Structure/Function Studies of Small Heat Shock Proteins

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。失败的sHSP功能与白内障、心肌病、运动神经病和神经退行性疾病相关。典型的人sHSP，α B-晶状体蛋白（“αB”），是眼睛透镜的主要蛋白质，并且与另一种相关的sHSP，α A-晶状体蛋白沿着，主要负责在人的一生中维持透镜的透明度。尽管它们对人类健康至关重要，但对sHSPs执行其功能的机制的理解仍然是基本的。关于sHSP结构的信息受到蛋白质作为大的、动态的、多分散的寡聚体组装体存在的事实的限制。使用固态NMR、小角X射线散射和电子显微镜的数据组合，解析了~600 kDa αB低聚物的结构，为低聚物的组装提供了见解。这个持续的项目寻求三个首要问题的答案：sHSPs中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通过细胞条件的变化来调节的？sHSP如何识别和结合客户蛋白？我们建议应用联合收割机结合溶液态和固态NMR、小角X射线散射、单粒子电子显微镜和串联质谱的数据的方法，用于确定最广为人知的αB突变体R120 G αB的结构，该突变体与白内障和心肌病有关（目标1）。目标2将研究代表与酸中毒条件相关的活化状态的αB突变体，以揭示pH激活sHSP的分子机制。目标3将研究αB/客户蛋白相互作用，使用肽和模型客户蛋白来定义sHSP客户识别的决定因素。拟议的研究建立在对αB的结构生物学和sHSPs的重要家族的理解的迅速进展的基础上，这些结构生物学和sHSPs的重要家族是由能够研究大型异质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, αB-crystallin ("αB"), is a major protein of the eye lens and along with another related sHSP, αA-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 αB 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 αB mutant, R120G αB, involved in cataracts and cardiomyopathies (Aim 1). A mutant of αB 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. αB/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 αB and the important family of sHSPs afforded by developments of techniques capable of studying large heterogenous species.