Structure/Function Studies of Small Heat Shock Proteins

描述（由申请人提供）：小热休克蛋白（sHSPs）与应激条件下的细胞存活密切相关。sHSPs在应激反应的早期阶段起作用，通过识别部分未折叠的蛋白质来抑制潜在毒性聚集体的形成。sHSP功能衰竭与白内障、心肌病、运动神经病和神经退行性疾病有关。典型的人类sHSP， ？B-crystallin(”?B")，是眼晶状体的主要蛋白质，与另一种相关的sHSP ？a -晶状体蛋白主要负责在人的一生中保持晶状体的透明度。尽管它们对人类健康至关重要，但对sHSPs发挥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蛋白质以大的、动态的、多分散的寡聚体存在这一事实的限制。~600 kDa ？利用固态核磁共振、小角度x射线散射和电子显微镜的数据组合解决了B低聚物的问题，为低聚物的组装提供了见解。这个正在进行的项目寻求三个首要问题的答案：sHSPs的遗传突变如何影响它们的结构，从而影响它们的功能？细胞条件的变化如何调节sHSP的结构和功能？sHSPs如何识别和结合客户蛋白？我们建议采用结合溶液态和固态核磁共振数据、小角度x射线散射、单粒子电子显微镜和串联质谱的方法来确定最广为人知的？B突变体，R120G ？B，参与白内障和心肌病（目的1）。的突变体？代表与酸中毒条件相关的激活状态的B将在Aim 2中进行研究，以揭示ph活化sHSP的分子机制。目标3将研究B/客户蛋白相互作用，使用多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结构生物学的迅速发展的理解上。B和由于能够研究大型异种种的技术的发展而提供的重要的sHSPs家族。

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.