Structure/Function Studies of Small Heat Shock Proteins

描述（由申请人提供）：小热休克蛋白（sHSPs）形成细胞伴侣网络的一个组成部分，在热、缺血和ph等应激条件下，其表达水平增加。sHSPs结合新生或应激诱导的未折叠蛋白，并将其维持在可溶性状态，直到被atp依赖性伴侣蛋白拯救。aB- crystallin （aB）和HSP27是人类发现的10种shsp中的两种，它们与许多疾病有关。aB和HSP27都参与了凋亡通路，它们已被证明对心脏缺血和再灌注有保护作用。aB和HSP27的遗传突变与肌肉疾病有关，如desmin2相关性肌病和远端遗传性运动神经病变（2型charco - marie - tooth病）。尽管关于aB和HSP27的生物学作用的信息越来越多，但对它们的结构和功能方面的了解仍然很少。aB和HSP27形成了多分散的大分子组合，是x射线晶体学测定结构的难降解目标，这种技术最适合于这种大小的分子。我们提出了一种分层方法来获取结构信息。所有sHSP共有的a-结晶蛋白结构域形成同型二聚体，被认为是sHSP低聚物的组成部分。我们建议通过溶液态核磁共振（Aims 1A和3A）研究aB和HSP27的二聚体a-结晶蛋白结构域。aB中高阶多聚体的结构信息将使用(i)固态核磁共振（Aim 1B）和（ii）可能适用于晶体学的蛋白质嵌合体（Aim 1C）获得。提出的sHSP作用模型包括在结合变性蛋白质底物之前sHSP低聚物分解成二聚体亚基。在Aim 2中，我们将通过蛋白质聚集试验、亚基交换的FRET测量和溶液状态NMR来研究aB伴侣活性的机制，以绘制变性蛋白在aB上的结合位点。HSP27在细胞凋亡中的作用（Aim 3）将通过研究其与细胞靶点、泛素和细胞色素c的结合来探索。通过比较突变蛋白与野生型蛋白的结构和功能特性，将深入了解与疾病相关的aB和HSP27遗传突变的后果。

DESCRIPTION (provided by applicant): Small Heat Shock Proteins (sHSPs) form an integral part of the cellular chaperone network whereby their levels of expression increase under conditions of stress such as heat, ischemia, and pH. sHSPs bind nascent or stress-induced unfolded proteins and maintain them in a soluble state until rescued by ATP-dependent chaperones. aB-Crystallin (aB) and HSP27 are two of the ten sHSPs found in humans and they are implicated in a number of diseases. Both aB and HSP27 are involved in the apoptotic pathway and they have been shown to protect against cardiac ischemia and reperfusion. Inherited mutations in aB and HSP27 are associated with muscular diseases such as desmin-related myopathy and distal hereditary motor neuropathy (Type 2 Charcot-Marie-Tooth disease). Despite growing information on their biological roles, structural and functional aspects of aB and HSP27 remain poorly understood. aB and HSP27 form polydisperse macromolecular assemblies and are refractory targets for structure determination by X-ray crystallography, the technique most suited for molecules of this size. We propose a hierarchical approach to obtain structural information. The a-crystallin domain, which is shared by all sHSPs, forms a homodimer, believed to be the building block of sHSP oligomers. We propose to study the dimeric a-crystallin domains from aB and HSP27 by solution state NMR (Aims 1A and 3A). Structural information on higher order multimers in aB will be obtained using (i) solid state NMR (Aim 1B) and (ii) protein chimeras that may be amenable to crystallography (Aim 1C). Proposed models for sHSP action include disassembly of sHSP oligomers into dimeric subunits before binding denatured protein substrates. In Aim 2, we will investigate the mechanism of aB chaperone activity by protein aggregation assays, FRET measurements of subunit exchange, and solution-state NMR to map the binding site of denatured protein on aB. The role of HSP27 in apoptosis (Aim 3) will be explored by investigating its binding to cellular targets, ubiquitin and cytochrome c. Finally, insights into the consequences of inherited mutations in aB and HSP27 associated with disease will be sought by comparison of the structures and functional properties of mutant proteins with their wild-type counterparts.