Allostery and Proteostasis of Regulated Actomyosin Systems in Human Cardiomyocytes

• Applicant: Professor Dr. Dietmar J. Manstein
• Subject Area: Biochemistry ; Cardiology, Angiology ; Structural Biology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314376469

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.

• DFG Programme: Research Grants