Modification of Alpha-Crystallin Chaperone Function

描述（由申请人提供）：儿童白内障是儿童失明的最常见形式，在临床和遗传上都是异质性的。迄今为止，已报道由近22个不同基因突变引起的常染色体显性和隐性形式的白内障。超过一半的突变体发生在晶体蛋白，在1？- 1、？主要是晶体蛋白该提议旨在研究1？？- 1、？晶状体蛋白通过引入显著的构象变化而引起白内障，所述构象变化将引发一系列事件，导致功能障碍的分子伴侣缺乏与天然1？？1、？晶体蛋白和各种蛋白质底物。在本研究中，我们选择了8个突变体的1？？-晶体蛋白和4个1？晶体蛋白，所有已知的负责先天性白内障。我们将研究1）这些突变体的结构，功能和流体力学性质的同源寡聚体和异源寡聚体的形式（目的1和2），2）的相互作用的突变体与1？- 重量和1？？- wt的荧光共振能量转移在体外系统（目的3），和3）研究突变体的表达和它们与1？Wt和1？wt使用FRET并评估蛋白质聚集体的存在，而每个突变体单独表达或与1？？wt或1？？-野生型在哺乳动物细胞中。这些研究有望揭示各种突变体在受影响个体中引起白内障的潜在机制。 公共卫生相关性：儿童白内障是儿童失明的最常见形式。全球约有2000万16岁以下的儿童患有白内障，其中约15%严重视力受损或失明。本研究旨在分析眼透镜中的突变蛋白，以揭示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.