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

描述(申请人提供)：UNC-45是一种分子伴侣，是横纹肌中肌球蛋白积累和肌原纤维组装所必需的。其C-端的UCS域直接与肌球蛋白相互作用，而其N-端的TPR结构域与伴侣Hsp90结合。尽管其作用机制尚不清楚，但UNC-45似乎对肌球蛋白在体内折叠和保护肌球蛋白免受应激诱导的变性都是至关重要的。此外，UNC-45水平的变化与骨骼肌包涵体肌病和心力衰竭相关，暗示UNC-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，我们解开了UNC-45的第一个晶体结构。这个建议建立在果蝇的黑腹果蝇结构上，以确定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与尚未确定的合作伙伴的S关系。目标1将绘制我们最近发现的UNC-45二聚体的结构和功能基础。我们将使用高分辨电子显微镜、分子模拟、交联研究和功能分析来验证UNC-45二聚化是其作用机制中的关键步骤的假设。Aim 2a将是UNC-45的第一个基于结构功能的突变，并将测试我们在UCS结构域中定义的高度保守的表面凹槽的作用。该蛋白的突变版本将通过肌球蛋白结合和聚集分析在体外进行分析，并在体内通过转基因果蝇的肌肉结构和功能分析进行分析。这将检验一种假设，即UNC-45的UCS结构域上的保守裂隙可以结合肌球蛋白，帮助肌球蛋白在肌肉中积累和/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UNC-45在不同肌肉类型的肌瘤中的差异定位，以及我们的电子显微镜结果显示UNC-45可以结合到肌球蛋白的颈部区域。我们将使用表达颈部转换区替代版本的转基因苍蝇系以及共聚焦显微镜来测试UNC-45与肌球蛋白颈部的转换域特异性结合并优先与该肌球蛋白结构域的特定版本结合的假设。AIM 3将使用遗传和生化方法来确定UNC-45的新伙伴，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UNC-45背景的苍蝇，结合强大的基因技术，如缺乏图谱和microRNA使能敲除，来定义这些合作伙伴。此外，我们将使用质谱仪通过基于UNC-45的蛋白质下拉来鉴定从发育中和应激肌肉中分离出来的蛋白质。我们将在体内通过基于RNAi的瞬时敲除来研究这些蛋白质在肌肉发育和应激过程中的作用。这一目标将检验UNC-45在肌球蛋白折叠、占据肌肉肌节和肌肉应激期间具有不同结合伙伴和功能的假设。总体而言，我们的综合分析将为了解UNC-45的作用机制及其在肌肉发育、停滞和应激中的作用提供重要的见解。

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.