Allostery and Proteostasis of Regulated Actomyosin Systems in Human Cardiomyocytes

在这个项目的框架内，我们的目标是表征肌动球蛋白复合体在人类心肌细胞中的机械力化学、变构和调节。肌球蛋白、肌动蛋白、肌钙蛋白和原肌球蛋白的翻译后修饰、异构体依赖的差异和致病突变的功能后果通过由正确匹配的组分重组的肌球蛋白复合体来表征。根据蛋白质-蛋白质相互作用、变构通讯和蛋白质稳定性的变化来评估基于小分子的方法的选择性和有效性。我和我的团队已经证明，小分子肌球蛋白57033可以恢复死亡肌球蛋白的活性，这些蛋白因应激诱导的错误折叠而变得不活跃。我们的工作首次展示了药理伴侣诱导的蛋白质折叠，随后恢复了蛋白质的功能。通过一系列的体外实验，我们进一步表明，肌球蛋白的稳定、复性和增加肌球蛋白力的产生可以通过其他化合物和化合物类来实现。在β-心肌肌球蛋白的情况下，我们观察到了噻二酮衍生物、小代谢物和多肽的类似效果。我们已经开始将我实验室开发的工具和方法与先进的基于先进仪器的工具相结合，以分析受调控的肌球蛋白复合体对不同类型的变构触发事件的响应，分辨率为亚纳米和亚毫秒。因此，我们现在能够全面研究运动活性或药物伴侣介导的蛋白质稳定和折叠事件的潜在机制。更好地理解潜在的过程将有助于设计和鉴定在稳定、折叠和激活力量产生方面比EMD 57033更有选择性的化合物。具有更高亲和力的肌球蛋白效应器具有改进的异构体特异性，有望促进强大的新治疗工具的开发，以治疗包括心力衰竭在内的遗传性和非遗传性横纹肌疾病。更好地了解和评价它们在酶周转和蛋白平衡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果的解释。此外，我们希望用肌球蛋白获得的结果可以作为开发基于小分子的方法的范例，以诱导其他类型的蛋白质在药理上由伴侣介导的折叠，例如那些与错误折叠的蛋白质在中枢神经系统中积累有关的折叠。

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.