An Unusual Membrane-Protein Topology for Sensing Bilayer Thickness and Triggering Bacterial Biofilm Formation

Abstract

Mutations in MYBPC3, the gene encoding the muscle regulatory protein cardiac myosin binding protein-C (cMyBP-C), are among the most common causes of hypertrophic cardiomyopathy (HCM) in both people and cats. However, despite the high prevalence of mutations in MYBPC3, relatively little is understood regarding how mutations lead to disease. One possibility is that some point mutations alter cMyBP-C protein structure leading to enhanced degradation and elimination of the mutant protein. If levels of cMyBP-C protein expression are reduced, then haploinsufficiency (lack of sufficient protein) can trigger disease. Here we tested this idea by analyzing the impact of the A31P mutation, linked to HCM in Maine Coon cats, on 1) the in vitro protein structure of the C0 domain of cMyBP-C, and 2) the total protein expression of cMyBP-C in myocardium of aged cats heterozygous for the A31P mutation. In vitro results demonstrated that the A31P mutation disrupts folding of the C0 domain as shown by three independent methods: altered epitope recognition on Western blots; changes in sensitivity to proteolytic degradation; and reduced b-sheet content assessed by circular dichroism. Western blots of endogenous cMyBP-C obtained from myocardial samples also suggested that C0 structure is altered in vivo because an antibody that preferentially recognizes C0 reacted less with A31P cMyBP-C compared to wild-type cMyBP-C. However, despite these significant structural differences, the A31P cMyBP-C was incorporated into sarcomeres and total cMyBP-C protein (wild-type plus mutant) was similar in wild type and heterozygous A31P cats. These results suggest that despite protein folding abnormalities, the A31P mutation does not lead to haploinsufficiency in the population of older heterozygous cats studied here.