Molecular Analysis of Microdissected Cataractous Human Lenses

描述（由申请方提供）：甲硫氨酸亚砜还原酶具有独特的通过直接还原氧化甲硫氨酸残基实际修复氧化应激损伤的能力，可能在透镜老化和白内障形成中发挥关键作用。已在人透镜中检测到MsrA活性，并且人透镜蛋白的氧化甲硫氨酸含量随年龄增加，相对于透明的人晶状体，在年龄相关性白内障中达到高达60%的水平。本提案将检验以下假设：甲硫氨酸亚砜还原酶保护透镜免受氧化应激损伤，以及Msr水平和/或活性降低与透镜甲硫氨酸氧化增加、透镜损伤和白内障相关。为了检验这一假设：（1）确定人和小鼠透镜的甲硫氨酸亚砜还原酶的特性、水平、活性和亚细胞定位模式;（2）确定甲硫氨酸亚砜还原酶保护透镜免受氧化应激的能力;（3）确定甲硫氨酸亚砜还原酶活性与透镜老化和年龄相关性白内障中甲硫氨酸氧化之间的关系。这些目标将使用功能测试体外和体内能力的综合方法来实现 保护透镜免受氧化应激损伤，并直接检查实际人类晶状体和白内障中的甲硫氨酸亚砜还原酶活性。这项工作的可行性是由三个独立的蛋氨酸亚砜还原酶基因在人类透镜的初步鉴定和空间表征的支持，并证明这些基因中的至少一个，称为MsrA，可以直接保护透镜细胞免受氧化应激损伤。

DESCRIPTION (provided by applicant): Methionine sulfoxide reductases are unique in their ability to actually repair oxidative stress damage through the direct reduction of oxidized methionine residues and are likely to play key roles in lens aging and cataract formation. MsrA activity has been detected in the human lens, and the oxidized methionine content of human lens proteins increases with age reaching levels as high as 60% in age-related cataract relative to clear human lenses. This proposal will test the hypothesis that methionine sulfoxide reductases defend the lens against oxidative stress damage and that decreased Msr levels and/or activities are associated with increased oxidation of lens methionines, lens damage and cataract. To test this hypothesis: (1) The identities, levels, activities and sub-cellular localization patterns of methionine sulfoxide reductases of the human and mouse lens will be established; (2) The ability of methionine sulfoxide reductases to defend the lens against oxidative-stress will be determined; and (3) the relationship between methionine sulfoxide reductase activity and methionine oxidation will be established in lens aging and age-related cataract. These aims will be accomplished using an integrative approach that functionally tests the in vitro and in vivo ability of methionine sulfoxide reductases to defend the lens against oxidative stress damage and directly examines methionine sulfoxide reductase activities in actual human lenses and cataracts. The feasibility of this work is supported by the initial identification and spatial characterization of three separate methionine sulfoxide reductase genes in the human lens and the demonstration that at least one of these genes, called MsrA, can directly defend lens cells against oxidative stress damage.