The UNC-45 Chaperone as a Modulator of Myosin Biogenesis and Function

 描述(申请人提供)：在这项提案中，我们的目标是阐明分子伴侣UNC-45和Hsp90帮助肌球蛋白头部折叠的确切分子机制，肌球蛋白头部折叠是成熟肌肉中应激诱导的肌节损伤的发展和修复过程中肌节组装的关键。对这些机制的理解是肌肉发育和功能的核心问题。它还与在非肌肉组织中表达的多种肌球蛋白分子的折叠和修复高度相关。我们最近发现，在UNC-45存在下，肌球蛋白获得了一种新的构象，它以正常的速度水解ATP，但极大地降低了肌动蛋白细丝的转运能力，并被Hsp90逆转，这引发了关于这些伴侣蛋白在肌球蛋白折叠、修复和肌丝组装中的确切作用的重要问题。根据我们最近发表的和初步的研究，我们假设在肌球蛋白组装或修复过程中，UNC-45帮助肌球蛋白头达到其天然构象，并随后将其锁定在一种防止早发性中风的状态；当成功组装到肌节或修复肌球蛋白头时，这种状态可被Hsp90缓解。这一假设将通过两个专家研究实验室之间的独特合作，使用体外和体内分析相结合的方法进行验证。在目标1中，我们将使用突变、生化和生物物理分析，包括肌球蛋白依赖的肌动蛋白细丝滑动分析，来建立介导UNC-45不同独特功能的UNC-45结构域和关键氨基酸区域。在目标2和目标3中，我们将使用线虫的力量作为一个模型系统来研究突变的UNC-45蛋白和肌节组装之间的功能关系(目标2)，以及在修复受损的肌球蛋白头部过程中UNC-45和Hsp90自身以及与肌球蛋白的结合动力学(目标3)。如果我们的假设得到证实，将代表肌球蛋白生物学的新范式，潜在的新治疗方法不仅适用于由肌节蛋白突变引起的横纹肌疾病(骨骼肌病和心肌病)，而且还适用于肿瘤侵袭性问题。

 DESCRIPTION (provided by applicant): In this proposal we aim to elucidate the precise molecular mechanisms by which the chaperones UNC-45 and Hsp90 assist in the folding of the myosin head which is critical for sarcomere assembly during development and repair of stress-induced damage to sarcomeres in mature muscle. Developing an understanding of these mechanisms is a problem at the core of muscle development and function. It is also highly relevant to the folding and repair of the many classes of myosin molecules that are expressed in non-muscle tissues. Our recent discovery that myosin attains a novel conformation, which hydrolyzes ATP at the normal rate but with a greatly reduced ability to translocate actin filaments, in the presence of UNC-45 and is reversed by Hsp90, raises important questions as to the exact roles of these chaperones in myosin folding and repair and myofilament assembly. Guided by our recent published and preliminary studies, we hypothesize that during myosin assembly or repair, UNC-45 assists the myosin head to attain its native conformation and subsequently locks it in a state that prevents premature powerstrokes; this state is relieved by Hsp90 upon successful assembly into the sarcomere or repair of myosin heads. This hypothesis will be tested by using a combination of in vitro and in vivo analyses through a unique collaboration between two expert research labs. In Aim 1 we will use mutagenesis, biochemical and biophysical assays, including myosin-dependent actin filament gliding assays, to establish the UNC-45 domains and key amino acid regions that mediate the different unique functions of UNC-45. In Aims 2 and 3 we will use the power of C. elegans as a model system to examine the functional relationships between mutant UNC-45 proteins and sarcomere assembly (Aim 2), and the dynamics of association of UNC-45 and Hsp90 with themselves and with myosin during repair of damaged myosin heads (Aim 3). Our hypothesis, if confirmed, will represent a new paradigm in the biology of myosins, with potential novel therapeutic approaches not only for striated muscle disorders stemming from mutations in sarcomeric proteins (skeletal myopathies and cardiomyopathies), but also for the problem of tumor invasiveness.