Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.
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PMID:Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure. 777 6

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.
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PMID:Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban. 878 31

Phospholamban is a regulator of the Ca(2+) affinity of the cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and of cardiac contractility. In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca(2+) affinity. To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban were generated. Quantitative immunoblotting revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric. The EC(50) value for Ca(2+) dependence of Ca(2+) uptake was 0.69 +/- 0.07 microM in mutant hearts, compared with 0.29 +/- 0.02 microM in wild-type hearts or 0. 43 +/- 0.03 microM in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca(2+) signal was 146%, compared with wild-types (100%). Langendorff-perfused mutant hearts also demonstrated depressed contractile parameters. Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca(2+) and of cardiac contractility in vivo.
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PMID:Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo. 1074 47

Because of safety issues, components of the beta-adrenergic signaling pathway cannot currently be viewed as attractive targets for human gene therapy. Rather, the balance of evidence supports strategies that will target gene products specifically and directly at diastolic regulation. Augmenting the activity of the SR Ca2+ ATPase by AAV-mediated delivery of the SERCA2a gene, directed by a cardiac-specific promoter with a tightly regulable on-off switch is perhaps the most attractive strategy. PLB and cTnI also are attractive targets but only if molecular techniques can be devised to modulate their activity specifically and conditionally. Such techniques may involve modifying the phosphorylation sites in vitro and replacing wild type proteins in the failing heart with the modified forms, again using regulated AAV vectors for gene delivery.
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PMID:Molecular aspects and gene therapy prospects for diastolic failure. 1098 93

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.
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PMID:Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban. 1283 55

The normally positive force- and Ca2+ -frequency responses (FFR and CaFR) are inverted in heart failure (HF); whether oxidative stress contributes to these abnormalities is unknown. We evaluated the impact of acute and prolonged oxidative stress on contraction and Ca2+ handling in adult rat cardiomyocytes. Acute (30 min) exposure to H2O2 (100 microM) induced a twofold increase (P<0.025) in intracellular oxyradicals together with contractile depression despite preservation of the Ca2+ transient and the FFR and CaFR to 3 Hz, indicating reduced myofilament Ca2+ responsiveness. In contrast, prolonged (24 h) exposure to the copper-zinc superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC, 1 microM) similarly augmented oxyradicals but also increased cell size, and contraction and Ca2+ transient duration (P<0.025). DDC-treated myocytes displayed inverted FFRs and attenuated (though still positive) CaFRs as compared to control, indicating reduced myofilament Ca2+ responsiveness coupled with altered Ca2+ handling. Protein levels of the Na+ -Ca2+ exchanger (NCX), sarcoplasmic reticular (SR) Ca2+ ATPase (SERCA2), and serine-16 phosphorylated phospholamban (pSer16-PLB) were increased (P<0.025), whereas dihydropyridine receptor abundance was decreased. Total PLB and ryanodine receptor protein expression were unchanged. Caffeine-induced Ca2+ release showed increased NCX activity (P<0.025) without changes in total releasable SR Ca2+, suggesting compensatory changes in SERCA2 and pSer16-PLB to maintain SR Ca2+ load. The superoxide scavenger Tiron attenuated these effects. Thus, acute oxyradical exposure rapidly depresses myofibrillar Ca2+ responsiveness. Prolonged oxidative stress further induces alterations in Ca2+ handling that combined with extant reductions in myofibrillar responsiveness invert the FFR. With regard to Ca2+ handling, reduced transsarcolemmal Ca2+ flux rather than reduced SR Ca2+ uptake was the primary determinant of a negative FFR. Analogous changes may be operative in HF, a state characterized by both oxidative stress and Ca2+ dysregulation.
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PMID:Prolonged oxidative stress inverts the cardiac force-frequency relation: role of altered calcium handling and myofilament calcium responsiveness. 1628 76

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.
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PMID:Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility. 1636 42

The sustained positive inotropic effect of alpha-adrenoceptor agonists in the heart is associated with a small increase in intracellular Ca(2+) transients together with a larger sensitization of myofilaments to Ca(2+). The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) could contribute to this effect, either by affecting the Ca(2+) release (ryanodine receptor) or by an uptake mechanism (via phospholamban [PLB] and SR Ca(2+) ATPase). Here we examined the role of CaMKII in the positive inotropic effect of the alpha-adrenoceptor agonist phenylephrine in left atria isolated from a genetic mouse model of cardiac CaMKII inhibition (AC3-I). Compared to atria from wild-type (WT) or AC3-C (scrambled peptide), AC3-I atria showed the following abnormalities. PLB phosphorylation at Thr17, a known CaMKII target, was significantly lower ( approximately 20%). Post-rest (30 s, 1 Hz, 37 degrees C) potentiation of force was absent (AC3-C, 190% of pre-rest amplitude). Basal force was approximately 20% lower at 1.8 mM Ca(2+), but normal at high Ca(2+) concentration (>4.5 mM). The maximal positive inotropic effect of phenylephrine, which was more pronounced at low frequencies in WT and AC3-C atria, lost its frequency dependence (1 Hz to 8 Hz). Thus, the effect of phenylephrine was reduced by approximately 50% at 1 Hz, but was normal at 8 Hz. All three groups showed a negative force-frequency relation, and did not differ in the frequency-dependent acceleration of relaxation. Our data indicate a role of CaMKII in post-rest potentiation and the positive inotropic effect of alpha-adrenergic stimulation at low frequencies.
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PMID:Reduced contractile response to alpha1-adrenergic stimulation in atria from mice with chronic cardiac calmodulin kinase II inhibition. 1729 91

Depressed sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) and Ca(2+)-release channels (ryanodine receptor RyR2) are involved in diabetic cardiomyopathy, however, the implication of intracellular calcium handling proteins in SR is undefined. It was hypothesized that the down-regulation of the intracellular calcium handling proteins of SR is closely related to an up-regulated endothelin (ET) system. Hydroxysafflor yellow A (HSYA) is expected to ameliorate cardiac insufficiency which is mediated by the depressed intracellular calcium handling system in diabetic rat heart. Diabetes was produced in male rats 8 weeks after an injection of streptozotocin (60 mg/kg i.p.) and HSYA was administered (100 mg/kg) by gavage in the last 4 weeks. Hemodynamic and echocardiographic changes, cardiac calcium handling proteins, serum biochemistry, ET system and redox were measured. The compromised cardiac function in diabetic rats was accompanied by a significant down-regulation of the expression of RyR2, FKBP12.6 as well as SERCA2a and PLB. These were closely linked with oxidative stress, an increased ET-1 and up-regulation of ECE, PropreET-1 and iNOS mRNA in diabetic cardiomyopathy. After a 4 week treatment with HSYA, all abnormalities were reversed significantly. In conclusion, diabetic cardiomyopathy was correlated with an abnormal expression of calcium handing proteins in SR and an activated ET-ROS (reactive oxygen species) system in the diabetic affected myocardium. HSYA significantly improved the cardiac function and down-regulated the ET system and ROS pathway, resulting in a reversal of the abnormalities of expression of calcium handing proteins and the cardiac performance in diabetic cardiomyopathy.
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PMID:Cardioprotective effects of hydroxysafflor yellow A on diabetic cardiac insufficiency attributed to up-regulation of the expression of intracellular calcium handling proteins of sarcoplasmic reticulum in rats. 1939 Nov 1

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) regulates the active transport of Ca(2+) into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca(2+) uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.
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PMID:Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function. 2085 Nov 1


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