Mechanisms of Activation for Human Small Heat Shock Proteins: An Integrated Approach

人类小热休克蛋白的激活机制：综合方法

• 批准号: 9304219
• 负责人: Rachel E Klevit
• 金额: $ 61.53万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304219
• 关键词: Affect; Affinity; Age related macular degeneration; Binding; Biological Assay; Blindness; Brain Hypoxia-Ischemia; Cataract; Cell Aging; Cells; Cellular Stress; Charge; Chronic; Client; Complex; Cornea; Crystalline Lens; Detection; Diabetic Retinopathy; Dimerization; Disease; Emerging Technologies; Equilibrium; Exhibits; Eye; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; HSPB1 gene; Health; Heat shock proteins; Heat-Shock Response; Histidine; Human; Inherited; Ions; Knowledge; Lead; Link; Maintenance; Mammals; Mass Spectrum Analysis; Metal Ion Binding; Metals; Modeling; Molecular; Molecular Chaperones; Mutation; Names; National Eye Institute; Negative Staining; Neurodegenerative Disorders; Oxidative Stress; Pathology; Physiological; Play; Property; Proteins; Refractory; Regulation; Resolution; Retina; Role; Stress; Structural Models; Structure; Temperature; Testing; Tissues; UV Radiation Exposure; Work; Zinc; base; dimer; disease phenotype; divalent metal; dynamic system; emergency service responder; experience; experimental study; insight; lens; monomer; protein aggregation; protein function; protein misfolding; protein structure; public health relevance; response; solid state nuclear magnetic resonance; structural biology

 DESCRIPTION (provided by applicant): Cells have numerous strategies to cope with the consequences of stresses that cause protein misfolding and aggregation, leading to formation of plaques, fibrils, and other aggregated species encountered in aging cells, cataract, and neurodegenerative diseases. The protein chaperones known as small heat shock proteins are the cell's first responders and are therefore key to maintenance of cellular health. Despite their name, human small heat shock proteins (sHSP) are rarely called up to respond to the stress of elevated temperature, as humans have effective and tight regulation of temperature. Nevertheless, much of the current knowledge of sHSPs has been obtained under elevated temperature conditions where they are highly active. In this project, we seek to understand the molecular mechanisms by which human sHSPs respond to physiologically relevant stresses and how they maintain cellular proteins in soluble forms. Of relevance to the National Eye Institute, the sHSPs to be focused on are highly expressed in ocular tissues (lens, retina, cornea) as well as in other tissues. Dysfunction and aberrant expression of HSPB1 and HSPB5 are associated with ocular diseases including cataract, diabetic retinopathy, and age-related macular degeneration that together account for 65% of blindness worldwide. Perhaps linked to their particular functional niche in which they must respond to small changes in cellular conditions such as pH and metal ion concentrations, human sHSPs have evolved properties that are unique in the protein world. They exist as large (> 100 kDa) polydisperse and dynamic assemblies that defy conventional structural biology approaches. We will apply emerging technologies capable of providing molecular and possible atomic-level information about heterogeneous and dynamic systems. Structural information obtained from solution- and solid-state NMR, negative-stain and cryo-EM, and native mass spectrometry will be integrated with dynamic information from FRET-based subunit dynamics and NMR and with functional information from activity assays and client-binding experiments to define the mechanism(s) by which HSPB5 and HSPB1 are activated by conditions brought about by stresses relevant to tissues of the eye such as hypoxia, ischemia, and UV exposure. Both wild-type sHSPs and forms carrying inherited disease mutations will be investigated.

 描述（由申请人提供）：细胞有多种策略来应对导致蛋白质错误折叠和聚集的应激后果，从而导致斑块、原纤维和细胞老化、白内障和神经退行性疾病中遇到的其他聚集物质的形成。被称为小热休克蛋白的蛋白质伴侣是细胞的第一反应者，因此是维持细胞健康的关键。尽管有这样的名字，人类小热休克蛋白（sHSP）很少被用来应对高温压力，因为人类对温度有有效和严格的调节。尽管如此，目前关于 sHSP 的大部分知识都是在高温条件下获得的，在高温条件下它们具有高度活性。在这个项目中，我们试图了解人类 sHSP 响应生理相关应激的分子机制，以及它们如何维持细胞蛋白质的可溶形式。与国家眼科研究所相关的是，要关注的 sHSP 在眼组织（晶状体、视网膜、角膜）以及其他组织中高度表达。 HSPB1 和 HSPB5 的功能障碍和异常表达与眼部疾病相关，包括白内障、糖尿病性视网膜病变和年龄相关性黄斑变性，这些疾病合计占全球失明的 65%。人类 sHSP 已经进化出蛋白质世界中独特的特性，这可能与它们必须对 pH 值和金属离子浓度等​​细胞条件的微小变化做出反应的特殊功能定位有关。它们以大型（> 100 kDa）多分散和动态组装体的形式存在，这违背了传统的结构生物学方法。我们将应用能够提供有关异构和动态系统的分子和可能的原子级信息的新兴技术。从溶液和固态 NMR、负染色和冷冻电镜以及天然质谱获得的结构信息将与来自基于 FRET 的亚基动力学和 NMR 的动态信息以及来自活性测定和客户结合实验的功能信息相结合，以定义 HSPB5 和 HSPB1 在与眼睛组织相关的应激（如缺氧、 缺血和紫外线照射。野生型 sHSP 和携带遗传性疾病突变的形式都将受到研究。