Structure/Function Studies of Small Heat Shock Proteins

小热激蛋白的结构/功能研究

• 批准号: 7415008
• 负责人: Rachel E Klevit
• 金额: $ 34.4万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7415008
• 关键词: Adult; Affect; Apoptosis; Apoptotic; Bacteria; Binding; Binding Sites; Biological; Biological Assay; Cardiac; Cellular Stress; Chaperonin 10; Charcot-Marie-Tooth Disease; Chimeric Proteins; Class; Condition; Crystallins; Crystallography; Desmin; Disease; Distal Muscular Dystrophies; Fluorescence Resonance Energy Transfer; HSPB1 gene; Heat shock proteins; Heating; Human; Human Genome; In Vitro; Inherited; Investigation; Ischemia; Length; Maps; Measurement; Missense Mutation; Modeling; Molecular Chaperones; Motor; Mutation; Myopathy; Neuropathy; Numbers; Organism; Pathway interactions; Peripheral Nerves; Physiological reperfusion; Property; Protein Binding; Proteins; Refractory; Reperfusion Therapy; Research Personnel; Resolution; Role; Solutions; Stress; Structure; Techniques; Ubiquitin; X-Ray Crystallography; cellular targeting; cytochrome c; dimer; disease-causing mutation; insight; macromolecular assembly; mutant; protein aggregation; protein protein interaction; size; solid state

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.

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，因此它们的表达水平在应激条件（例如热、缺血和pH）下增加。 sHSP 结合新生或应激诱导的未折叠蛋白，并将其保持在可溶状态，直到被 ATP 依赖性伴侣拯救。 aB-晶状体蛋白 (aB) 和 HSP27 是人类中发现的 10 种 sHSP 中的两种，它们与多种疾病有关。 aB 和 HSP27 都参与细胞凋亡途径，并且已被证明可以防止心脏缺血和再灌注。 aB 和 HSP27 的遗传性突变与肌肉疾病有关，例如结蛋白相关性肌病和远端遗传性运动神经病（2 型腓骨肌萎缩症）。尽管关于 aB 和 HSP27 的生物学作用的信息越来越多，但人们对 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装体，是 X 射线晶体学结构确定的难点靶标，X 射线晶体学技术最适合这种尺寸的分子。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-晶状体蛋白结构域形成同二聚体，被认为是 sHSP 寡聚体的构建模块。我们建议通过溶液态 NMR 研究 aB 和 HSP27 的二聚 a-晶状体蛋白结构域（目标 1A 和 3A）。 aB 中高阶多聚体的结构信息将使用 (i) 固态 NMR（目标 1B）和 (ii) 可能适合晶体学的蛋白质嵌合体（目标 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将 sHSP 寡聚物分解成二聚体亚基。在目标 2 中，我们将通过蛋白质聚集测定、亚基交换的 FRET 测量以及溶液态 NMR 来研究 aB 伴侣活性的机制，以绘制变性蛋白质在 aB 上的结合位点。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（目标 3）。最后，通过比较突变蛋白与其野生型对应物的结构和功能特性，可以深入了解与疾病相关的 aB 和 HSP27 遗传突变的后果。