Dissecting Transient Protein Interactions Implicated in Cardiovascular Disease: G Protein-Coupled Receptors and Cardiac Myosin-Binding Protein C
2021-10
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Dissecting Transient Protein Interactions Implicated in Cardiovascular Disease: G Protein-Coupled Receptors and Cardiac Myosin-Binding Protein C
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2021-10
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Weak, transient protein-protein interactions in the cell are being increasingly appreciated, yet characterization of these interactions presents a unique challenge. We have used protein engineering techniques, including ER/K α-helical linkers and DNA nanotechnology, to characterize G protein-coupled receptor (GPCR) and cardiac myosin-binding protein C (cMyBP-C) interactions.The cellular environment can have a significant impact on GPCR signaling and functional selectivity. Our lab has found that GPCR interactions with non-cognate G-proteins can enhance, or ‘prime’, signaling through the canonical pathway. To investigate the impact of non-cognate interactions on signaling in two promiscuous Gi-coupled receptors, adenosine type 1 (A1R) and cannabinoid type 1 (CB1), we utilized a variation of the Systematic Protein Affinity Strength Modulation (SPASM) approach to observe the impact on downstream signaling in live cells. To the C-terminus of intact A1R or CB1, we tethered native G-peptides (s-pep, i-pep, and q-pep) derived from the Gα subunit of G-proteins. We found that i-pep and q-pep enhanced Gi signaling while suppressing Gq signaling. This study provides an initial model for the impact of G-peptide interactions in Gi-coupled receptors, and highlights the potential of G-peptide interactions to enhance receptor specificity.
CMyBP-C is an important regulator of cardiac muscle contraction and is commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanism of regulation by cMyBP-C remains unclear due to experimental challenges in dissecting these weak, transient interactions. In this study we utilized a nanosurf assay, containing a synthetic β-cardiac myosin thick filament, to systematically probe cMyBP-C interactions with actin and myosin. We recapitulated inhibition of β-cardiac myosin HMM nanotube motility by C0-C2 and C1-C2 N-terminal fragments. Equivalent inhibition of an β-cardiac myosin S1 construct suggests the actin-cMyBP-C interaction dominates this inhibitory mechanism. We found that a C0-C1f fragment lacking the majority of the M-domain did not inhibit β-cardiac myosin nanotube motility, confirming the importance of the M-domain in regulatory interactions. Release of inhibition by phosphomimetic fragments further highlights the importance of the phosphorylatable serines in the regulatory M-domain. These results shed light on the mechanism of cMyBP-C and highlight the utility of the nanosurf assay for precisely manipulating and defining transient protein interactions.
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University of Minnesota Ph.D. dissertation. October 2021. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Sivaraj Sivaramakrishnan. 1 computer file (PDF); xi, 104 pages.
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Touma, Anja. (2021). Dissecting Transient Protein Interactions Implicated in Cardiovascular Disease: G Protein-Coupled Receptors and Cardiac Myosin-Binding Protein C. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/225873.
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