Allostery and Proteostasis of Regulated Actomyosin Systems in Human Cardiomyocytes

在这个项目的框架内，我们的目标是表征的机械化学，变构，并调节肌动球蛋白复合物，因为它们发生在人类心肌细胞。肌球蛋白、肌动蛋白、肌钙蛋白和原肌球蛋白的翻译后修饰、亚型依赖性差异和致病突变的功能后果的特征在于肌动球蛋白复合物的帮助下，所述肌动球蛋白复合物从正确匹配的组分重构。在蛋白质-蛋白质相互作用、变构通信和蛋白质稳定性的变化方面评价基于小分子的方法的选择性和功效。我和我的团队已经证明，小分子EMD 57033可以恢复因应力诱导的错误折叠而失去活性的死亡肌球蛋白的活性。我们的工作代表了药理学伴侣诱导的蛋白质重折叠的第一个演示，随后恢复蛋白质的功能。通过一系列体外实验，我们进一步表明，稳定，重折叠，并增加肌球蛋白力的生产可以介导的其他化合物和化合物类。在β-心肌肌球蛋白的情况下，我们观察到噻二嗪酮衍生物，小代谢物和肽的类似作用。我们已经开始联合收割机在我的实验室开发的工具和方法与先进的新仪器为基础的先进的分析调节肌动球蛋白复合物的响应不同类型的变构触发事件与亚纳米和亚毫秒的分辨率。因此，我们现在能够全面研究运动活动或药理学伴侣介导的蛋白质稳定和重折叠事件的潜在变化机制。更好地了解潜在的过程将有助于设计和鉴定比EMD 57033更有选择性地稳定，重折叠和激活力产生的化合物。具有改进的同种型特异性的更高亲和力的肌球蛋白效应物有望促进强大的新治疗工具的开发，以治疗遗传和非遗传形式的横纹肌疾病，包括心力衰竭。更好地理解和评价它们在酶周转和蛋白质稳定方面的作用将有助于改进临床研究的设计，并促进对试验中观察到的临床效应的解释。此外，我们预计，肌球蛋白获得的结果可以作为一个范例，为发展的小分子为基础的方法，以诱导药理学伴侣介导的其他类型的蛋白质，如与错误折叠的蛋白质在中枢神经系统中的积累的重折叠。

Within the framework of this project, we aim to characterize the mechanochemistry, allostery, and regulation of actomyosin complexes as they occur in human cardiomyocytes. The functional consequences of post-translational modifications, isoform-dependent differences, and disease-causing mutations of myosin, actin, troponin, and tropomyosin are characterized with the help of actomyosin complexes that are reconstituted from correctly matched components. The selectivity and efficacy of small molecule-based approaches is evaluated in regard to changes in protein-protein interactions, allosteric communication, and protein stability. My team and I have shown that the small molecule EMD 57033 can restore the activity to dead myosin protein that has been rendered inactive by stress-induced misfolding. Our work represents the first demonstration of a pharmacological chaperone-induced protein refolding, with subsequent restoration of the function of the protein. Through a series of in vitro experiments, we have further shown that stabilization, refolding, and increase of myosin force production can be mediated by other compounds and compound classes. In the case of beta-cardiac myosin, we observed similar effects with thiadiazinone derivatives, small metabolites, and peptides. We have started to combine the tools and methods developed in my laboratory with sophisticated new instrument-based advanced to analyze the response of regulated actomyosin complexes to different types of allosteric trigger events with sub-nanometer and sub-millisecond resolution. Therefore, we are now in a position to comprehensively investigate the mechanisms underlying changes in motor activity or pharmacological chaperone-mediated protein stabilization and refolding events. A better understanding of the underlying processes will aid in the design and identification of compounds that act more selectively in regard to stabilization, refolding, and activation of force production than EMD 57033. Higher affinity myosin effectors with improved isoform-specificity are expected to foster the development of powerful new therapeutic tools to treat genetic and non-genetic forms of striated muscle diseases including heart failure. Better understanding and appreciation of their effects in regard to enzymatic turnover and proteostasis will help to improve the design of clinical studies and facilitate the interpretation of the clinical effects observed in trials. Moreover, we expect that the results obtained with myosin can serve as a paradigm for the development of small molecule-based approaches to induce the pharmacological chaperone-mediated refolding of other types of proteins, such as those associated with the accumulation of misfolded protein in the central nervous system.