Functional Elements in Alpha Crystallin Chaperone

Alpha Crystallin Chaperone 中的功能元素

• 批准号: 7586128
• 负责人: KRISHNA K SHARMA
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586128
• 关键词: Accounting; Adult; Alcohol dehydrogenase; Amino Acid Sequence; Binding; Binding Sites; Biological Assay; Blindness; Cataract; Chemicals; Complex; Crystallins; Cysteine; Event; Eye Lens Protein; Fluorescence; Goals; Grant; Heating; Human; Hydrophobicity; In Vitro; Indium; Investigation; Knowledge; Label; Methods; Modification; Molecular; Molecular Chaperones; Molecular Weight; Mutagenesis; Peptides; Physiological; Play; Progress Reports; Proteins; Research Personnel; Role; Scanning; Site; Site-Directed Mutagenesis; Structure; Temperature; Water; age related; alpha-Crystallins; insight; lens; lens protein; lens transparency; macromolecule; meetings; mutant; novel; orientation selectivity; prevent; programs; tool

Cataract, a major cause of blindness in the world, develops as a result of age-related modifications and aggregation of the eye lens proteins. a-Crystallin accounts for nearly 40% of the adult lens proteins but its structure-function is yet to be fully understood. The chaperone-like activity of a-crystallin is believed to play a central role in maintaining lens transparency. We propose the following specific aims to increase our understanding of chaperone function of a-crystallin and its subunit organization to meet our long-term goal of understanding structure-function of a-crystallin. 1) Confirm that residues 70-88 in aA-crystallin and residues 73-92 in aB-crystallin are the major chaperone sites. Determine the amino acid sequences (binding site) in aB-crystallin that contribute to the enhanced hydrophobicity and chaperone function at 37¿C following deletion of 54-61 sequence. 2) Identify the a-crystallin binding site(s) in p- and y-crystallins during an in vitro chaperone assay at 37¿C. Identify the interaction sites in a-p and a-y complexes in human lens high-molecular-weight aggregates with the use of novel cross- linkers and mass spectrometric analysis. 3) Determine the directional preference and orientation of ADH peptide (YSGVCHTDLHAWHGDWPLPVK) during its interaction with aA- crystallin by site-directed fluorescence labeling and quenching studies. 4) Identify and characterize the aB-aB-; aB-aA- and aA-aA- crystallin interaction sites using cysteine scanning mutagenesis and chemical modification. We plan to accomplish these specific aims by site-directed mutagenesis studies and the use of novel cross-linkers and mass spectrometric methods. Understanding the structure of a-crystallin and its mechanisms of its action, including its interaction with other lens proteins, is likely to provide us better tools to delay or prevent cataractogenesis.

白内障是世界上导致失明的主要原因之一，其发病原因与年龄有关 眼晶状体蛋白质的修饰和聚集。A-晶体蛋白占近40% 但其结构和功能尚不完全清楚。这个 A-晶状体蛋白的伴侣样活性被认为在维持晶状体的过程中起着核心作用。 透明度。我们提出以下具体目标，以增加我们对 A-晶体蛋白及其亚基组织的伴侣功能，以满足我们的长期目标 了解α-晶状体蛋白的结构和功能。 1)确认AA-晶状体蛋白中70-88位残基和AB-晶状体蛋白中73-92位残基为 主要的陪护地点。AB-晶状体蛋白中氨基酸序列(结合部位)的测定 这有助于在37℃时增强疏水性和伴侣功能 54-61序列缺失。2)鉴定p-和y-晶状体蛋白中的a-晶状体蛋白结合部位(S) 在37℃的体外伴侣试验中，确定a-p和a-y中的相互作用部位 新型交联剂在人晶状体高分子聚集体中的应用 连接物和质谱分析。3)确定方向偏好和 ADH多肽(YSGVCHTDLHAWHGDWPLPVK)与AA-相互作用时的定位 晶体蛋白的定点荧光标记和猝灭研究。4)识别和 用半胱氨酸表征AB-AB-；AB-AA-和AA-AA-晶状体蛋白相互作用部位 扫描诱变和化学修饰。我们计划实现这些具体目标 通过定点突变研究和新型交联剂和MASS的使用 光谱分析方法。 了解a-晶状体蛋白的结构及其作用机制，包括其 与其他晶状体蛋白的相互作用，很可能为我们提供更好的工具来延迟或预防 白内障的发生。