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 -结晶蛋白（sHsps）的作用机制和基本功能。sHsps（16至42 kDa）是一类普遍存在的伴侣蛋白，由大约90个氨基酸的c端α -结晶蛋白结构域定义，通常根据蛋白质的不同组装成9至30个亚基的天然低聚物。目前的模型表明，sHsps通过结合底物并使底物保持适合atp依赖性伴侣蛋白重折叠的构象状态，从而防止底物不可逆变性。因此，它们可以调节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尚不清楚，sHsps的明确靶标仍需确定。了解sHsp的功能对应激反应、疾病状态、正常发育和衰老都有重要意义，这些都与sHsp的表达有关。与其伴侣活性模型一致，sHsps越来越多地与蛋白质聚集发生的病理状况联系在一起。我们已经确定了真核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。在实验室中已经开发出了对sHsps伴侣活性的体外检测方法。此外，我们在蓝细菌Synechocystis sp. 6803中建立了一个遗传系统，其中单个sHsp基因的缺失或点突变导致温度敏感。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，并为研究sHsp在保护细胞免受应激中的作用提供了一种新的方法。有了这些生物化学和遗传资源，以下具体目标将得到解决。目的1：定义sHsp-底物结合位点和sHsp/底物配合物的组织结构。目的2：确定sHsps在体内如何影响底物功能。目的3：明确sHsp的底物结合种类。目标4：通过检查sHsp家族不同成员的结构和活性来扩展我们对sHsp机制的了解。本实验将极大地促进我们对sHsp功能的理解，并为sHsp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.