Structure/Function of Small Heat Shock Proteins

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-晶状体蛋白（sHsps）的作用机制和基本功能。sHsps（16至42 kDa）是一类普遍存在的分子伴侣，其由大约90个氨基酸的C-末端α-晶状体蛋白结构域限定，并且通常组装成9至> 30个亚基的天然寡聚体，这取决于蛋白质。目前的模型提出，sHsps的行为，以防止不可逆的底物变性结合底物，并保持它们在一个构象状态，适合于重折叠的ATP依赖性伴侣。因此，它们可以介导对多种形式的压力的保护。这种与底物的保护性相互作用的机制知之甚少，并且sHsps的限定靶点仍需要鉴定。了解sHsp功能对于应激反应、疾病状态、正常发育和衰老都很重要，所有这些都涉及sHsp表达。与其分子伴侣活性的模型一致，sHsps已越来越多地与其中发生蛋白质聚集的病理条件相关联。我们已经确定了第一个真核sHsp的X射线结构（2.65埃），为进一步的机制研究提供了结构背景。已经在实验室中开发了用于sHsps的伴侣蛋白活性的稳健的体外测定。此外，我们已经建立了一个遗传系统的蓝藻集胞藻sp.6803，其中单一的sHsp基因的缺失或点突变引起的温度敏感性。在这种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，并提供了一种新的方法来研究sHsps在保护细胞免受应激中的作用。利用这些生物化学和遗传资源，将实现以下具体目标。目的1：确定sHsp-底物结合位点及sHsp/底物复合物的结构。目的2：研究sHsps在体内对底物功能的影响。目的3：确定sHsp的底物结合种类。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。

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.