Characterization of the Interaction between the N-Terminal Extension of Human Cardiac Troponin I and Troponin C

Ward, Douglas G.; Brewer, Susan; Calvert, Melanie; Gallon, Clare E.; Gao, Yuan; Trayer, Ian P.

doi:10.1021/bi036128l

Cited by 33 publications

(33 citation statements)

References 40 publications

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“…Consistently, NMR studies demonstrated that the NH 2 -terminal extension of cTnI in the absence of phosphorylation interacted with the N-domain of cardiac TnC via residues immediately downstream of the phosphorylation sites (40). Phosphorylation of Ser 23/24 weakened this interaction (40,41).…”

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confidence: 69%

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The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Akhter

Zhang

Jin

2012

American Journal of Physiology-Heart and Circulatory Physiology

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Akhter S, Zhang Z, Jin JP. The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I. Am J Physiol Heart Circ Physiol 302: H923-H933, 2012. First published December 2, 2011; doi:10.1152/ajpheart.00637.2011.-In addition to the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an ϳ30 amino acids NH 2-terminal extension. This peptide segment is a heart-specific regulatory structure containing two Ser residues that are substrates of PKA. Under ␤-adrenergic regulation, phosphorylation of cTnI in the NH 2-terminal extension increases the rate of myocardial relaxation. The NH 2-terminal extension of cTnI is also removable by restrictive proteolysis to produce functional adaptation to hemodynamic stresses. The molecular mechanism for the NH 2-terminal modifications to regulate the function of cTnI is not fully understood. In the present study, we tested a hypothesis that the NH 2-terminal extension functions by modulating the conformation of other regions of cTnI. Monoclonal antibody epitope analysis and protein binding experiments demonstrated that deletion of the NH 2-terminal segment altered epitopic conformation in the middle, but not COOH-terminal, region of cTnI. PKA phosphorylation produced similar effects. This targeted longrange conformational modulation corresponded to changes in the binding affinities of cTnI for troponin T and for troponin C in a Ca 2ϩ -dependent manner. The data suggest that the NH2-terminal extension of cTnI regulates cardiac muscle function through modulating molecular conformation and function of the core structure of cTnI.troponin I phosphorylation; proteolytic NH 2-terminal truncation; TnITnT interface; cardiac muscle regulation; protein conformation analysis THE CONTRACTION OF CARDIAC muscle is regulated by binding of cytosolic Ca 2ϩ to troponin, which activates cross bridge cycling between sarcomeric myosin and actin filaments. The troponin complex consists of three protein subunits: the Ca 2ϩ -binding subunit troponin C (TnC), the tropomyosin-binding subunit troponin T (TnT), and the inhibitory subunit troponin I (TnI) (19). The function of TnI is essential to cardiac muscle contraction (34).Three homologous genes are present in vertebrate species encoding the fast skeletal muscle, slow skeletal muscle, and cardiac isoforms of TnI (20,29). Cardiac TnI (cTnI) is the newest member evolved in the family of TnI isoform genes (7). In addition to the ϳ180 amino acids core structure that is highly conserved in the three TnI isoforms in all vertebrates, cTnI has a unique NH 2 -terminal extension of ϳ30 amino acids, which is not found in the two skeletal muscle TnI isoforms.Showing functional conservation and exchangeability among the TnI isoforms, embryonic cardiac muscle utilizes solely slow skeletal muscle TnI. During development, it is completely replaced by cTnI in the adult heart (22, 36). Therefore, the NH 2 -terminal extension of cTnI is not an essential structure for the basic contractility of c...

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confidence: 69%

“…Phosphorylation of Ser 23/24 weakened this interaction (40,41). Another model proposed that phosphorylation of Ser 23/24 increased the affinity of the switch peptide of cTnI to the N-domain of cardiac TnC (2).…”

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confidence: 99%

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Akhter

Zhang

Jin

2012

American Journal of Physiology-Heart and Circulatory Physiology

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“…Ser22 and Ser23 in the unique NH 2 -terminal region of cTnI are substrates for all these kinases. There is evidence that the unphosphorylated NH 2 -terminal peptide of cTnI binds to the NH 2 lobe of cTnC and is released when Ser22 and Ser23 are phosphorylated (47). The release of the peptide is associated with a decreased Ca 2ϩ sensitivity of myofilament activation, a decrease in the affinity of cTnC for Ca 2ϩ (52), and an increase in cross-bridge kinetics (21).…”

Section: Discussionmentioning

confidence: 99%

Specific enhancement of sarcomeric response to Ca²⁺protects murine myocardium against ischemia-reperfusion dysfunction

