aA- and aB-crystallins in which different number of C-terminal residues have been deleted are present in human and rat lenses. These are aA1-172, aA1-168, aA1-162, aB1-174, and aB1-170 in human lenses and aA1-168, aA1-163, aA1-162, aA1-157, aA1-151 and aB1-170 in rat lenses. We have shown increased levels of the truncated aA- and aB-crystallins in diabetic human and rat lenses. We have shown that rat aA1-168 and aA1-163 have nearly normal chaperone activity and oligomeric size whereas aA1-162, aA1-157, and aA-151 have lost 60-100% chaperone activity and their oligomeric size decreased 75%. Human aA1-172 has lost 40% chaperone activity with no change in oligomeric size. It is noteworthy that cleavage of Arg-163, which forms aA1-162 resulted in substantial decrease in oligomeric size. Truncated aA- and aB-crystallins could be present in the lens mainly as homoaggregates and as heteroaggregates with normal aA and aB. So, the chaperone function, structure and oligomeric size will be influenced by the formation of various forms of aggregates. The relative strength of the homointeractions and heterointeractions will dictate the level of the various forms of aggregates. This proposal is aimed: 1) to explore the role of Arg-163 in the oligomerization of human and rat aA- and aB-crystallins by generating and characterizing site-directed mutants of Arg-163, 2) to study the effect of truncations of C-terminal residues of human and rat aA- and aB-crystallins while the mutants exist in the form of homo-aggregates and heteroaggregates, 3)to study the homogeneous and heterogeneous interactions of the truncated mutants by determining the rates of subunit exchange using fluorescence resonance energy transfer, and 4) to determine the extent of homogeneous and heterogeneous interactions of the truncated mutants in vivo utilizing a mammalian two-hybrid assay. These studies are expected to show the consequence of the generation of the various aA- and aB-crystallin C-terminal truncated mutants and how it could influence lens pathology, in diabetic lenses in particular.
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