Gamma-crystallin modifications & mechanisms of lens opacity

描述（由申请人提供）：我们的长期目标是了解γ-晶状体蛋白的特定翻译后化学修饰和基因突变如何导致透镜混浊。我们假设并非所有的蛋白质修饰都是有害的，只有那些导致光散射增加的蛋白质修饰才是有害的。我们设计用于测试该假设的策略如下：我们将在体外模拟γ-晶状体蛋白的给定修饰或突变的白内障发生性，通过将所需的变化引入蛋白质并确定对蛋白质聚集体、含有富含蛋白质和缺乏蛋白质相的液滴和蛋白质晶体（即，蛋白质衍生的凝聚相）。增强这种凝聚相形成的蛋白质修饰（所有这些都导致光散射增加）将是潜在的致白内障的。我们将比较天然和修饰的γ-晶体蛋白的相行为（液-液和固-液相分离），以评估给定的修饰是否增强或抑制冷凝物的形成。同时，我们还将使用生物化学和光谱学方法（色谱法，HPLC，凝胶电泳，拉曼光谱，圆二色性，荧光和X射线晶体学）检查蛋白质结构和构象。我们提出以下具体目标： (1)评估发现的7种晶体蛋白的哪些生理学相关的化学修饰增强了凝聚相的形成-（1a）检查半胱氨酸残基的修饰（即，巯基化）与（i）谷胱甘肽（ii）半胱氨酸（iii）半胱氨酰甘氨酸和（iv）γ-谷氨酰半胱氨酸，（1b）检查天冬酰胺和谷氨酰胺残基的脱酰胺作用，和（1c）检查γ-晶状体蛋白多肽链截短的效果（2）评估γ-晶状体蛋白的哪些遗传变体增强凝聚相的形成-（2a）确定由与人类或动物模型白内障相关的（文献中）γ-晶状体蛋白基因表达的突变蛋白引起的混浊机制。（2b）确定由单核苷酸多态性引起的γ-晶状体蛋白基因变体表达的突变蛋白对混浊有什么影响。 这项工作的目的是提供合理的机制，透镜混浊由于γ-晶体蛋白的修改和突变。了解这些机制是开发试剂以延迟混浊发生的重要第一步，混浊是由于（a）常见的年龄性白内障和（B）在儿童和年轻人中观察到的罕见遗传源性核混浊。 鉴于文献中最近的证据表明年龄性白内障可能具有遗传成分，本文提出的研究有可能预测由于一般人群中γ-晶体蛋白的正常变体而导致的个体对这种疾病的易感性。

DESCRIPTION (provided by applicant): Our long-term objectives are to understand how specific post-translational chemical modifications and genetic mutations of the gamma-crystallin proteins lead to lens opacity. We hypothesize that not all protein modifications are deleterious - only those that lead to increased light scattering. Our strategy designed to test this hypothesis is as follows: We will model the cataractogenicity of a given modification or mutation of a gamma-crystallin in vitro, by introducing the required change into the protein and determining the effect on the formation of protein aggregates, liquid droplets containing protein-rich and protein-poor phases, and protein crystals (i.e., protein-derived condensed phases). Protein modifications that enhance the formation of such condensed phases (all of which lead to increased light scattering) will be potentially cataractogenic. We will compare the phase behavior (liquid-liquid and solid-liquid phase separation) of native and modified gamma-crystallins to evaluate if a given modification enhances or suppresses the formation of condensates. In parallel, we will also examine protein structure and conformation using biochemical and spectroscopic methods (chromatography, HPLC, gel electrophoresis, Raman spectroscopy, circular dichroism, fluorescence, and x-ray crystallography). We propose the following Specific Aims: (1) Evaluate which physiologically relevant chemical modifications of the 7 crystallins found enhance the formation of condensed phases - (1 a) Examine the effect of modifications of cysteine residues (i.e., thiolations) with (i) glutathione (ii) cysteine (iii) cysteinylglycine and (iv) gamma-glutamyl cysteine, (1b) Examine the effect of deamidation of asparagine and glutamine residues, and (1c) Examine the effect of truncations of the 7 crystallin polypeptide chain (2) Evaluate which of the genetic variants of the gamma-crystallins enhance the formation of condensed phases - (2a) Determine the mechanisms of opacification due to mutant proteins expressed from gamma-crystallin genes associated (in the literature) with human or animal model cataracts, (2b) Determine what effect the mutant proteins expressed from gamma-crystallin gene variants due to single-nucleotide polymorphisms, have an effect on opacification. This work is designed to provide plausible mechanisms for lens opacification due to gamma-crystallin modifications and mutations. Understanding these mechanisms is an essential first step towards the development of reagents to delay the onset of opacities due to (a) the commonly encountered age-onset cataract and (b) the rare nuclear opacities of genetic origin seen in children and young adults. In view of the recent evidence in the literature that suggests that age-onset cataract may have a genetic component, the studies proposed here have the potential for predicting the susceptibility of individuals towards this disease due to normal variants of the gamma-crystallins in the general populations.