STRUCTURE/FUNCTION/ORGANIZATION--LENS ALPHA A-CRYSTALLIN

结构/功能/组织--LENS ALPHA A-Crystallin

• 批准号: 6342682
• 负责人: BERNARD K FUNG
• 金额: $ 26.15万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342682
• 关键词: cataract; crystallins; fluorescence resonance energy transfer; fluorescent dye /probe; lens proteins; molecular chaperones; pathologic process; protein protein interaction; protein structure function; cataract; crystallins; fluorescence resonance energy transfer; fluorescent dye /probe; lens proteins; molecular chaperones; pathologic process; protein protein interaction; protein structure function

AlphaA-crystallin, a member of the small heat-shock protein family (sHsp), is known to play a pivotal role in inhibiting protein aggregation in the lens. This finding has led to the exciting possibility that its chaperone- like properties may be critical in preventing the progression of age-related cataracts. A hallmark of alphaA-crystallin and other sHsps is their tendency to form large oligomers. This unique structural feature of sHsps is thought to be important for their chaperone function. The overall goal of this research proposal is to elucidate the quaternary structure of alphaA- crystallin and to determine the molecular mechanism underlying the assembly of its subunits. Three interrelated research programs are proposed. (1) The specific aim of the first program is to define the subunit organization of the alphaA-crystallin. In this program, site-directed labeling will be employed to determine the orientation of the alphaA- crystallin subunits and the proximity of the N- and C-terminus of neighboring subunits in the oligomeric complex. Knowledge of the subunit organization will provide a starting point to study the accessibility of other important sites. (2) The specific aim of the second program is to uncover the molecular mechanism underlying the formation of the oligomeric complex. A panel of chimeras between alphaA-crystallin and Hsp27 will be constructed. The differences in size, conformation, chaperone activity, and subunit exchange rate between Hsp27, alphaA- crystallin and the chimeric proteins will be used to determine which regions of alphaA-crystallin are involved in oligomer formation. (3) Subunit exchange of alphaA-crystallin is known to involve the breaking of hydrophobic and ionic interactions between neighboring subunits. The specific aim of the final program is to determine what kind of effects do cationic and anionic detergents have on the subunit dissociation rate constant of alphaA-crystallin. Studies will also be carried out to define how raising the temperature or increasing the chain-length of the cationic detergents may affect the rate of subunit exchange. Together, the findings obtained from these research programs will provide a better understanding of the structure of alphaA-crystallin and its interaction with other lens proteins.

众所周知，小型热蛋白家族（SHSP）的成员α-crystallin在抑制镜头中蛋白质聚集方面起着关键作用。这一发现导致了令人兴奋的可能性，即其类似伴侣的特性对于防止与年龄相关的白内障的发展至关重要。 Alphaa-Crystallin和其他SHSP的标志是它们形成大型低聚物的趋势。 SHSP的这种独特的结构特征被认为对其伴侣功能很重要。该研究建议的总体目标是阐明α-晶蛋白的第四纪结构，并确定其亚基组装的基础机制。提出了三个相互关联的研究计划。 （1）第一个程序的具体目的是定义α-Crystallin的亚基组织。在该程序中，将采用以网站为导向的标签来确定α-结晶蛋白亚基的方向以及寡聚络合物中相邻亚基的N-和C端的接近度。亚基组织的知识将为研究其他重要站点的可访问性提供一个起点。 （2）第二个程序的具体目的是揭示寡聚复合物形成的分子机制。将构建α-Crystallin和Hsp27之间的嵌合体面板。 Hsp27，α-晶体蛋白和嵌合蛋白之间的大小，构象，伴侣活性和亚基汇率的差异将用于确定哪些αA-crystallin的区域参与低聚物形成。 （3）已知α-晶状体的亚基交换涉及邻近亚基之间疏水和离子相互作用的破坏。最终程序的具体目的是确定阳离子和阴离子洗涤剂对αA-crystallin的亚基解离速率常数有什么样的影响。还将进行研究以定义如何升高温度或增加阳离子洗涤剂的链长度可能会影响亚基交换的速率。从这些研究计划中获得的发现将共同了解α-晶状体蛋白的结构及其与其他晶状体蛋白的相互作用。