Distinct Regions of Troponin I Regulate Ca2+-dependent Activation and Ca2+ Sensitivity of the Acto-S1-TM ATPase Activity of the Thin Filament

Eyk, Jennifer E. Van; Thomas, L.; Tripet, Brian; Wiesner, Rudolf J.; Pearlstone, Joyce R.; Farah, Chuck S.; Reinach, Fernando C.; Hodges, Robert S.

doi:10.1074/jbc.272.16.10529

Cited by 78 publications

(75 citation statements)

References 40 publications

(26 reference statements)

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“…Exon 3 includes the sequence for the functionally important serines which are phosphorylated by protein kinase A [30]. The ' inhibitory ' region of the protein (amino acids 129-149 in the rat cDNA sequence), which as a peptide is capable of inhibiting actomyosin ATPase activity [31], is contained within exon 7 in all members of the gene family. The cysteines that interact with troponin T (TnT) [32] are split between exons 5 and 6.…”

Section: Gene Structure and Putative Regulatory Motifsmentioning

confidence: 99%

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

Murphy

Thompson

Peng

et al. 1997

Biochemical Journal

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Troponin I is a thin-filament contractile protein expressed in striated muscle. There are three known troponin I genes which are expressed in a muscle-fibre-type-specific manner in mature animals. Although the slow skeletal troponin I isoform is expressed in fetal and neonatal heart, the cardiac isoform is restricted in its expression to the myocardium at all developmental stages. To study the regulation of this cardiac-specific and developmentally regulated gene in vitro, the rat cardiac troponin I gene was cloned. Transient transfection assays were performed with troponin I–luciferase fusion plasmids to characterize the regulatory regions of the gene. Proximal regions of the upstream sequence were sufficient to support high levels of expression of the reporter gene in cardiocytes and relatively low levels in other cell types. The highest luciferase activity in the cardiocytes was noted with a plasmid that included the region spanning -896 to +45 of the troponin I genomic sequence. Co-transfection of GATA-4, a recently identified cardiac transcription factor, with troponin I–luciferase constructs permitted high levels of luciferase expression in non-cardiac cells. Electrophoretic mobility-shift assays demonstrated specific binding of GATA-4 to oligonucleotides representative of multiple sites of the troponin I sequence. Mutation of a proximal GATA-4 DNA-binding site decreased transcriptional activation in transfected cardiocytes. These results indicate that the proximal cardiac troponin I sequence is sufficient to support high levels of cardiac-specific gene expression and that the GATA-4 transcription factor regulates troponin I–luciferase expression in vitro.

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Section: Gene Structure and Putative Regulatory Motifsmentioning

confidence: 99%

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

Murphy

Thompson

Peng

et al. 1997

Biochemical Journal

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“…Reconstituted TnI fragments containing the inhibitory region of TnI (residues 96 -116) plus either the NH 2 -terminal (TnI-(1-116)) or COOHterminal (TnI-(96 -148)) region of TnI were shown to be responsible for either maintaining the maximal level of actomyosin ATPase activity or the Ca 2ϩ dependence of ATPase, respectively (20). In summary, the regulatory complex containing TnT, TnC, and TnI-(96 -148) retained all of the full regulatory properties of troponin, suggesting that TnI-(96 -148) contains the major sequence of TnI responsible for inhibitory activity (17,20). Based upon these experiments, an extended inhibitory region of TnI has been proposed, containing residues 96 -148 (17, 19 -22).…”

mentioning

confidence: 99%

“…Several regions of TnI, including its inhibitory region (residues 96 -116), have been identified as interacting with actin-Tm and TnC (12)(13)(14)(15)(16). Sequence 104 -115 of TnI has been shown to share both the actin-Tm-and TnC-binding sites.…”

mentioning

confidence: 99%

“…Several studies have shown that residues 96 -116 of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC and residues 117-148 for the binding of TnI to the NH 2 -terminal domain of TnC (19,20). Reconstituted TnI fragments containing the inhibitory region of TnI (residues 96 -116) plus either the NH 2 -terminal (TnI-(1-116)) or COOHterminal (TnI-(96 -148)) region of TnI were shown to be responsible for either maintaining the maximal level of actomyosin ATPase activity or the Ca 2ϩ dependence of ATPase, respectively (20). In summary, the regulatory complex containing TnT, TnC, and TnI-(96 -148) retained all of the full regulatory properties of troponin, suggesting that TnI-(96 -148) contains the major sequence of TnI responsible for inhibitory activity (17,20).…”

