Modification of Alpha-Crystallin Chaperone Function

性状（由申请人提供）：人和大鼠晶状体中存在C-末端残基缺失数量不同的aA-和aB-晶状体蛋白。在人晶状体中为aA 1 -172、aA 1 -168、aA 1 -162、aB 1 -174和aB 1 -170，在大鼠晶状体中为aA 1 -168、aA 1 -163、aA 1 -162、aA 1 -157、aA 1 -151和aB 1 -170。我们已经证明糖尿病人类和大鼠晶状体中截短的aA-和aB-晶状体蛋白水平增加。我们已经表明，大鼠aA 1 -168和aA 1 -163具有几乎正常的伴侣蛋白活性和寡聚体大小，而aA 1 -162、aA 1 -157和aA-151丧失了60-100%的伴侣蛋白活性，它们的寡聚体大小减少了75%。人aA 1 -172已经失去了40%的伴侣活性，而寡聚体大小没有变化。值得注意的是，形成aA 1 -162的Arg-163的裂解导致寡聚体大小的显著降低。截短的aA-和aB-晶体蛋白可以主要作为同质聚集体和作为与正常aA和aB的异质聚集体存在于透镜中。因此，分子伴侣的功能、结构和寡聚体的大小将受到各种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。这项建议的目的是：1）通过产生和表征Arg-163的定点突变体来探索Arg-163在人和大鼠aA-和aB-晶体蛋白的寡聚化中的作用，2）研究当突变体以同源聚集体和异源聚集体的形式存在时，人和大鼠aA-和aB-晶体蛋白的C-末端残基的截短的影响，3）研究Arg-163的同源和异源相互作用， 通过使用荧光共振能量转移测定亚基交换速率来确定截短的突变体，和4）使用哺乳动物双杂交测定来确定截短的突变体在体内的同质和异质相互作用的程度。预期这些研究将显示各种α A-和α B-晶状体蛋白C-末端截短突变体的产生的结果，以及它如何影响透镜病理学，特别是在糖尿病晶状体中。

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.