Publications (4)17.97 Total impact
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Article: PMCA differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation
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ABSTRACT: The exposure of plasma membrane calcium pump (PMCA) to surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analogue [125I]TID-PC/16 to the protein. In the presence of Ca2+ both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in 3 main fragments: N which includes transmembrane segments M1 and M2, M which includes M3 and M4 and C which includes M5 to M10. CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, while phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured RET between PMCA labeled with eosin-isothiocyanate at the ATP-binding site and the phospholipid Rho-PE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes while PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.Journal of Biological Chemistry 03/2011; · 4.77 Impact Factor -
Article: Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation.
[show abstract] [hide abstract]
ABSTRACT: The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.Journal of Biological Chemistry 03/2011; 286(21):18397-404. · 4.77 Impact Factor -
Article: The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.
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ABSTRACT: Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.Journal of Biological Chemistry 12/2009; 285(7):4544-53. · 4.77 Impact Factor -
Article: A New Conformation in SERCA and PMCA Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe
Biophysical Journal 01/2009; 96:614. · 3.65 Impact Factor
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Institutions
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2011
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University of Buenos Aires
- Facultad de Farmacia y Bioquímica
Buenos Aires, Buenos Aires F.D., Argentina
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2009
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Fundación Instituto Leloir
Buenos Aires, Buenos Aires F.D., Argentina
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