The long term objective of this project is to understand the role of proteolysis in the formation of cataract. It is hypothesized that partial degradation of beta-crystallins initiates protein insolubilization in the lens. This proteolytic insolubilization proceeds at a slow rate in normal lenses during maturation and aging and is compatible with lens transparency. However, during cataract formation there is a loss of protease regulation which accelerates the rate of insolubilization, leading to lens opacification. The proposed work will test this hypothesis by characterizing the proteolysis occurring in insolubilized lens crystallins during both aging and cataract formation.
The specific aims are to: 1. Determine the cleavage sites in the partially degraded alpha and beta- crystallins appearing in the insoluble fraction of rat lens during cataract formation and lens maturation. 2. Determine if protein insolubilization in bovine and rabbit lenses during maturation and cataract formation is associated with calpain- induced proteolysis. 3. Determine the identity and cleavage sites of partially degraded crystallins appearing in human lens during maturation. These experiments will utilize two-dimensional electrophoresis to purify crystallins and their degradation products. The purified crystallins will then be analyzed using both direct Edman sequencing of crystallins from blotted membranes, and a combination of fast atom bombardment and electrospray mass spectroscopy of electroeluted crystallins. The precise cleavage sites on the N- and C-terminal regions of partially degraded crystallins will be determined and comparisons made between the naturally occurring cleavage sites found in the insoluble crystallins of aged lenses and cleavage sites found in insoluble crystallins from cataractous lenses. The cleavage site information will also determine which proteases are responsible for crystallin degradation. These studies are significant, because how post-translational changes in crystallins lead to their insolubilization is poorly understood, and crystallin insolubilization maybe a major mechanism by which cataracts form.
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