Structure/Function Studies of Small Heat Shock Proteins

描述(由申请人提供)：小热休克蛋白(SHSP)与细胞在应激条件下的生存密切相关。热休克蛋白在应激反应的早期阶段，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功能衰竭与白内障、心脏肌病、运动神经病和神经退行性疾病有关。人类SHSP的原型β-晶状体蛋白是眼晶状体的主要蛋白质，与另一种与之相关的SHSP-A-晶状体蛋白在人的一生中主要负责维持晶状体的透明度。尽管它们对人类健康至关重要，但对sHSP执行其功能的机制的了解仍处于初级阶段。关于SHSP结构的信息一直受到这样一个事实的限制，即蛋白质以大的、动态的、多分散的寡聚体的形式存在。结合固体核磁共振、小角X射线散射和电子显微镜的数据，解析了~600 kDa？B齐聚物的结构，为低聚物的组装提供了深入的了解。这个持续进行的项目寻求三个首要问题的答案：SSSP的遗传突变如何影响它们的结构，从而影响它们的功能？SHSP的结构和功能是如何被细胞条件的变化所调节的？热休克蛋白是如何识别和结合客户蛋白的？我们建议应用结合溶液和固态核磁共振、小角X射线散射、单粒子电子显微镜和串联质谱学数据的方法来确定最广为人知的B突变体R120G？B的结构，该突变体与白内障和心肌病有关(目标1)。在目标2中，将研究一个代表与酸化条件相关的激活状态的突变体？B，以揭示pH激活SHSP的分子机制。？B/客户蛋白质的相互作用将在目标3中进行研究，使用多肽和一个模型客户蛋白来确定SHSP客户识别的决定因素。拟议的研究建立在对？B和重要的热休克蛋白家族的结构生物学理解方面的迅速进展的基础上，这些进展是由于能够研究大型异源物种的技术的发展所提供的。

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.