CATARACT RELATED MODIFICATIONS OF LENS CRYSTALLINS

大量证据支持透镜混浊是由于晶体蛋白聚集的假设。 为了确定这种聚集的可能原因，我们目前的研究计划已被定向到确定所有的主要共价修饰在体内发生的人透镜晶体蛋白，重点是区分透明和白内障晶状体。更广泛地说，这项研究的目标是阐明引发或传播白内障的化学物质，其中包括衰老的化学物质。 我们使用质谱法在鉴定这些共价修饰方面取得了重大进展。 例如，已经可以明确区分导致蛋白质更酸性的各种年龄相关修饰，如磷酸化，脱酰胺和赖氨酸修饰，并容易识别多种降解产物。 透镜水溶性部分的所有主要基因产物中的所有主要修饰位点都已鉴定。更新申请包括四个具体目标。 目的1将扩展我们目前的研究，包括明确的和白内障的人晶状体的水不溶性组分中存在的晶体蛋白的共价修饰的鉴定。 有效追求这一目标包括与Larry大卫教授（俄勒冈州健康科学大学）的合作。 大卫博士要求对这种合作进行审查，以代替直接竞争，并更新他的R 01赠款。 在目标2-4中，我们将探索已知的人透镜晶体蛋白的体内修饰与其聚集倾向之间的联系。 具体目标2将确定β-和γ-晶体蛋白的修饰对其溶解度的影响。 在目标3中，将获得关于α-晶状体蛋白溶解γ-晶状体蛋白的机制的新的和高度详细的结构信息。 目的4将解决的影响，具体修改α-晶体蛋白，抑制他们的能力，执行这一伴侣功能。 基于酰胺氢交换和赖氨酸反应性的新分析方法将用于实现目标3和4。

A large body of evidence supports the hypothesis that lens opacity is due to crystallin aggregation. To determine possible causes of this aggregation, our current research program has been directed towards identifying all of the major covalent modifications occurring in vivo to human lens crystallins with emphasis on distinguishing between clear and cataractous lenses. More broadly, the goal of this research has been to elucidate the chemistry that initiates or propagates cataract, which includes the chemistry of aging. Our use of mass spectrometry has led to major advances in identifying these covalent modifications. For example, it has been possible to unequivocally distinguish among a variety of age-related modifications that cause the proteins to be more acidic, such as phosphorylation, deamidation and lysine modification, and to readily identify multiple degradation products. All the major sites of modification in all the major gene products of the water-soluble portion of the lens have been identified. The renewal application includes four Specific Aims. Aim 1 will extend our present studies to include identification of covalent modifications to crystallins present in the water-insoluble fractions of clear and cataractous human lenses. Effective pursuit of this goal includes collaboration with Prof. Larry David (Oregon Health Sciences University). Dr. David requests a subcontract for this collaboration in lieu of direct competition with renewal of his R01 grant. In Aims 2-4, we shall explore linkages between known in vivo modifications of human lens crystallins and their propensity to aggregate. Specific Aim 2 will determine the effect of modifications of beta- and gamma- crystallins on their solubility. In Aim 3, new and highly detailed structural information will be obtained about the mechanisms through which alpha-crystallins solubilize gamma- crystallins. Aim 4 will address the effects of specific modifications to alpha-crystallins that inhibit their ability to perform this chaperone function. New analytical methods based on amide hydrogen exchange and lysine reactivity will be used to accomplish Aims 3 and 4.