Modification of Alpha-Crystallin Chaperone Function

描述(申请人提供)：儿童白内障是最常见的儿童失明形式，在临床和遗传上都是不同的。到目前为止，据报道，常染色体显性和隐性白内障是由近22种不同基因的突变引起的。超过一半的突变体出现在晶体蛋白中，主要是1？-和1-？-晶体蛋白。本研究旨在探讨1？？-和1？？-晶状体蛋白基因突变可能通过引入显著的构象变化而导致一系列事件，从而导致功能失调的分子伴侣缺乏与天然的1？？-和1？？晶状体蛋白和各种蛋白质底物相互作用的能力。在这项研究中，我们选择了8个1？？-晶体蛋白突变和4个1？？-晶体蛋白突变，它们都是导致先天性白内障的已知原因。我们将研究1)这些突变体的结构、功能和流体动力学性质(目标1和2)，2)在体外系统中利用FRET研究突变体与1？wt和1？wt的相互作用(目标3)，3)利用FRET研究突变体的表达及其与1？wt和1？wt的相互作用，并评估蛋白质聚集体的存在，而每个突变体在哺乳动物细胞中单独表达或与1？wt或1？wt共表达。这些研究有望揭示各种突变导致受影响个体白内障的潜在机制。 与公共卫生相关：儿童白内障是最常见的儿童失明形式。在全球范围内，约有2000万16岁以下儿童患有白内障，其中约15%严重视力受损或失明。目前的研究是关于分析眼睛晶状体中的突变蛋白质，目的是揭开白内障发生的潜在机制。

DESCRIPTION (provided by applicant): Pediatric cataract is the most common form of childhood blindness and is both clinically and genetically heterogeneous. Autosomal dominant and recessive forms of cataract have been reported to be caused by mutations in nearly 22 different genes so far. More than half of the mutants occur in crystallins, in 1?- and 1??-crystallins mostly. This proposal is aimed to investigate the possibility that mutations in 1??- and 1??-crystallins cause cataract by introducing significant conformational changes which will initiate a cascade of events leading to dysfunctional molecular chaperones lacking the ability to interact with native 1??- and 1??-crystallins and the various protein substrates. For this study, we have selected 8 mutants of 1??-crystallin and 4 mutants of 1??-crystallin, all known to be responsible for congenital cataracts. We will study 1) the structural, functional and hydrodynamic properties of these mutants in the homooligomeric and heterooligomeric forms (Aims 1 & 2), 2) the interaction of the mutants with 1?? -wt and 1??-wt by using FRET in an in vitro system (Aim 3), and 3) study the expression of the mutants and their interaction with 1?? -wt and 1?-wt using FRET and asses the presence of protein aggregates, while each mutant is expressed alone or co-expressed with 1??-wt or 1??-wt in mammalian cells. These studies are expected to show the underlying mechanism by which various mutants cause cataract in the affected individuals. PUBLIC HEALTH RELEVANCE: Pediatric cataract is the most common form of childhood blindness. Globally, about 20 million children under the age of 16 suffer from cataract and among them about 15% are severely visually impaired or blind. The present study is about analyzing the mutated proteins in the eye lens with a goal to unravel the underlying mechanism of cataract development.