14 research outputs found
Characterisation of a novel interaction between the cardiac myosin binding protein-C and the ryanodine receptor/calcium release channel
Muscle excitation-contraction coupling (ECC) is a fundamental physiological process by which electrically-evoked sarcoplasmic reticulum (SR) Ca2+ release triggers cardiomyocyte contraction. Human ryanodine receptor 2 (RyR2) is an ion channel present in the SR membrane of cardiac myocytes, responsible for mediating calcium efflux. Cardiac myosin binding protein-C (cMyBP-C) is a modular protein anchored to the thick filament through its C-terminal region, whereas the N-terminal region of cMyBP-C is thought to regulate myocardial contractility by modifying actin-myosin association. Here, we present several lines of evidence indicating potential RyR2:cMyBP-C interaction, which could provide a novel retrograde regulation of SR Ca2+ release by the sarcomere.
Firstly, co-immunoprecipitation (co-IP) experiments indicated that the detected putative association between RyR2 and cMyBP-C can be applicable to large mammalian species as well as to recombinant human proteins heterologously expressed in a mammalian cell line, with RyR2 N-terminus and cMyBP-C C-terminus being responsible for the binding. A 3D co-localisation immunocytochemistry study supported the possibility for the proteins to be readily available for protein-protein interaction, with recombinant human RyR2 and cMyBP-C shown to co-localise in HEK293 cells.
Secondly, with the aid of two complementary techniques, namely co-IP and yeast 2-hybrid (Y2H), we showed that RyR2 residues 1-906 comprise the main cMyBP-C site, with residues 1-346 (corresponding to subdomain A and B of RyR2 NTD) and 654-906 (domain SPRY1 and P1) being important determinants for the binding with cMyBP-C C-terminus. Further mapping studies implied that cMyBP-C residues 820-972 and 1061-1274 are involved in the interaction with RyR2 N-terminus, with cMyBP-C Fn domain/s proposed to be directly responsible for the binding. Additionally, parallel experiments with the skeletal muscle RyR1 demonstrated that the corresponding N-terminus region (RyR2 1-906 amino acids) of mammalian isoform RyR1 can also interact with MyBP-C, which could indicate that the detected association can also be relevant in the regulation of ECC in skeletal muscle.
Thirdly, we pursued the possible functional significance of RyR2:cMyBP-C interaction. The results from single cell Ca2+ imaging indicated that cMyBP-C interaction with RyR2 may have an inhibitory effect on Ca2+ release channel function, with cMyBP-C binding diminishing RyR2 channel activity. In particular, HEK293 cells co-expressing RyR2 and cMyBP-C had a reduced frequency of spontaneous intracellular calcium oscillations compared to cells expressing only RyR2.
Lastly, further co-IP data demonstrated that cMyBP-C:RyR2 binding is not affected by four different RyR2 single point mutation variants (S166C, R176Q, R420Q and L433P) associated with different pathological phenotypes. Thus, while a physical and functional association between RyR2 and cMyBP-C was shown to possibly regulate normal cardiac physiology, it is unlikely to be involved in RyR2-related cardiac disease. In order to unravel the pathological significance of the detected cMyBP-C:RyR2 association, more studies are needed to understand what role cMyBP-C plays in RyR2 channel dysfunction that could potentially affect muscle contraction/relaxation
Genetic and Biochemical Approaches for <em>In Vivo</em> and <em>In Vitro</em> Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins
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Elucidating mechanisms of genetic cross-disease associations at the PROCR vascular disease locus
AbstractMany individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte–endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.</jats:p
Effects of Coronary Artery Disease-Associated Variants on Vascular Smooth Muscle Cells
10.1161/CIRCULATIONAHA.121.058389CIRCULATION14612917-92