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

 描述（由申请人提供）：细胞具有多种策略来科普引起蛋白质错误折叠和聚集的应激的后果，导致形成斑块、原纤维和在老化细胞、白内障和神经退行性疾病中遇到的其他聚集物质。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。尽管它们的名字，人类小热休克蛋白（sHSP）很少被调用来应对高温的压力，因为人类对温度有有效和严格的调节。尽管如此，目前对sHSP的大部分了解都是在高温条件下获得的，在高温条件下它们是高度活性的。在这个项目中，我们试图了解人类sHSPs响应生理相关压力的分子机制，以及它们如何保持可溶性形式的细胞蛋白。与国家眼科研究所相关的是，所关注的sHSP在眼组织（透镜、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包括白内障、糖尿病性视网膜病变和年龄相关性黄斑变性在内的眼部疾病相关，这些眼部疾病一起占全球失明的65%。也许与它们特定的功能生态位有关，它们必须对细胞条件（如pH值和金属离子浓度）的微小变化做出反应，人类sHSPs已经进化出蛋白质世界中独特的特性。它们以大（> 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EM以及天然质谱法获得的结构信息将与来自基于FRET的亚基动力学和NMR的动力学信息以及来自活性测定和客户结合实验的功能信息整合，以定义HSPB 5和HSPB 1被与眼睛组织相关的应激（例如缺氧）引起的条件激活的机制，局部缺血和紫外线照射。将研究野生型sHSP和携带遗传性疾病突变的形式。

 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.