Stress-induced myosin folding and assembly mechanisms

肌肉的功能和维持是由结构蛋白和运动蛋白组成收缩肌节的时空组织决定的。鉴于肌肉组织的持续使用和机械应力暴露，肌节的完整性不断受到蛋白质折叠和降解途径的复杂网络的保护。肌节形成和肌肉功能的一个重要调节因子是HSP90辅助伴侣UNC-45。根据UNC-45在肌球蛋白组装中的中心作用，UNC-45蛋白的数量和定位与机械应力条件或诱导的肌纤维损伤密切相关。我们对肌球蛋白导向的UNC-45伴侣网络特别感兴趣，该网络协调从秀丽线虫到人类的肌原纤维形成。除了阐明人类同源基因UNC-45B与疾病的相关性外，我们还在第一个资助期成功地开发了强大的实验方法，用于深入研究肌肉特定的质量控制途径。为了确定肌球蛋白导向的应激反应机制，我们在蠕虫和小鼠肌管中建立了光遗传学方法、转基因报告分析和电脉冲刺激，并结合优化的下拉和质谱学方案。此外，我们确定了一条迄今未被发现的肌肉特定废物管理途径，该途径受UNC-45调控。这项拟议研究的中心目标是了解在机械应力条件下，蛋白质折叠和降解网络如何与肌肉组织和修复的动力学相协调。建议的项目将解决肌球蛋白导向的应激反应计划，并系统分析：UNC-45在蛋白质合成、蛋白质降解和外膜形成中的作用(目标1)，机械应激诱导的UNC-45调控和肌球蛋白导向的质量控制(目标2)，以及UNC-45功能的保守调节(目标3)。为此，将进行机械应力的光遗传诱导、邻近标记技术、CRISPR/Cas9基因编辑、自动运动活动测量和大规模遗传筛查。鉴于我们最近发现的与肌病相关的UNC-45突变和一种新的肌肉特定废物管理系统，再加上在第一个资助期建立的最先进的技术，我们处于理想的地位，可以定义在急性体育锻炼或持续使用期间保护肌肉功能的机械应激反应机制。

Muscle function and maintenance is governed by the spatiotemporal organization of structural and motor proteins into contractile sarcomeres. Given the constant use and mechanical stress exposure of the muscle tissue, the sarcomeric integrity is continuously protected by a complex network of protein folding and degradation pathways. An important regulator of sarcomere formation and muscle function is the HSP90 co-chaperone UNC-45. According to its central role in myosin assembly, UNC-45 protein amount and localization are tightly coordinated with mechanical stress conditions or induced damage to the myofiber. We are particularly interested in the myosin-directed UNC-45 chaperone network, orchestrating myofibrillogenesis from Caenorhabditis elegans to man. Besides elucidating the disease relevance of the human orthologue UNC-45B, we successfully developed powerful experimental approaches during the first funding period for the in-depth investigation of muscle-specific quality control pathways. In order to define myosin-directed stress response mechanisms, we have established optogenetic methods, transgenic reporter assays, and electrical pulse stimulation both in worms and murine myotubes in combination with optimized pull-down and mass spectrometry protocols. Moreover, we identified a so far undiscovered muscle-specific waste management pathway that is regulated by UNC-45. The central objective of the proposed research is to understand how protein folding and degradation networks are coordinated with the dynamics of muscle organization and repair in the context of mechanical stress conditions. The suggested project will address myosin-directed stress response programs and systematically analyze: the role of UNC-45 in protein synthesis, protein degradation, and exopher formation (Aim 1), mechanical stress-induced UNC-45 regulation and myosin-directed quality control (Aim 2), and the conserved regulation of UNC-45 function (Aim 3). To this end, optogenetic induction of mechanical stress, proximity labeling technologies, CRISPR/Cas9 gene editing, automated locomotor activity measurement, and large-scale genetic screenings will be performed. Given our recent discoveries of myopathy-related UNC-45 mutations and a novel muscle-specific waste management system, combined with state-of-the-art technologies established during the first funding period, we are ideally positioned to define mechanical stress response mechanisms that protect muscle function during acute physical exercise or continued use.