Archives of Biochemistry and Biophysics xxx (2016) 1e8 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency Sabine J. van Dijk a, * , Kristina Bezold Kooiker b, 1 , Stacy Mazzalupo a , Yuanzhang Yang a , Alla S. Kostyukova c , Debbie J. Mustacich a , Elaine R. Hoye b , Joshua A. Stern d , Mark D. Kittleson d , Samantha P. Harris a a Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA, USA Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA d Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA, USA b c a r t i c l e i n f o a b s t r a c t Article history: Received 27 November 2015 Received in revised form 31 December 2015 Accepted 7 January 2016 Available online xxx Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mu- tation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degra- dation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. © 2016 Elsevier Inc. All rights reserved. Keywords: cMyBP-C Hypertrophic cardiomyopathy Missense mutation Animal models of cardiac disease 1. Introduction Hypertrophic cardiomyopathy (HCM) is the most common ge- netic cause of cardiomyopathy and is estimated to affect 1 in 500 people [1e3]. Hundreds of mutations in genes encoding sarcomeric proteins are thought to cause HCM [3]. However, the majority of mutations occur in two genes, MYH7 and MYBPC3. MYH7 encodes the b -myosin heavy chain, the primary force generating protein of cardiac muscle, while MYBPC3 encodes cardiac myosin binding protein C (cMyBP-C), an essential regulatory protein that modulates the force and speed of cardiac contraction. Although the majority of mutations in MYH7 cause single amino acid substitutions, most * Corresponding author. Department of Cellular and Molecular Medicine, Uni- versity of Arizona, 1656 East Mabel Street, Tucson, AZ 85724, USA. E-mail address: sjvandijk@email.arizona.edu (S.J. van Dijk). Present address: Division of Pediatric Cardiology, Stanford University, CA, USA. MYBPC3 mutations are non-sense or frameshift mutations that encode premature termination codons and are thus predicted to cause early termination of cMyBP-C. However, truncated cMyBP-C proteins have not been detected in myocardium from HCM pa- tients [4e6], most likely because cell quality control mechanisms either efficiently degrade mRNAs that contain premature termi- nation codons or because truncated or misfolded proteins are rapidly degraded thus preventing their accumulation [4,7,8]. The apparent lack of expression of mutant truncated proteins combined with observations that the total amount of cMyBP-C protein is reduced in some patients with HCM has led to the hypothesis that haploinsufficiency causes sub-stoichiometric amounts of cMyBP-C within the sarcomere which impair contractile function and cause disease. Because reduced cMyBP-C protein expression was also found in myocardium of some patients with cMyBP-C missense mutations that cause single amino acid substitutions [5], both truncation and missense mutations could cause disease through a http://dx.doi.org/10.1016/j.abb.2016.01.006 0003-9861/© 2016 Elsevier Inc. All rights reserved. Please cite this article in press as: S.J. van Dijk, et al., The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency, Archives of Biochemistry and Biophysics (2016), http://dx.doi.org/10.1016/j.abb.2016.01.006
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Jun 13, 2022
R John Solaro 
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