mentioning

confidence: 99%

“…Furthermore, Tripet et al (17) demonstrated that residues 116 -131 are not important for inhibition, but are critical for the interaction with TnC and designated this region to be the second TnC-binding site (17). Several studies have shown that residues 96 -116 of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC and residues 117-148 for the binding of TnI to the NH 2 -terminal domain of TnC (19,20). Reconstituted TnI fragments containing the inhibitory region of TnI (residues 96 -116) plus either the NH 2 -terminal (TnI-(1-116)) or COOHterminal (TnI-(96 -148)) region of TnI were shown to be responsible for either maintaining the maximal level of actomyosin ATPase activity or the Ca 2ϩ dependence of ATPase, respectively (20).…”

mentioning

confidence: 99%

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The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Szczêsna

Zhang

Zhao

et al. 1999

Journal of Biological Chemistry

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The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(⌬96 -116)), 2) the COOH-terminal domain (TnI-(⌬105-115)), and 3) the NH 2 -terminal domain (TnI-(⌬95-106)). Measurements of Ca 2؉ -regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI⅐TnC complex restored the Ca 2؉ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(⌬96 -116)⅐TnC and TnI-(⌬105-115)⅐TnC. The TnI-(⌬95-106)⅐TnC mutant retained ϳ55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca 2؉ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca 2؉ , and two of them (TnI-(⌬96 -116) and TnI-(⌬105-115)) gave significant activation in the absence of Ca 2؉ . These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH 2 -terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca 2؉ -sensitive molecular switch during muscle contraction.Contraction of skeletal muscle is initiated by the binding of Ca 2ϩ to the regulatory sites of troponin (Tn) 1 C, the Ca 2ϩ -binding subunit of Tn, which causes an interaction with TnI, the inhibitory subunit of Tn, releasing its inhibition of actomyosin ATPase activity. For full biological function, Tn requires a third subunit, TnT, which anchors the TnI⅐TnC complex to the actin-tropomyosin (Tm) filaments and also plays a role in the Ca 2ϩ -mediated activation of actomyosin ATPase activity and/or contraction (1-5).The inhibitory function of Tn has been studied in many different ways, yet the structure-function of the inhibitory region of TnI, responsible for this, is still under investigation. The role of the putative inhibitory region of TnI, which consists of 21 amino acids (residues 96 -116), has been studied previously utilizing synthetic peptides as well as proteolytic and recombinant fragments of TnI. The cyanogen bromide fragment (CN4) of TnI (residues 96 -116) was originally found to possess all of the inhibitory properties of intact TnI (6). Studies with synthetic peptides have demonstrated that residues 105-114 represent the minimal sequence necessary to produce inhibition of actomyosin ATPase activity and to retain TnC binding (7-10); however, the CN4 fragment (TnI-(96 -116)) has been shown to have ...

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Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle

Neverova¹,

Eyk

2002

PROTEOMICS

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The application of protein separation methodologies, such as reversed phase chromatography, should allow differential separation of the proteome, or at least specific subproteomes, comparable to that achieved by two-dimensional electrophoresis (2-DE). A rapid sequential protein extraction method (termed "IN Sequence") was developed to isolate three distinct subproteomes of cardiac muscle. Two subproteomes, those enriched for the cytoplasmic or myofilament proteins, can be separated by either reversed phase high performance liquid chromatography (RP-HPLC) or 2-DE. Reversed phase HPLC of the myofilament protein enriched extract was optimized for resolution and peak numbers by altering flow rate, gradient rate and the organic modifiers, isopropanol and acetronitrile. The myofilament protein enriched extract from failing swine heart, due to coronary artery ligation (LAD), was compared to the extract from a sham operated animal (SHAM). The HPLC chromatograms of these extracts were similar, but distinctive in many regions. The HPLC fractions, collected within some of these distinct regions of the chromatograms were analyzed using peptide mass fingerprinting - mass spectrometry and immunoblot analysis. Two myofilament proteins, troponin T and myosin heavy chain, were identified and found differentially modified in the SHAM and LAD hearts. Both troponin T and myosin heavy chain are problematic proteins for 2-DE, but yet they were resolved by reversed phase chromatography. Therefore, RP-HPLC can be used in conjunction with 2-DE to enhance protein separation of myofilament protein subproteome.

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Distinct Regions of Troponin I Regulate Ca2+-dependent Activation and Ca2+ Sensitivity of the Acto-S1-TM ATPase Activity of the Thin Filament

Cited by 78 publications

References 40 publications

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle

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