Modification of Alpha-Crystallin Chaperone Function

描述(申请人提供)：人和大鼠晶状体中存在不同数量的C-末端残基缺失的AA-和AB-晶体蛋白。在人类晶状体中为Aa1-172、Aa1-168、Aa1-162、AB1-174和AB1-170，在大鼠晶状体中为Aa1-168、Aa1-163、Aa1-162、Aa1-157、Aa1-151和AB1-170。我们发现糖尿病人和大鼠晶状体中截短的AA-和AB-晶状体蛋白水平升高。我们发现，大鼠Aa1-168和Aa1-163具有接近正常的伴侣活性和低聚物大小，而Aa1-162、Aa1-157和AA-151失去了60-100%的伴侣活性，其低聚物大小减少了75%。人Aa1-172失去了40%的伴侣活性，低聚物大小没有变化。值得注意的是，Arg-163的切割形成Aa1-162，导致低聚物大小显著减小。截短的AA-和AB-晶状体蛋白主要以均聚体和杂聚体的形式存在于晶状体中，而正常的AA和AB则以杂聚体的形式存在。因此，分子伴侣的功能、结构和寡聚体的大小会受到不同形式聚集体形成的影响。同质相互作用和异质相互作用的相对强度将决定各种形式的聚集体的水平。本研究的目的是：1)通过构建和鉴定Arg-163的定点突变体，探讨Arg-163在人和大鼠AA-和AB-晶体蛋白齐聚中的作用；2)研究当突变体以均聚体和异聚体的形式存在时，人和大鼠AA-和AB-晶体蛋白C末端残基截断的影响；3)研究Arg-163的均相和异相相互作用 通过使用荧光共振能量转移来确定亚基交换速率来确定截短突变体，以及4)使用哺乳动物双杂交试验来确定截断突变体在体内的同质和异质相互作用的程度。这些研究有望揭示各种AA和AB晶体蛋白C末端截断突变的产生的后果，以及它如何影响晶状体病理，特别是糖尿病晶状体。

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.