Disease-Causing Mutations in Cardiac Troponin T: Identification of a Critical Tropomyosin-Binding Region

Palm, Thomas; Graboski, Sarah; Hitchcock‐DeGregori, Sarah E.; Greenfield, Norma J.

doi:10.1016/s0006-3495(01)75924-3

Cited by 128 publications

(214 citation statements)

References 71 publications

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“…The results confirm that a 1:1 complex of TnT or TnT 1 with Tm binds to actin in both cases. Under these conditions TnT or TnT 1 only causes a comparatively small (ϳ2-fold) increase in the K 50% of Tm for actin similar to that reported for a peptide consisting of residues 70 -150 of cardiac TnT (25) and for the effects of TnT 1 and TnT 2 fragments on non-polymerizable Tm (26). This is a far smaller effect than the 2-3 orders of magnitude change seen for the whole Tn complex (27)(28)(29).…”

Section: Resultssupporting

confidence: 79%

A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

Maytum¹,

Geeves²,

Lehrer³

2002

Journal of Biological Chemistry

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In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnITnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT 1 tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT 1 producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C-to M-state equilibrium, K T , for TnT 1 . This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.

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Section: Resultssupporting

confidence: 79%

A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

Maytum¹,

Geeves²,

Lehrer³

2002

Journal of Biological Chemistry

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“…TNNT2 mutations are located in regions essential for anchoring the troponin-tropomyosin complex onto the thin filament. 18 In 2 unrelated patients, a termination codon (W287X) involving the last residue of the protein was identified. All TNNI3 mutations were located in the carboxyterminus part of troponin I, which is the first binding site to cardiac troponin C. MYL2 mutations are predicted to alter the phosphorylation site and the Ca ϩϩ binding properties.…”

Section: Discussionmentioning

confidence: 99%

Hypertrophic Cardiomyopathy

et al. 2003

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MD; for the EUROGENE Heart Failure ProjectBackground-Hypertrophic cardiomyopathy is an autosomal-dominant disorder in which 10 genes and numerous mutations have been reported. The aim of the present study was to perform a systematic screening of these genes in a large population, to evaluate the distribution of the disease genes, and to determine the best molecular strategy in clinical practice. Methods and Results-The entire coding sequences of 9 genes (MYH7, MYBPC3, TNNI3, TNNT2, MYL2, MYL3, TPM1, ACTC, and TNNC1) were analyzed in 197 unrelated index cases with familial or sporadic hypertrophic cardiomyopathy. Disease-causing mutations were identified in 124 index patients (Ϸ63%), and 97 different mutations, including 60 novel ones, were identified. The cardiac myosin-binding protein C (MYBPC3) and ␤-myosin heavy chain (MYH7) genes accounted for 82% of families with identified mutations (42% and 40%, respectively). Distribution of the genes varied according to the prognosis (Pϭ0.036). Moreover, a mutation was found in 15 of 25 index cases with "sporadic" hypertrophic cardiomyopathy (60%). Finally, 6 families had patients with more than one mutation, and phenotype analyses suggested a gene dose effect in these compound-heterozygous, double-heterozygous, or homozygous patients. Conclusion-These results might have implications for genetic diagnosis strategy and, subsequently, for genetic counseling. First, on the basis of this experience, the screening of already known mutations is not helpful. The analysis should start by testing MYBPC3 and MYH7 and then focus on TNNI3, TNNT2, and MYL2. Second, in particularly severe phenotypes, several mutations should be searched. Finally, sporadic cases can be successfully screened. (Circulation.

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“…Determination of the enthalpy of folding/unfolding using direct fitting or van't Hoff plots should give results within experimental error of one another and the T M values of folding determined from the direct fits, and from the 1st derivative of the melts should be similar to each other. Figures 2 and 3 illustrates the use of circular dichroism to examine the interaction of model proteins containing the C-and N-terminal domains (NTD and CTD, respectively) of tropomyosin, an actin binding protein, with a fragment of the tropomyosin binding domain of troponin (TnT 70-170 ), a protein that regulates contraction of striated muscles in response to calcium [39][40][41][42][43] . The NTD and CTD model proteins form a binary complex, which binds the troponin T fragment to form a ternary complex.…”

Section: Anticipated Resultsmentioning

confidence: 99%

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

2006

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Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy (ΔH) and entropy (ΔS) of unfolding, the midpoint of the unfolding transition (T M ) and the free energy (ΔG) of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength takes approximately eight hours.

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Disease-Causing Mutations in Cardiac Troponin T: Identification of a Critical Tropomyosin-Binding Region

Cited by 128 publications

References 71 publications

A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

Hypertrophic Cardiomyopathy

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

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