Modification of Alpha-Crystallin Chaperone Function

描述（由申请人提供）：人类和大鼠晶状体中存在不同数量的c端残基被删除的aA-和ab -晶体蛋白。人晶状体为a1 -172、a1 -168、a1 -162、a1 -174和a1 -170，大鼠晶状体为a1 -168、a1 -163、a1 -162、a1 -157、a1 -151和a1 -170。我们发现，在糖尿病人和大鼠晶状体中，截断的aA-和ab -晶体蛋白水平增加。我们已经证明大鼠a1 -168和a1 -163具有接近正常的伴侣活性和寡聚物大小，而a1 -162、a1 -157和aA-151失去了60-100%的伴侣活性，其寡聚物大小减少了75%。人类a1 -172失去了40%的伴侣活性，寡聚物大小没有变化。值得注意的是，Arg-163的裂解形成a1 -162，导致寡聚物尺寸大幅减小。截短的aA-和aB-晶体蛋白在晶状体中主要以同聚集体和异聚集体的形式存在，与正常的aA和aB存在异聚集体。因此，不同形式聚集体的形成会影响伴侣蛋白的功能、结构和寡聚物的大小。同质相互作用和异质相互作用的相对强度决定了各种形式聚集体的水平。本课题旨在：1)通过生成和表征Arg-163位点定向突变体，探索Arg-163在人和大鼠aA-和ab -结晶蛋白寡聚化中的作用；2)研究突变体以同质聚集体和异质聚集体形式存在时，人和大鼠aA-和ab -结晶蛋白c端残基截断的影响

DESCRIPTION (provided by applicant): 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.