RESUMEN
Fluorescence resonance energy transfer and native PAGE analytical techniques were employed to assess the quaternary structure of ABCA1, an ATP binding cassette transporter playing a crucial role in cellular lipid handling. These experimental approaches support the conclusion that ABCA1 is associated in dimeric structures that undergo transition into higher order structures, i.e. tetramers, during the ATP catalytic cycle. Our data hence underline molecular assembly as a crucial parameter in ABCA1 function and the advantage of native PAGE as analytical tool for intractable membrane proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Catálisis , Dimerización , Electroforesis en Gel de Poliacrilamida , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Cinética , Sustancias Macromoleculares , Plásmidos , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The ATP-binding cassette transporter A1 (ABCA1) modulates the transbilayer distribution of phosphatidylserine at the outer leaflet of the plasma membrane. This external exposure of phosphatidylserine is a hallmark of microparticle production and is impaired in ABCA1(-/-) mice. In this study, we report about the complete resistance to cerebral malaria of these mice. On analysis of histological and systemic parameters we evidenced an impairment of cellular responses to Plasmodium berghei ANKA infection in ABCA1(-/-) mice, as shown by lower plasma tumor necrosis factor levels, a weaker up-regulation of endothelial adhesion molecules in brain microvessels, a reduced leukocyte sequestration, as well as an ablated platelet accumulation. Besides, the number and the procoagulant activity of microparticles were dramatically reduced in the plasma of ABCA1(-/-) compared to ABCA1(+/+) mice. Moreover, microparticles derived from Plasmodium berghei ANKA-infected ABCA1(+/+) mice induced a significant increase of tumor necrosis factor release by noninfected macrophages. In ABCA1(-/-) mice platelet and macrophage responses to vesiculation agonists were ablated and reduced, respectively. Altogether, by pointing out the ABCA1 transporter as a major element controlling cerebral malaria susceptibility, these data provide a novel insight into its pathophysiological mechanisms and are consistent with a pathogenic role of microparticles in this neurological syndrome.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Eliminación de Gen , Malaria Cerebral/patología , Malaria Cerebral/prevención & control , Transportador 1 de Casete de Unión a ATP , Animales , Humanos , Inmunohistoquímica , Malaria Cerebral/genética , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Plasmodium berghei , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The loss of ABCA1 function leads to Tangier dyslipidemia in humans and to a Tangier-like phenotype in mice, by impairing the transformation of nascent apolipoproteins into mature HDL particles. Mechanistically this ensues from the inability of cells to release membrane lipids and cholesterol. Whereas the ability of ABCA1 to promote phospholipid effluxes, surface binding of apolipoproteins and outward flip of membrane lipids has been documented, the relationship between this series of ABCA1-dependent events is still elusive. Here we provide evidence that i) lipid effluxes require both flip of membrane lipids and binding of apolipoproteins to the cell surface, ii) apolipoprotein A-I binding depends on structural determinants on ABCA1, and iii) phospholipid effluxes can be modulated by engineered mutations on the structural determinants identified on ABCA1.