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Site-specific Regulation of Myosin Binding Protein-C

Beiersdorfer, Alex
http://orcid.org/0000-0002-0505-5137
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511856330493573

Abstract Details

2017, MS, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
Cardiovascular disease (CVD) is a devastating condition, contributing to nearly 1 in 3 deaths and costing more than $300 billion annually in the United States alone. Development of CVD may be avoided in many cases by minimizing risk factors, such as smoking and unhealthy diet, however a considerable portion of CVD are inherited. Hypertrophic cardiomyopathy (HCM) is one such inherited diseased defined as thickening of the ventricular wall resulting in the inability of the heart to pump blood efficiently. HCM is caused by mutations in a range of sarcomeric genes such as cardiac myosin binding protein C (MYBPC3). MYBPC3 encodes for a thick filament accessory protein (cMyBP-C), a primary modulator of contractility through the phosphorylation of cMyBP-C, which regulates myosin cross bridge formation with actin. Currently, the mechanism of regulation is not well understood. Recent investigations suggest that phosphorylation increases the number of myosin heads available for actin interactions, increasing cross bridging and ultimately contractility. Recent investigations have identified that the fully phosphomimetic state with three aspartic acid residues (DDD) allows for less cross bridging between actin and myosin than the fully phosphodeficient state with three alanine residues (AAA), resulting in less contractility of the sarcomere in DDD. These phosphomimetic studies help to understand the mechanism through which phosphorylation can regulate cMyBP-C function. Although the fully phosphomimetic state has been investigated, there is substantially less known about the partially phosphomimetic states containing both aspartic acid and alanine mutations within the sample protein and whether differential phosphorylation affects cMyBP-C regulation. In this thesis, I tested the hypothesis that phosphorylation states of cMyBP-C affect its binding to myosin in a site-specific manner, thereby regulating the number of heads available for cross bridging. To investigate this, cosedimentation assays were performed using full length myosin and N-terminal fragments of cMyBP-C containing the C0 through C2 domains (C0C2). The C0C2 fragment contains the 3 phosphorylation sites, which were modified with site-specific mutations to replace endogenous serine residues with phospho-ablated alanine (A) or phospho-mimetic aspartic acid (D), respectively. Focus was placed on the second phosphorylation site Ser282, therefore the constructs created contained the amino acid sequence ADA and DAD. The cosedimentation assay resulted in increased binding as well as Kd of the DAD state and decreased binding of the ADA. Interestingly, these constructs display binding similar to the control with identical A or D on the Ser282 site, AAA and DDD respectively. These data taken together suggest the phosphorylation site do not affect cMyBP-C regulation in an equal manner and the Ser282 site has the greatest regulatory effect on cMyBP-C inhibition of myosin and actin cross bridging.
Sakthivel| Sadayappan, Ph.D. (Committee Chair)
William Miller, Ph.D. (Committee Member)
Paul Rosevear, Ph.D. (Committee Member)
55 p.
Molecular Biology
Cardiac myosin binding protein C; phosphomimetic; Cardiovascular Disease; Hypertrophic Cardiomyopathy; MYBPC3

Recommended Citations

Citations

  • Beiersdorfer, A. (2017). Site-specific Regulation of Myosin Binding Protein-C [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511856330493573

    APA Style (7th edition)

  • Beiersdorfer, Alex. Site-specific Regulation of Myosin Binding Protein-C. 2017. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511856330493573.

    MLA Style (8th edition)

  • Beiersdorfer, Alex. "Site-specific Regulation of Myosin Binding Protein-C." Master's thesis, University of Cincinnati, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1511856330493573

    Chicago Manual of Style (17th edition)

ucin1511856330493573
149
© 2017, some rights reserved.Creative Commons License Site-specific Regulation of Myosin Binding Protein-C by Alex Beiersdorfer is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.