RESUMO
Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Miosinas/metabolismo , Varredura Diferencial de Calorimetria , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The intronic SNP rs7903146 in the T-cell factor 7-like 2 gene (TCF7L2) is the common genetic variant most highly associated with Type 2 diabetes known to date. The risk T-allele is located in an open chromatin region specific to human pancreatic islets of Langerhans, thereby accessible for binding of regulatory proteins. The risk T-allele locus exhibits stronger enhancer activity compared to the non-risk C-allele. The aim of this study was to identify transcriptional regulators that bind the open chromatin region in the rs7903146 locus and thereby potentially regulate TCF7L2 expression and activity. Using affinity chromatography followed by Edman sequencing, we identified one candidate regulatory protein, i.e. high-mobility group protein B1 (HMGB1). The binding of HMGB1 to the rs7903146 locus was confirmed in pancreatic islets from human deceased donors, in HCT116 and in HEK293 cell lines using: (i) protein purification on affinity columns followed by Western blot, (ii) chromatin immunoprecipitation followed by qPCR and (iii) electrophoretic mobility shift assay. The results also suggested that HMGB1 might have higher binding affinity to the C-allele of rs7903146 compared to the T-allele, which was supported in vitro using Dynamic Light Scattering, possibly in a tissue-specific manner. The functional consequence of HMGB1 depletion in HCT116 and INS1 cells was reduced insulin and TCF7L2 mRNA expression, TCF7L2 transcriptional activity and glucose stimulated insulin secretion. These findings suggest that the rs7903146 locus might exert its enhancer function by interacting with HMGB1 in an allele dependent manner.