Structure/Function Studies of Small Heat Shock Proteins

描述（由申请人提供）：小热休克蛋白（sHSP）形成细胞伴侣网络的组成部分，由此它们的表达水平在应激条件下增加，所述应激条件例如热、缺血和pH。sHSP结合新生或应激诱导的未折叠蛋白并将它们维持在可溶状态，直到被ATP依赖性伴侣拯救。aB-晶体蛋白（aB）和HSP 27是在人类中发现的十种sHSP中的两种，并且它们与许多疾病有关。aB和HSP 27都参与细胞凋亡途径，并且它们已经显示出对心脏缺血和再灌注的保护作用。aB和HSP 27的遗传突变与肌肉疾病相关，如结蛋白相关肌病和远端遗传性运动神经病（2型Charcot-Marie-Tooth病）。尽管越来越多的信息，他们的生物学作用，结构和功能方面的aB和HSP 27仍然知之甚少。aB和HSP 27形成多分散的大分子组装体，并且是通过X射线晶体学（最适合于这种尺寸的分子的技术）进行结构测定的难熔目标。我们提出了一个分层的方法来获得结构信息。由所有sHSP共享的α-晶状体蛋白结构域形成同源二聚体，被认为是sHSP寡聚体的结构单元。我们提出通过溶液状态NMR研究来自aB和HSP 27的二聚体α-晶状体蛋白结构域（目的1A和3A）。aB中高阶多聚体的结构信息将使用（i）固态NMR（Aim 1B）和（ii）可能适用于晶体学的蛋白质嵌合体（Aim 1C）获得。所提出的sHSP作用模型包括在结合变性蛋白质底物之前将sHSP寡聚体分解成二聚体亚基。在目标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分子伴侣活性的机制，以绘制变性蛋白在aB上的结合位点。HSP 27在细胞凋亡中的作用（目的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。最后，深入了解与疾病相关的aB和HSP 27的遗传突变的后果将寻求通过比较突变蛋白与野生型对应物的结构和功能特性。

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.