Structure/Function Studies of Small Heat Shock Proteins

小热激蛋白的结构/功能研究

• 批准号: 8437511
• 负责人: Rachel E Klevit
• 金额: $ 44.85万
• 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 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8437511
• 关键词: Acidosis; Address; Affect; Architecture; Binding; Binding Sites; Brain Hypoxia-Ischemia; C-terminal; Cardiac; Cardiomyopathies; Cataract; Cell Survival; Cells; Cellular Stress; Charcot-Marie-Tooth Disease; Charge; Client; Crystalline Lens; Crystallins; Crystallization; Data; Disease; Distal; Electron Microscopy; Eye Lens Protein; Family; Functional disorder; Genes; Grant; HSPB1 gene; Health; Heart; Heat shock proteins; Histidine; Human; Human Genome; Inherited; Investigation; Kinetics; Learning; Length; Link; Lysine; Magic; Measures; Micrococcal Nuclease; Modeling; Molecular; Molecular Chaperones; Motor; Mutation; Myopathy; Neurodegenerative Disorders; Neuropathy; Organ; Peptides; Property; Protein Binding; Proteins; Reagent; Solutions; Staging; Stress; Structure; Syndrome; Thermodynamics; Tissues; Variant; biological adaptation to stress; dimer; human disease; in vitro activity; insight; isoleucylvaline; lens transparency; member; mutant; particle; protein B; protein activation; protein function; protein structure; protein structure function; public health relevance; response; solid state; solid state nuclear magnetic resonance; structural biology; tandem mass spectrometry; technique development; tool

DESCRIPTION (provided by applicant): Small heat shock proteins (sHSPs) are intimately linked to cell survival under conditions of stress. sHSPs act at an early stage of the stress response, by recognizing partly unfolded proteins to inhibit formation of potentially toxic aggregates. Failed sHSP function is associated with cataract, cardiac myopathies, motor neuropathies, and neurodegenerative disease. The archetypal human sHSP, ?B-crystallin ("?B"), is a major protein of the eye lens and along with another related sHSP, ?A-crystallin, is largely responsible for maintaining lens transparency throughout one's lifetime. Despite their critical importance to human health, understanding of the mechanisms by which sHSPs perform their functions remains rudimentary. Information on sHSP structure has been limited by the fact that the proteins exist as large, dynamic, polydisperse oligomeric assemblies. A structure of the ~600 kDa ?B oligomer has been solved using a combination of data from solid- state NMR, small-angle x-ray scattering, and electron microscopy, providing insights into the assembly of the oligomer. This continuing project seeks answers to three overarching questions: How do inherited mutations in sHSPs affect their structure and consequently, their function? How is sHSP structure and function modulated by changes in cellular conditions? and How do sHSPs recognize and bind client proteins? We propose to apply approaches that combine data from solution-state and solid-state NMR, small-angle x-ray scattering, single particle electron microscopy, and tandem mass spectrometry used to determine the structure of the most widely known ?B mutant, R120G ?B, involved in cataracts and cardiomyopathies (Aim 1). A mutant of ?B that represents the activated state associated with acidosis conditions will be investigated in Aim 2 to uncover the molecular mechanism of sHSP activation by pH. ?B/client protein interactions will be studied in Aim 3, using peptides and a model client protein to define the determinants of sHSP client recognition. The proposed studies build on the burgeoning progress towards understanding the structural biology of ?B and the important family of sHSPs afforded by developments of techniques capable of studying large heterogenous species.

描述（由申请人提供）：小热休克蛋白（sHSP）与应激条件下的细胞存活密切相关。 sHSP 在应激反应的早期阶段发挥作用，通过识别部分未折叠的蛋白质来抑制潜在有毒聚集体的形成。 sHSP 功能失败与白内障、心肌病、运动神经病和神经退行性疾病有关。人类 sHSP 原型 βB-晶状体蛋白 (“βB”) 是眼睛晶状体的主要蛋白质，与另一种相关的 sHSP βA-晶状体蛋白一起，在人的一生中主要负责维持晶状体透明度。尽管小热休克蛋白对人类健康至关重要，但对小热休克蛋白发挥其功能的机制的了解仍然处于初级阶段。有关 sHSP 结构的信息受到以下事实的限制：这些蛋白质以大型、动态、多分散的寡聚体形式存在。结合固态核磁共振、小角 X 射线散射和电子显微镜的数据，已经解析了约 600 kDa βB 低聚物的结构，从而提供了对低聚物组装的深入了解。这个持续的项目寻求三个首要问题的答案：sHSP 的遗传突变如何影响它们的结构，从而影响它们的功能？ sHSP 结构和功能如何通过细胞条件的变化进行调节？以及 sHSP 如何识别并结合客户蛋白？我们建议应用结合溶液态和固态核磁共振、小角度 X 射线散射、单粒子电子显微镜和串联质谱数据的方法来确定与白内障和心肌病有关的最广为人知的 βB 突变体 R120G βB 的结构（目标 1）。代表与酸中毒条件相关的激活状态的 ?B 突变体将在目标 2 中进行研究，以揭示 pH 激活 sHSP 的分子机制。 ?B/客户蛋白相互作用将在目标 3 中进行研究，使用肽和模型客户蛋白来定义 sHSP 客户识别的决定因素。拟议的研究建立在理解 βB 结构生物学和重要的 sHSP 家族的迅速进展的基础上，这些进展是由能够研究大型异质物种的技术的发展所提供的。