PROTEIN CLEAVAGE SITES IN EXPERIMENTAL CATARACTOUS LENSE

实验性白内障晶状体中的蛋白质裂解位点

• 批准号: 2161684
• 负责人: LARRY L DAVID
• 金额: $ 9.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2161684
• 关键词: aminopeptidase; cataract; chemical cleavage; chromatography; electrophoresis; endopeptidases; laboratory mouse; laboratory rat; lens proteins; protease inhibitor; protein sequence; proteolysis

Cataracts are the third leading cause of blindness in the United States. However, the underlying biochemical mechanisms of most cataracts are unknown. One alteration in lenses that may contribute to cataract formation is accumulation of partially degraded protein fragments. Many experimental cataracts studied to date contain significant amounts of partially degraded lens proteins. The role these proteins play in cataract formation and which lens proteases are responsible for protein degradation are unknown. The two goals of this project are to better understand the significance of proteolysis in cataractous lenses, and to determine which endogenous lens proteases are activated during cataract formation. Partially degraded lens proteins, from selenite and galactose induced cataracts and rom hereditary mouse cataracts, will be isolated by electrophoresis. These partially degraded polypeptides will be analyzed using protein micro-sequencing techniques to identify their origin, and to determine their N - and C-terminal cleavage sites. Intact lens proteins will then be incubated in vitro with three different major lens endopeptidases (high molecular neutral protease complex, trypsin-like protease, and calpain II). The cleavage sites occurring in vitro will be compared to cleavage sites occurring in vivo during cataract formation. Endopeptidase cleaved lens proteins will also be incubated with two major lens exopeptidases (aminopeptidase III and leucine aminopeptidase). This will determine if endopeptidase cleavage makes lens proteins better substrates for exopeptidases. Proteolysis may contribute to cataract formation by significantly altering the properties of lens proteins. Comparison of in vivo cleavage sites found in cataractous lenses with cleavage sites produced by proteases in vitro may identify the proteases that are activated in the lens. Determining which lens proteases are active during experimental cataract formation may suggest ways of treating human cataract. For instance, inhibitors of proteases could be used as therapeutic agents to slow the progression of cataract.

白内障是美国第三大致盲原因 States. 然而，大多数的潜在生化机制 白内障未知。 镜片的一个变化可能 白内障形成的原因是 降解的蛋白质片段。 许多实验性白内障研究 迄今为止含有大量的部分降解的透镜 proteins. 这些蛋白质在白内障形成中的作用， 哪些透镜蛋白酶负责蛋白质降解， 未知 本项目的两个目标是更好地了解 白内障晶状体蛋白水解的意义，并确定 其中内源性透镜蛋白酶在白内障期间被激活 阵 部分降解的透镜蛋白，来自亚硒酸盐和 半乳糖诱导的白内障和ROM遗传性小鼠白内障， 将通过电泳分离。 这些部分降解的 将使用蛋白质微测序分析多肽 技术，以确定其来源，并确定其N -和 C-末端裂解位点。 完整的透镜蛋白将被 与三种不同的主要透镜内肽酶体外孵育 (high分子中性蛋白酶复合物，胰蛋白酶样蛋白酶， 和钙蛋白酶II）。 在体外发生的切割位点将是 与在白内障期间体内发生的切割位点相比， 阵 内肽酶切割的透镜蛋白也将被 与两种主要的透镜外肽酶（氨肽酶III和 亮氨酸氨肽酶）。 这将决定内肽酶 切割使透镜蛋白质成为外肽酶的更好底物。 蛋白质水解可能通过显著增加白内障的发生而促进白内障的形成。 改变透镜蛋白质的性质。 体内比较 在具有卵裂位点的白内障晶状体中发现的卵裂位点 通过体外蛋白酶产生的蛋白酶可以鉴定 在透镜中被激活。 确定哪些透镜蛋白酶是活性的 在实验性白内障形成过程中， 人类白内障 例如，蛋白酶的抑制剂可以是 用作治疗剂以减缓白内障的进展。