IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Mechanismof molecular aluminium inhibitiononCa2+-ATPasesand itseffectontheintracellularhomeostasis of calcium
Autor/es:
SAFFIOTI NICOLÁS; ROSSI JPFC; FERREIRA GOMES, MARIELA; ROLANDO C ROSSI; DE SAUTU, MARILINA; RINALDI, DEBORA; MANGIALAVORI IRENE
Lugar:
Buenos Aires
Reunión:
Congreso; Protein Biophysics at the End of the World v2.0; 2017
Resumen:
Aluminium (Al3+) is a metal widely distributed in the environment. It is involved with the pathophysiology of neurodegenerative disorders, such as Parkinsonism dementia and Alzheimer?s disease. The mechanisms that have been proposed to explain the toxicity of Al3+ are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets. PMCA is a P-ATPase involved in the regulation of the calcium homeostasis. Its function is to transport Ca2+ from cytoplasm towards the extracellular medium against the electrochemical gradient modulating the cytoplasmic Ca2+ concentration. Like all P-ATPases, PMCA forms a phosphorylated intermediate from ATP during its reaction cycle. The aims of this work were to characterize the molecular inhibitory mechanism of Al3+ on the plasma membrane Ca2+-ATPase (PMCA), and to study the toxic effect of Al3+ at cellular level. We tested the Al3+ effect on Ca2+-ATPase activity and on the levels of PMCA phosphorylated intermediate on purified preparations of PMCA. In order to obtain structural information about the PMCA-Al3+ inhibited complex, we used proteinase-K cleavage and hydrophobic photolabeling with [125I]TID/PC16 to study the cytoplasmic and transmembrane region of PMCA, respectively. To characterize the effect of Al3+ on PMCA at cellular level, we measured the intracellular calcium in HEK293T cells at different concentration of AlCl3, using fluorescent probes. Furthermore, we studied the amount and location of Al3+ in cells using Lumogallion as a direct fluorescence molecular probe for aluminium, detected by confocal fluorescence microscopy and fluorometry.Our results show that: (i) Al3+ binds strongly to PMCA inhibiting the Ca2+-ATPase activity by preventing the dephosphorylation of the pump, (ii) The proteinase-K cleavage in the presence of calcium and ATP shows that Al3+ fixes PMCA in an analogue conformation to the intermediary of ATP hydrolysis E2P (E2PAl3+), (iii) At cellular level, Al3+ inhibits calcium efflux mediated by PMCA in HEK293T cells, (iv) Time-course fluorescence shows an increase of Al3+ in cells as a function of time and concentration, and (v) Confocal fluorescence microscopy indicates that Al3+ first interacts with the membrane lipids and afterwards is located inside the cell.This work was supported by Agencia Nacional de Promoción Científica y Tecnológica PICT 2014 0065, Consejo Nacional de Investigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad de Buenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B