Cataracts are the third leading cause of blindness in the United States. However, the underlying biochemical mechanisms of most cataracts are unknown. One alteration in lenses that may contribute to cataract formation is accumulation of partially degraded protein fragments. Many experimental cataracts studied to date contain significant amounts of partially degraded lens proteins. The role these proteins play in cataract formation and which lens proteases are responsible for protein degradation are unknown. The two goals of this project are to better understand the significance of proteolysis in cataractous lenses, and to determine which endogenous lens proteases are activated during cataract formation. Partially degraded lens proteins, from selenite and galactose induced cataracts and rom hereditary mouse cataracts, will be isolated by electrophoresis. These partially degraded polypeptides will be analyzed using protein micro-sequencing techniques to identify their origin, and to determine their N - and C-terminal cleavage sites. Intact lens proteins will then be incubated in vitro with three different major lens endopeptidases (high molecular neutral protease complex, trypsin-like protease, and calpain II). The cleavage sites occurring in vitro will be compared to cleavage sites occurring in vivo during cataract formation. Endopeptidase cleaved lens proteins will also be incubated with two major lens exopeptidases (aminopeptidase III and leucine aminopeptidase). This will determine if endopeptidase cleavage makes lens proteins better substrates for exopeptidases. Proteolysis may contribute to cataract formation by significantly altering the properties of lens proteins. Comparison of in vivo cleavage sites found in cataractous lenses with cleavage sites produced by proteases in vitro may identify the proteases that are activated in the lens. Determining which lens proteases are active during experimental cataract formation may suggest ways of treating human cataract. For instance, inhibitors of proteases could be used as therapeutic agents to slow the progression of cataract.
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