Mechanism of Myosin Chaperone UNC-45: Structural, Functional & Genetic Approaches

肌球蛋白伴侣 UNC-45 的机制：结构、功能

• 批准号: 8489071
• 负责人: Sanford I Bernstein
• 金额: $ 31.77万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8489071
• 关键词: Binding; Biochemical; Biological Assay; Biological Models; C-terminal; Chemicals; Cleaved cell; Confocal Microscopy; Contractile Proteins; Crystallography; Development; Dimerization; Disease; Drosophila genus; Drosophila melanogaster; Electron Microscopy; Future; Genetic; Genetic Screening; Genetic Techniques; Goals; Heart failure; Heat Stress Disorders; Image; Image Analysis; In Vitro; Inclusion Bodies; Knowledge; Maps; Mass Spectrum Analysis; Mediating; Methods; MicroRNAs; Minor; Molecular; Molecular Chaperones; Molecular Models; Molecular Motors; Muscle; Muscle Development; Muscle function; Mutagenesis; Myocardium; Myofibrils; Myopathy; Myosin ATPase; N-terminal; Neck; Phylogenetic Analysis; Preparation; Protein Isoforms; Proteins; RNA Interference; Resolution; Role; Sarcomeres; Scheme; Site; Skeletal Muscle; Spectrometry; Stress; Striated Muscles; Structure; Structure-Activity Relationship; Surface; Symptoms; Tertiary Protein Structure; Testing; Therapeutic; Transgenic Organisms; base; crosslink; design; dimer; fly; human disease; in vivo; insight; molecular modeling; muscle stress; muscular structure; mutant; prevent; protein misfolding; public health relevance; yeast protein

DESCRIPTION (provided by applicant): UNC-45 is a molecular chaperone that is required for myosin accumulation and myofibril assembly in striated muscle. Its C-terminal UCS domain interacts directly with myosin, while its N-terminal TPR domain binds the chaperone Hsp90. Although its mechanism of action is unknown, UNC-45 appears to be critical both for myosin folding in vivo and for protecting myosin from stress-induced denaturation. Further, changes in UNC-45 levels correlate with skeletal muscle inclusion body myopathy and cardiac failure, implicating UNC-45 in human disease. To begin to understand structure-function relationships in this enigmatic protein, we solved the first crystal structure of UNC-45. This proposal builds upon this Drosophila melanogaster structure to identify the molecular mechanisms and consequences of UNC-45 dimerization, UNC-45 interaction with myosin and UNC-45's relationship with yet to be identified partners. Aim 1 will map the structural and functional basis of our recent discovery that UNC-45 dimerizes. We will employ high-resolution electron microscopy, molecular modeling, cross-linking studies and functional analyses to test the hypothesis that dimerization of UNC-45 is a critical step in its mechanism of action. Aim 2a will be the first structure-functio based mutagenesis of UNC-45 and will test the role of a highly-conserved surface groove that we defined in the UCS domain. Mutant versions of the protein will be analyzed in vitro through myosin-binding and aggregation assays, and in vivo by muscle structure and function analysis in transgenic Drosophila. This will test the hypothesis that the conserved cleft in the UCS domain of UNC-45 binds myosin, aids in myosin accumulation in muscle and/or protects myosin from stress-induced denaturation. Aim 2b will explore our observed differential localization of UNC-45 within sarcomeres of different muscle types along with our electron microscopy results showing that UNC-45 can bind to the neck region of myosin. We will use transgenic fly lines expressing alternative versions of the neck converter region along with confocal microscopy to test the hypothesis that UNC-45 binds specifically to the converter domain of the myosin neck and preferentially binds to specific versions of this myosin domain. Aim 3 will employ both genetic and biochemical approaches to define new partners for UNC-45 and test their importance in muscle structure and function. We will use flies with a depleted UNC-45 background in conjunction with the powerful genetic techniques of deficiency mapping and microRNA-enabled knockdown to define these partners. Further, we will use mass spectrometry to identify proteins isolated from developing and stressed muscles by UNC-45-based protein pull-down. We will examine the roles of these proteins during muscle development and stress by RNAi-based transient knockdown in vivo. This aim will test the hypothesis that UNC-45 has different binding partners and functions during myosin folding, during its occupancy of the muscle sarcomere and during muscle stress. Overall, our integrative analysis will provide important insights into the mechanism of action of UNC-45 and its role in muscle development, stasis and stress.

描述（由申请人提供）：UNC-45是一种分子伴侣，是肌球蛋白积累和肌纤维组件在横纹肌中所必需的。其C末端UCS结构域与肌球蛋白直接相互作用，而其N末端TPR结构域结合了伴侣HSP90。 尽管其作用机理尚不清楚，但UNC-45对于体内肌球蛋白折叠和保护肌球蛋白免受压力诱导的变性而似乎都是至关重要的。此外，UNC-45水平的变化与骨骼肌纳入身体肌病和心脏衰竭相关，这意味着人类疾病中的UNC-45。为了开始理解这种神秘蛋白质中的结构功能关系，我们解决了UNC-45的第一个晶体结构。该建议建立在果蝇的结构上，以识别UNC-45二聚化的分子机制和后果，UNC-45与肌球蛋白的相互作用以及UNC-45与尚未识别的伴侣的关系。 AIM 1将绘制我们最近发现UNC-45二聚体的结构和功能基础。我们将采用高分辨率电子显微镜，分子建模，交联研究和功能分析来检验以下假设：unc-45的二聚化是其作用机理的关键步骤。 AIM 2A将是第一个基于结构 - 功能的UNC-45诱变，并将测试我们在UCS域中定义的高度保存的表面凹槽的作用。将通过肌球蛋白结合和聚集分析在体外分析该蛋白的突变版本，并通过转基因果蝇中的肌肉结构和功能分析在体内分析。这将检验以下假设：UNC-45 UC-45的UCS结构域的保守裂缝结合肌球蛋白，有助于肌球员在肌肉中的积累和/或保护肌球蛋白免受压力诱导的变性。 AIM 2B将探索我们观察到的UNC-45在不同肌肉类型的肉瘤中的差异定位，以及我们的电子显微镜结果，表明UNC-45可以与肌球蛋白的颈部区域结合。 我们将使用表达颈部转换器区域的替代版本的转基因飞行线以及共聚焦显微镜来检验以下假设：UNC-45专门与肌球蛋白颈的转换器结构结合，并优先与该肌球蛋白结构域的特定版本结合。 AIM 3将采用遗传和生化方法来定义UNC-45的新伴侣，并测试其在肌肉结构和功能中的重要性。 我们将使用具有耗尽的UNC-45背景的苍蝇，并结合强大的缺陷映射和支持MicroRNA的敲低的遗传技术来定义这些伴侣。此外，我们将使用质谱法来鉴定通过UNC-45基蛋白下拉从发育和压力肌肉中分离出来的蛋白质。我们将在体内通过基于RNAi的瞬时敲低敲击在肌肉发育和应激中的这些蛋白质的作用。该目标将检验以下假设：UNC-45在肌球蛋白折叠期间，在肌肉肌肉肌肉肌肉占据和肌肉胁迫期间具有不同的结合伴侣和功能。 总体而言，我们的综合分析将为UNC-45的作用机理及其在肌肉发育，停滞和压力中的作用提供重要见解。