An investigation of myosin binding protein C mutations in South Africa and a search for ligands binding to myosin binding protein C

[摘要] ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease.The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and anincreased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring inthirteen genes, have been identified. As the vast majority of HCM-causing mutations occur incomponents of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere.Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed thatHCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausingmutations in the gamma two subunit of adenosine monophosphate activated protein kinasehighlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports ahypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion.Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second mostprevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomericthick filament, where it acts as a regulator of thick filament structure and cardiac contractility.Although cMyBPC has been studied extensively, the mechanisms through which it fulfill thesefunctions have remained elusive, largely due to a lack of a comprehensive understanding of itsinteractions with other sarcomeric components and its quaternary structure.The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in apanel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC inwhich HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individualswas screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)-based single-strand conformation polymorphism method, as well as restriction enzyme digestion,DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands ofdomains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandandlibrary-assays were performed.Three novel and two previously described putative HCM-causing mutations were identified inMYBPC3. Data generated in this and other studies, however, suggest that two of these 'mutationsare likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausingmutations.Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This findingidentified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specificC7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 anddomain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no knownsarcomeric ligands of the N-terminal region of cMyBPC.Identification of the ligands of specific domains of cMyBPC led to the development of detailedmodels of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fullyphosphorylated. The integration of these models into an existing model of thick filament quaternarystructure allows new insights into the functioning of cMyBPC as a regulator of both thick filamentstructure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM.

[发布机构] Stellenbosch University