Crystallin proteolysis in lens during aging and cataract formation Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/fb494c054

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  • The major purpose of this research was to characterize partially degraded proteins appearing in the ocular lens during aging and cataract, and to identify the responsible proteolytic activities. This research is significant, because increased protein degradation is associated with lens opacification and cataract. Determining the sites where lens proteins become truncated and identification of the responsible proteases is important because this information could be used to develop anticataract agents, such as new protease inhibitors. The methods used in this research include the application of several techniques employed in the field of proteomics. Two-dimensional electrophoresis (2-DE) was used to separate crystallins, the major proteins of the lens, and the modifications to these proteins were then determined by electrospray ionization mass spectrometry (ESI-MS). Lenses from mice of increasing age were used for these studies. Lp82 and m-calpain, the proteases hypothesized to cause the observed truncation of mouse crystallins, were purified from fetal calf lenses or expressed recombinantly, and their enzymatic properties compared. The cleavage sites produced in α-crystallin by Lp82 and m-calpain in vitro were then compared to truncated α-crystallins from normal rat lens, and rat lens with selenite-induced cataract. The results included data showing that crystallins in mouse lenses underwent extensive truncation after 6-weeks of age that was associated with their insolubilization. Characteristics of purified Lp82 included: a lower calcium requirement for activation, a decreased sensitivity to the endogenous inhibitor calpastatin, and a greater resistance to autolysis than m-calpain. Analysis of truncated α-crystallins isolated from both normal and cataractous rat lens indicated that Lp82 was more active in young rat lens than was m-calpain. Lp82 specifically removed 5 residues from the C-terminus of αA-crystallin. This αA degradation product was far more abundant in the lenses of young rats than was the m-calpain specific aA product missing 11 residues from its C-terminus. In conclusion, this study showed that insolubilization of crystallins and cataract may result in animal lenses from uncontrolled crystallin degradation by the protease Lp82. This has led to a fundamental change in our view of experimental cataract formation, since activation of the protease m-calpain was previously believed to cause cataract.
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  • description.provenance : Approved for entry into archive by Patricia Black(patricia.black@oregonstate.edu) on 2012-08-24T16:52:41Z (GMT) No. of bitstreams: 1 UedaYoji2001.pdf: 7753785 bytes, checksum: e902bd055dc0c2bed45fd0f8224e9ca2 (MD5)
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