Structure/Function of Small Heat Shock Proteins

小热激蛋白的结构/功能

• 批准号: 7924933
• 负责人: Elizabeth Vierling
• 金额: $ 4.95万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924933
• 关键词: Address; Affect; Aging; Amino Acids; Binding; Binding Sites; Biochemical Genetics; C-terminal; Cells; Cellular Stress; Characteristics; Collaborations; Complex; Crystallins; Crystallography; Cyanobacterium; Data; Deuterium; Development; Disease; Eating; Exhibits; Family; Genes; Genetic; Genetic screening method; Goals; Heat shock proteins; Heat-Shock Response; Heating; Hydrogen; Knowledge; Laboratories; Link; London; Maps; Mass Spectrum Analysis; Mediating; Methods; Modeling; Modification; Molecular Chaperones; Molecular Conformation; Mutation; Point Mutation; Proteins; Proteolysis; Research; Roentgen Rays; Role; Solubility; Stress; Structure; Substrate Interaction; Surface; Synechocystis; System; Temperature; Testing; Variant; alpha-Crystallins; biological adaptation to stress; cofactor; college; crosslink; dimer; genetic resource; in vitro Assay; in vitro activity; in vivo; insight; member; mutant; novel; novel strategies; prevent; protein aggregation; research study

DESCRIPTION (provided by applicant): The long term goals of the proposed research are to define the mechanism of action and essential function of small heat shock/alpha-crystallin proteins (sHsps). The sHsps (16 to 42 kDa) are a ubiquitous class of chaperones that are defined by approximately 90 amino acid C-terminal alpha-crystallin domain and that typically assemble into native oligomers of 9 to > 30 subunits, depending on the protein. Current models propose that sHsps act to prevent irreversible substrate-denaturation by binding substrates and keeping them in a conformational state suitable for refolding by ATP-dependent chaperones. Thereby they can mediate protection against many forms of stress. The mechanism of this protective interaction with substrates is poorly understood, and defined targets of the sHsps still need to be identified. Understanding sHsp function is important to stress responses, disease states, normal development and aging, all of which involve sHsp expression. Consistent with the model for their chaperone activity, sHsps have been increasingly linked to pathological conditions in which protein aggregation occurs. We have determined the first X-ray structure (2.65 Angstrom) of an eukaryotic sHsp, providing a structural context for further mechanistic studies. Robust in vitro assays for chaperone activity of sHsps have been developed in the laboratory. In addition, we have established a genetic system in the cyanobacterium Synechocystis sp. 6803, in which deletion or point mutation of the single sHsp gene causes temperature sensitivity. The ability to perform in vivo functional studies in this homologous system is unique to the field of sHsp/alpha-crystallin research and provides a new approach to studying the role of sHsps in protection of cells from stress. With these biochemical and genetic resources, the following specific aims will be addressed. Aim 1: To define the sHsp-substrate binding sites and the organization of sHsp/substrate complexes. Aim 2: To determine how sHsps affect substrate function in vivo. Aim 3: To define the substrate binding species of the sHsp. Aim 4: To extend our knowledge of sHsp mechanisms by examining the structure and activity of diverse members of the sHsp family. The proposed experiments will greatly advance our understanding of sHsp function and provide new insight into the role of sHsps in disease and normal development.

描述(由申请人提供)：拟议研究的长期目标是确定小热休克/α-晶体蛋白(SHsps)的作用机制和基本功能。SHSP(16到42 kDa)是一类普遍存在的伴侣蛋白，由大约90个氨基酸组成的C-末端α-晶体蛋白结构域定义，通常组装成9到30个亚基的天然低聚物，具体取决于蛋白质。目前的模型认为，sHSPs通过结合底物并使其处于适合于ATP依赖的伴侣进行复性的构象状态来防止不可逆的底物变性。因此，它们可以调节对多种形式的压力的保护。这种保护性与底物相互作用的机制尚不清楚，sHSP的明确靶点仍需确定。了解SHSP的功能对于应激反应、疾病状态、正常发育和衰老都很重要，所有这些都与SHSP的表达有关。与其伴侣活性的模型一致，sHSPs越来越多地与蛋白质聚集发生的病理条件联系在一起。我们已经确定了真核生物SHSP的第一个X射线结构(2.65埃)，为进一步的机制研究提供了结构背景。实验室已经建立了可靠的体外检测sHsps伴侣活性的方法。此外，我们还建立了蓝藻聚球藻的遗传系统。6803，其中单个SHSP基因的缺失或点突变导致温度敏感性。在这个同源系统中进行体内功能研究的能力是SHSP/α-晶体蛋白研究领域所独有的，并为研究sHSP在细胞应激保护中的作用提供了一种新的途径。利用这些生化和遗传资源，将实现以下具体目标。目的1：确定SHSP与底物的结合部位和SHSP/底物复合体的组织结构。目的2：研究热休克蛋白在体内对底物功能的影响。目的3：确定SHSP的底物结合物种。目的4：通过研究SHSP家族不同成员的结构和活性来扩展我们对SHSP机制的认识。这些实验将极大地促进我们对sHSP功能的理解，并为sHsps在疾病和正常发育中的作用提供新的见解。