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: 8683640
• 负责人: Sanford I Bernstein
• 金额: $ 3.62万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683640
• 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; 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）：α-45是横纹肌中肌球蛋白积聚和肌原纤维组装所需的分子伴侣。其C-末端UCS结构域直接与肌球蛋白相互作用，而其N-末端TPR结构域结合伴侣Hsp 90。 虽然其作用机制尚不清楚，但α-45似乎对肌球蛋白在体内折叠和保护肌球蛋白免受应力诱导的变性都至关重要。此外，β-45水平的变化与骨骼肌包涵体肌病和心力衰竭相关，暗示β-45与人类疾病有关。为了开始了解这种神秘蛋白质的结构-功能关系，我们解决了第一个晶体结构的β-45。该建议建立在这种果蝇结构的基础上，以确定α-45二聚化的分子机制和后果，α-45与肌球蛋白的相互作用以及α-45与尚未确定的伙伴的关系。目的1将绘制我们最近发现的α-45二聚化的结构和功能基础。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验证以下假设：UNC-45的二聚化是其作用机制的关键步骤。目标2a将是第一个基于结构-功能的突变的突变体，并将测试我们在UCS结构域中定义的高度保守的表面沟的作用。蛋白质的突变形式将通过肌球蛋白结合和聚集测定在体外进行分析，并通过转基因果蝇的肌肉结构和功能分析在体内进行分析。这将检验以下假设：β-45的UCS结构域中的保守裂缝结合肌球蛋白，有助于肌球蛋白在肌肉中的积累和/或保护肌球蛋白免受应激诱导的变性。 目的2b将探索我们观察到的不同肌肉类型的肌节内的差异定位，沿着我们的电子显微镜结果显示，<$-45可以结合到肌球蛋白的颈部区域。 我们将使用转基因苍蝇线表达的颈部转换器区域的替代版本沿着与共聚焦显微镜测试的假设，即α-45特异性结合到转换器域的肌球蛋白颈部，并优先结合到该肌球蛋白域的特定版本。Aim 3将采用遗传和生物化学方法来定义新的伴侣，并测试它们在肌肉结构和功能中的重要性。 我们将使用具有耗尽的α-45背景的果蝇，结合缺陷作图和microRNA激活的敲除的强大遗传技术来定义这些伴侣。此外，我们将使用质谱法来鉴定从发育和应激肌肉中分离出的蛋白质，这些蛋白质是通过基于α-45的蛋白质下拉来分离的。我们将通过基于RNAi的体内瞬时敲低来研究这些蛋白在肌肉发育和应激过程中的作用。这一目标将测试的假设，即在肌球蛋白折叠过程中，在其占用的肌肉肌节和肌肉应力过程中，β-45有不同的结合伙伴和功能。 总的来说，我们的综合分析将提供重要的见解的作用机制的α-45及其在肌肉发育，停滞和压力的作用。