βB1-crystallin was as stable as wild-type. However, without the extensions, truncated βB1-crystallin caused accelerated precipitation in a complex with αA-crystallin, suggesting that the extensions may contribute to proper association with other crystallins and to stability of the soluble complexes. In summary, proteolysis of proteins by calpains was more pronounced than protein oxidation in lens epithelium of selenite cataract. Deamidation and truncation caused instability of βB1-crystallin and abnormal association with αA-crystallin. Thus, proteolysis and deamidation may increase susceptibility of lenses to cataract.
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  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-08-20T21:11:29Z (GMT) No. of bitstreams: 1 KimYungHae2003.pdf: 10502703 bytes, checksum: 0b86c8b541504db3ca18c6cb0e7c09ce (MD5)
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