AMINOPEPTIDASES IN NORMAL AND CATARACT LENSES

正常晶状体和白内障晶状体中的氨基肽酶

• 批准号: 6329536
• 负责人: KRISHNA K SHARMA
• 金额: $ 17.07万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329536
• 关键词: aging; aminopeptidase; animal tissue; cataract; chemical binding; crystallins; disease /disorder etiology; electrofocusing; enzyme activity; enzyme substrate; high performance liquid chromatography; human tissue; lens; molecular chaperones; molecular pathology; personal computers; protein engineering; protein purification; proteolysis

In spite of biochemical characterization of normal and cataract lenses and decades of research, the etiology of cataract is not clearly understood. The past investigations have shown the occurrence of proteolysis in aged and cataract lenses. However, the proteolytic enzymes identified by their ability to hydrolyze chromogenic, fluorescence or protein substrates have thus far provided only limited insight to the in vivo cleavage mechanisms of alpha-, beta- and gamma-crystallins. We hypothesize that novel proteases, which recognize a specific peptide sequence or tertiary structure around the cleavage site, are responsible for some of the crystallin cleavages occurring during aging and cataract development. We propose to continue our studies on proteolysis in lens with following specific aims. 1) Determine if the in vivo cleavages seen during the characterization of alpha-, beta- and gamma-crystallins from aging and cataract lenses are due to sequence specific proteases. Additionally, to evaluate whether non-enzymatic cleavages are also important mechanisms for the generation of truncated crystallins in vivo, we intend to study the susceptibility of peptides containing in vivo cleavage site sequences to proteolysis by free radical generating systems. 2) Purification and characterization of novel sequence specific proteases identified in lens. 3) Continue the studies on lens acylaminopeptidase, and evaluate its role in the proteolysis of crystallins in vivo. We also hypothesize that the crystallin fragments may play a decisive role in cataractogenesis by interfering in the chaperone activity of alpha-crystallin in vivo. Therefore we propose to continue to studies on the low molecular weight crystallin fragments from lens to investigate their origin and properties including interaction with purified alpha-, beta- and gamma- crystallins.

尽管对正常和白内障透镜的生化表征以及数十年的研究，但白内障的病因尚未清楚地了解。 过去的研究表明，老化和白内障透镜中蛋白水解的发生。 然而，蛋白水解酶通过水解发色，荧光或蛋白质底物的能力仅提供了有限的洞察力，使其对α-，β-和gamma-晶体的体内切割机制有限。 我们假设新型蛋白酶识别裂解位点周围特定的肽序列或第三纪结构，是造成在衰老和白内障发育过程中发生的某些结晶蛋白裂解的原因。 我们建议继续对具有以下特定目的的晶状体蛋白水解研究。 1）确定在衰老和白内障镜片中α-，β-和γ-晶状蛋白表征期间看到的体内裂解是否是由于序列特异性蛋白酶所致。 此外，为了评估非酶裂解是否也是体内截短晶体蛋白的重要机制，我们打算研究通过自由基产生系统中含有体内切割位点序列对蛋白水解的肽的敏感性。 2）在晶状体中鉴定出的新型序列特异性蛋白酶的纯化和表征。 3）继续研究晶状体酰胺肽酶，并评估其在体内结晶蛋白蛋白水解中的作用。我们还假设，通过干扰体内α-晶状体的伴侣活性，结晶蛋白片段可能在白内障中起决定性作用。 因此，我们建议继续研究晶状体的低分子量晶体片段，以研究其起源和特性，包括与纯化的α-，β-和γ-晶体蛋白的相互作用。