Structure/Function Studies of Small Heat Shock Proteins

描述(申请人提供)：小的热休克蛋白(SHSP)形成细胞伴侣网络的组成部分，在热、缺血和pH等应激条件下，它们的表达水平增加。SHSP结合新生或应激诱导的未折叠蛋白，并将它们保持在可溶状态，直到被依赖于ATP的伴侣拯救。AB-Crystallin(AB)和HSP27是人类发现的十种sHSPs中的两种，它们与许多疾病有关。AB和HSP27都参与了细胞凋亡的途径，并已被证明对心肌缺血和再灌流具有保护作用。AB和HSP27的遗传突变与肌肉疾病有关，如结蛋白相关性肌病和远端遗传性运动神经病(2型Charcot-Marie-Tooth病)。尽管对AB和HSP27的生物学作用有越来越多的了解，但AB和HSP27的结构和功能方面仍然知之甚少。AB和HSP27形成多分散的大分子组件，是通过X射线结晶学确定结构的难熔目标，这项技术最适合这种大小的分子。我们提出了一种层次化的方法来获取结构信息。A-晶体蛋白结构域由所有sHSP共享，形成一个同源二聚体，被认为是SHSP寡聚体的构建块。我们建议用溶液状态核磁共振研究AB和HSP27的二聚体a-晶体蛋白结构域(目标1A和3A)。关于AB中更高阶多聚体的结构信息将使用(I)固体核磁共振(Aim 1B)和(Ii)可能符合结晶学的蛋白质嵌合体(Aim 1C)。所提出的SHSP作用模型包括在结合变性蛋白底物之前将SHSP低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分析、亚基交换的FRET测量和溶液状态核磁共振来研究AB伴侣活性的机制，以定位变性蛋白在AB上的结合位置。将通过研究HSP27与细胞靶标泛素和细胞色素c的结合来探索HSP27在细胞凋亡中的作用(AIM 3)。最后，将通过比较突变蛋白和野生型蛋白的结构和功能特性来寻求对AB和HSP27遗传突变与疾病相关的后果的见解。

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.