Arteaga

Warren

Milutinović

et al. 2005

American Journal of Physiology-Heart and Circulatory Physiology

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Alteration in myofilament response to Ca 2ϩ is a major mechanism for depressed cardiac function after ischemia-reperfusion (I/R) dysfunction. We tested the hypothesis that hearts with increased myofilament response to Ca 2ϩ are less susceptible to I/R. In one approach, we studied transgenic (TG) mice with a constitutive increase in myofilament Ca 2ϩ sensitivity in which the adult form of cardiac troponin I (cTnI) is stoichiometrically replaced with the embryonic/neonatal isoform, slow skeletal TnI (ssTnI). We also studied mouse hearts with EMD-57033, which acts specifically to enhance myofilament response to Ca 2ϩ . We subjected isolated, perfused hearts to an I/R protocol consisting of 25 min of no-flow ischemia followed by 30 min of reperfusion. After I/R, developed pressure and rates of pressure change were significantly depressed and end-diastolic pressure was significantly elevated in nontransgenic (NTG) control hearts. These changes were significantly blunted in TG hearts and in NTG hearts perfused with EMD-57033 during reperfusion, with function returning to nearly baseline levels. Ca 2ϩ -and cross bridge-dependent activation, protein breakdown, and phosphorylation in detergent-extracted fiber bundles were also investigated. After I/R NTG fiber bundles exhibited a significant depression of cross bridge-dependent activation and Ca 2ϩ -activated tension and length dependence of activation that were not evident in TG preparations. Only NTG hearts demonstrated a significant increase in cTnI phosphorylation. Our results support the hypothesis that specific increases in myofilament Ca 2ϩ sensitivity are able to diminish the effect of I/R on cardiac function.

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“…Ward et al (53) also showed that peptide residues 1-18 of cTnI do not bind to cTnC. NMR studies (53,54), as well as previous studies (37), demonstrated that PKA phosphorylation at Ser 23 and Ser 24 produced only localized structural effects in segment residues ϳ25-35.…”

Section: Phosphorylation Of Ctni: Cardiac-specific N-terminal Extensionmentioning

confidence: 92%

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

Solaro

Kobayashi

2011

Journal of Biological Chemistry

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Homeostasis of cardiac function requires significant adjustments in sarcomeric protein phosphorylation. The existence of unique peptides in cardiac sarcomeres, which are substrates for a multitude of kinases, strongly supports this concept (1). We focus here on the troponin complex of the thin filaments, which contain two major proteins that participate in these phosphoryl group transfer reactions: the inhibitory protein (cardiac troponin (cTn) 2 I) and the tropomyosin (Tm)-binding protein (cTnT). We describe the relatively new understanding of the molecular mechanisms of thin filament-based control of the heartbeat and how these mechanisms are altered by phosphorylation. We discuss new concepts regarding the relation between the beat of the heart and the location of thin filament proteins and their long-and short-range interactions. We also discuss elucidation of mechanisms by which these phosphorylations exacerbate or ameliorate effects of mutations in the myofilament proteins that are linked to familial cardiomyopathies. Fig. 1 depicts an A-band region of the cardiac thin filament functional unit in the diastolic and systolic states. In diastole, force-generating reactions of cross-bridges with actin are inhibited, ATP hydrolysis is relatively low, and the sarcomere is relatively extensible (2). Properties of the giant protein titin dominate the compliance of the relaxed sarcomere (3). Interactions of thin filament regulatory proteins, the troponin heterotrimeric complex, and Tm hinder the actin-cross-bridge reaction and establish the B-state. Calcium binding to a single regulatory site on cTnC triggers a release from this inhibited state by modifications of interactions among actin, Tm, and Tn. Thin Filaments during the Relaxed StateEvidence derived from the core crystal structure of cardiac Tn (4), from elucidation of the structures by NMR (5), from biochemical investigations of protein-protein interactions (6, 7), and from reconstructions and single-particle analysis of electron micrographs of reconstituted myofilament preparations (8, 9) provided the basis for the illustration in Fig. 1 (see Ref. 6 for a review). Apart from the lack of a thin filament lattice in the Tn core crystal structure, there was no structural information on significant regions, including the tail region of cTnT, an inhibitory peptide (Ip; which tethers cTnI to actin), the unique N-terminal peptide (ϳ30 amino acids), and portions of the far C-terminal domain of cTnI. Thus, Fig. 1 (upper) shows binding of cTnI to actin via two regions, the highly basic Ip and a second actin-binding region. Importantly, these regions flank a switch peptide, which binds to cTnC when Ca 2ϩ binds to the N-terminal lobe of cTnC, which houses the regulatory Ca 2ϩ -binding site, thereby participating in the mechanism by which Tn releases the thin filament from inhibition.The C-terminal mobile domain of cTnI beyond the second actin-binding site may also participate in establishing the relaxed state. Two observations point to this possibility. The first is a ...

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Characterization of the Interaction between the N-Terminal Extension of Human Cardiac Troponin I and Troponin C

Cited by 33 publications

References 40 publications

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Specific enhancement of sarcomeric response to Ca²⁺protects murine myocardium against ischemia-reperfusion dysfunction

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

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Characterization of the Interaction between the N-Terminal Extension of Human Cardiac Troponin I and Troponin C

Cited by 33 publications

References 40 publications

The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I

The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I

Specific enhancement of sarcomeric response to Ca2+protects murine myocardium against ischemia-reperfusion dysfunction

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

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The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Specific enhancement of sarcomeric response to Ca²⁺protects murine myocardium against ischemia-reperfusion dysfunction