AGGRECAN FOLDING, CHAPERONES, AND INTRACELLULAR ROUTING

Aggrecan 折叠、伴侣和细胞内路由

• 批准号: 6171722
• 负责人: MARVIN L TANZER
• 金额: $ 30.94万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171722
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; chondroitin sulfates; confocal scanning microscopy; electron microscopy; green fluorescent proteins; intracellular transport; molecular chaperones; nucleic acid sequence; polymerase chain reaction; protein binding; protein folding; protein isoforms; proteoglycan; site directed mutagenesis; stress proteins; tissue /cell culture

Aggrecan imparts resilience to articular cartilage, enabling cartilage to withstand compression, thereby protecting the bony surfaces of joints. Aggrecan's protective properties emanate from its molecular structure which is assembled via a complicated cellular pathway. A critical element early in that pathway is quality control, imposed by molecular chaperones which assist aggrecan core protein to properly fold before it can continue on its route. Until now, the molecular chaperones responsible for assisting aggrecan core protein to fold have not been identified; recent studies in this lab have implicated chaperone Hsp25 as a principal participant in that process. It is postulated that Hsp25 binds to the nascent core protein's G3 domain in the cytosol and accompanies the core protein into the lumen of the endoplasmic reticulum where it assists G3 folding; once folded, G3, as an intramolecular chaperone, binds to the core protein's nascent G1 domain, releasing Hsp25 which recycles to the cytosol; G3, while adhering to G1, enters the Golgi zones where glycosaminoglycan chains are added, followed by secretion of completed aggrecan into the matrix. The long term objective of this proposal is to comprehend the molecular details of this proposed aggrecan-chaperone pathway; critical features of Hsp25-G3 association and dissociation will be investigated, including diversion of Hsp25/G3 complexes into the nucleus and culture medium under certain circumstances. Three specific aims will focus on: 1) which amino acids in nascent G3 interact with Hsp25 and vice-versa; 2) the nature of the interactions of G1, G3 and Hsp25; 3) which isoform(s) of Hsp25 interact with G3. Cellular transfection with informative constructs will be used to explore each specific aim; normal and mutant Hsp25 and G3 proteins will be expressed, as will hybrid proteins formed between G1, G3, Hsp25 and unique colored isoforms of green fluorescent protein (GFP). GFP labeling will enable tracking of expressed proteins through intracellular compartments; simultaneous tracking of two different proteins will be accomplished as they will be emitting separate colors. Other studies will utilize expressed G1, G3 and Hsp25 proteins containing a polyhistidine tag; proteins will be labeled using radioactive precursors, then harvested and purified by immunocapture and/or by nickel chelation. The composite results will further define a unique system for aggrecan core protein folding and routing. Cartilage repair, including aggrecan production, is impaired in arthritic disease. Understanding the chaperone-based pathway of aggrecan formation and its regulation will potentially provide new insights into the way in which arthritic diseases alter cartilage physiology.

聚集蛋白聚糖赋予关节软骨弹性，使软骨 以承受压力，从而保护骨表面， 接头. 聚集蛋白聚糖的保护特性源于其分子 通过复杂的细胞途径组装的结构。 一 在这条道路的早期，关键因素是质量控制， 协助聚集蛋白聚糖核心蛋白正确折叠的分子伴侣 才能继续它的路线 到目前为止， 负责协助聚集蛋白聚糖核心蛋白折叠的伴侣蛋白具有 尚未确定;本实验室最近的研究表明， 伴侣蛋白Hsp 25作为该过程的主要参与者。 是 假设Hsp 25与新生核心蛋白的G3结构域结合， 的 胞质溶胶和陪同核心蛋白进入管腔的 内质网，在那里它帮助G3折叠;一旦折叠，G3，如 一种分子内伴侣，与核心蛋白的新生G1结合 结构域，释放热休克蛋白25，其粘附到胞质溶胶; G3，而 粘附于G1，进入糖胺聚糖链所在的高尔基区 随后将完成的聚集蛋白聚糖分泌到基质中。 这项提案的长期目标是理解分子 该建议的聚集蛋白聚糖-伴侣蛋白途径的细节;关键特征 研究Hsp 25-G3结合和解离的机制，包括 将Hsp 25/G3复合物转移到细胞核和培养基中 在某些情况下。 三个具体的目标将集中在：1）哪些氨基酸在新生G3 与Hsp 25相互作用，反之亦然; 2）相互作用的性质 G1、G3和Hsp 25的相互作用; 3）Hsp 25的哪种同种型与G3相互作用。 使用信息构建体的细胞转染将用于 探索每个特定的目标;正常和突变的Hsp 25和G3蛋白将 G1、G3、Hsp 25和Hsp 26之间形成的杂合蛋白也将被表达。 绿色荧光蛋白（GFP）的独特颜色同种型。 GFP 标记将能够通过以下方式跟踪表达的蛋白质 细胞内室;同时跟踪两种不同的 蛋白质将被完成，因为它们将发出不同的颜色。 其他研究将利用表达的G1、G3和Hsp 25蛋白 含有多组氨酸标签;蛋白质将使用 放射性前体，然后收获并通过免疫捕获纯化 和/或通过镍螯合。 综合结果将进一步确定 一种独特的聚集蛋白聚糖核心蛋白折叠和路由系统。 软骨修复，包括聚集蛋白聚糖的产生， 关节炎 了解基于分子伴侣的 聚集蛋白聚糖的形成及其调节将潜在地提供新的 深入了解关节炎疾病改变软骨的方式 physiology.