Expression in Escherichia coli and a Functional Study of a β-Troponin T 25 kDa Fragment of Rabbit Skeletal Muscle

Fujita, Setsuko; Maéda, Kayo; Maéda, Yuichiro

doi:10.1093/oxfordjournals.jbchem.a123896

Cited by 23 publications

(18 citation statements)

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“…Previous results suggested that the first 45 residues of skeletal TnT are not essential for anchoring the Tn complex to the thin filament and do not play a crucial role in the cooperative response of actomyosin ATPase to Ca 2ϩ . Others have also shown (20,21) that removal of the N-terminal region of TnT does not affect the Ca 2ϩ sensitivity of actomyosin ATPase activity. However, we now know that TnT is more complex than previously thought, and changes in the N-terminal region have recently been shown to affect the structure of the C-terminal region of TnT (22, 23).…”

Section: Discussionmentioning

confidence: 97%

Cardiac Troponin T Isoforms Affect the Ca2+Sensitivity and Inhibition of Force Development

Gomes¹,

Guzman

Zhao

et al. 2002

Journal of Biological Chemistry

119

137

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At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca 2؉ sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher ␣-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca 2؉ sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca 2؉ sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.

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Section: Discussionmentioning

confidence: 97%

Cardiac Troponin T Isoforms Affect the Ca2+Sensitivity and Inhibition of Force Development

Gomes¹,

Guzman

Zhao

et al. 2002

Journal of Biological Chemistry

119

137

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“…Deletion of rabbit residues 1-69 has at most a twofold effect on troponin-tropomyosin binding (14), troponin-induced tropomyosin-actin binding (12)(13)(14), and the cooperativity of thin filament assembly (12, 13). Fujita et al (15), reported normal function for a truncated form of rabbit skeletal muscle TnT that was missing residues 1-58, and includes the amino acid corresponding to Ile79 in human cardiac TnT. These previous studies are consistent with the present results, in which the Ile to Asn mutation at this site has no effect on troponin-tropomyosin binding, troponin-induced tropomyosin-actin binding, Ca 2ϩ activation of the myosin-thin filament ATPase rate, the degree of cooperativity and apparent Ca 2ϩ affinity for this activation, or cooperative myosin S-1 binding to the thin filament.…”

Section: Discussionmentioning

confidence: 99%

“…Numerous structure-function studies of TnT have been reported, providing valuable characterization of the properties of TnT fragments and regions (see reviews in 9-11), but the functions of individual residues have not been investigated. Furthermore, two of the eight reported HCM-associated TnT mutations are within an NH 2 -terminal region that can be deleted with little loss of troponin function (12)(13)(14)(15). To address how one of these TnT mutations, Ile79Asn, causes HCM, this report characterizes the functional properties of the homologous mutation in recombinant rat cardiac TnT.…”

Section: Introductionmentioning

confidence: 99%

Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy.

et al. 1996

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Familial hypertrophic cardiomyopathy (HCM) can be caused by dominant missense mutations in cardiac troponin T (TnT), ␣ -tropomyosin, C-protein, or cardiac myosin heavy chain genes. The myosin mutations are known to impair function, but any functional consequences of the TnT mutations are unknown. This report describes the in vitro function of troponin containing an Ile91Asn mutation in rat cardiac TnT, corresponding to the HCM-causing Ile79Asn mutation in man. Mutant and wild-type TnT cDNAs were expressed in bacteria and the proteins purified and reconstituted with the other troponin subunits. The mutation had no effect on troponin's affinity for tropomyosin, troponininduced binding of tropomyosin to actin, cooperative binding of myosin subfragment 1 to the thin filament, Ca 2 ϩ -sensitive regulation of thin filament-myosin subfragment 1 ATPase activity, or the Ca 2 ϩ concentration dependence of this regulation. However, the mutation resulted in 50% faster thin filament movement over a surface coated with heavy meromyosin in in vitro motility assays. The increased sliding speed suggests an unexpected role for the amino terminal region of TnT in which this mutation occurs. The relationship between this faster motility and altered cardiac contraction in patients with HCM is discussed. ( J. Clin. Invest. 1996. 97:2842-2848.)

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“…The aberrant splicing occurs in the modulatory NH 2 -terminal variable region of TnT (Fig. 3) and the alterations do not destroy the core structure (44,45) and basic activity (13) of TnT. In an isolated system such as the reconstituted myofilaments, the exclusion of exon 8 from turkey cTnT even produced higher Ca 2ϩ sensitivity in comparison with the wild type control (13).…”

Section: The Correlation Of Aberrant Ctnt Splicing With Dilatedmentioning

confidence: 98%

“…The primary structural maps of the embryonic (the asterisk indicates that the embryonic turkey cTnT map was predicted using the adult turkey cTnT sequence plus the chicken embryonic exon 5 segment, assuming the turkey exon 5 segment is identical), and adult turkey cTnT isoforms and the low molecular weight variants are also shown. The previously characterized TnT fragments T1, T2, CB3 (8) and 26-kDa (44,45) are outlined at the top of the figure to demonstrate the location of the alternatively spliced exons to the NH 2 -terminal variable region. b Calculated using the adult sequences plus the chicken embryonic exon 5 segment, assuming the turkey exon 5 segment is identical to the chicken counterpart.…”

Section: Figmentioning

confidence: 99%

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

Biesiadecki

Elder

et al. 2002

Journal of Biological Chemistry

100

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Adult cardiac muscle normally expresses a single cardiac troponin T (cTnT). As a potential pathogenic mechanism for turkey dilated cardiomyopathy, the splice-out of a normally constitutive exon generates an additional low molecular weight cTnT with altered conformation and function. We further found that aberrant splicing of cTnT also occurs in several mammals correlating to dilated cardiomyopathy. Skipping of the same exon as that in the turkey was found in the canine cTnT. Spliceout of the adjacent exon 6 occurred in the guinea pig cTnT. Retention of the embryonic exon 5 was found in the cTnT of cat, dog, and guinea pig. These aberrant splicing variants significantly altered the structure of cTnT to sustain functional effects as that in the myopathic turkey cTnT. The genomic sequence of canine cTnT gene shows no specific alterations. However, the alternative splicing patterns of canine cTnT are different in developing cardiac and skeletal muscles, suggesting abnormality of trans-regulatory factors. Transgenic expression of the aberrant cTnT variants resulted in contractile changes in mouse cardiomyocytes. The findings support the hypothesis that thin filament heterogeneity due to the co-expression of alternatively spliced cTnT variants may desynchronize myocardial contraction and contribute to the pathogenesis and pathophysiology of cardiomyopathy and heart failure.

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Expression in Escherichia coli and a Functional Study of a β-Troponin T 25 kDa Fragment of Rabbit Skeletal Muscle

Cited by 23 publications

References 0 publications

Cardiac Troponin T Isoforms Affect the Ca2+Sensitivity and Inhibition of Force Development

Cardiac Troponin T Isoforms Affect the Ca2+Sensitivity and Inhibition of Force Development

Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy.

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

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