Dynamique des membranes hétérogènes et effets des molécules d'asymétrie stérique positive (étude sur des vésicules géantes)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity.

Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis

Arrangement physique des lipides membranaires susceptibles d’être utilisés par les processus d’adressage cellulaire des protéines

Le rôle des domaines lipidiques distincts au sein de la matrice membranaire capables de servir de zone d’ancrage pour certaines protéines, est évoqué par de nombreuses observations biologiques. Les protéines glypiées (liées au glycosyl-phosphatidylinositol), palmitoylées et myristoylées présentent des chaînes grasses saturées susceptibles d’interagir avec la phase lamellaire ordonnée (Lo) constituée par les sphingolipides saturés et le cholestérol membranaire. La distribution focalisée de ces protéines sur la surface cellulaire, leur localisation identique à celle des sphingo(glyco)lipides saturés ou au contraire la dispersion de leur distribution hétérogène lorsque le cholestérol membranaire est extrait par un traitement à la méthylcyclodextrine ont confirmé l’existence in vivo de ces domaines membranaires.L’étude par diffraction des rayons X d’un mélange quaternaire de lipides d’origine naturelle (phosphatidylsérine, phosphatidyléthanolamine, sphingomyéline et cholestérol dans le rapport molaire 1/4/3/3) permet de conforter l’hypothèse de la ségrégation d’un domaine lamellaire distinct produit par l’interaction préférentielle de la sphingomyéline et du cholestérol. L’arrangement interdigité de ce domaine Lo est évoqué par la faible distance répétitive. Les conditions physiques (température de l’ordre de la température physiologique) et chimiques (la composition du mélange lipidique riche en phosphatidylsérine et phosphatidyléthanolamine, la présence favorable de calcium, la stéréospécificité de l’interaction entre le stérol et la sphingomyéline, l’asymétrie des chaînes grasses de ce dernier lipide) et la réversibilité de la ségrégation de phases suggèrent fortement la formation in vivo de tels domaines à la face interne de la membrane plasmique et leur interdigitation avec les domaines correspondant à la face extracellulaire

Cytosolic phospholipase A2-p11 interaction controls arachidonic acid release as a function of epithelial cell confluence.

Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E2 in response to various stimuli when analysed after cell confluence. In contrast, non-confluent Madin-Darby canine kidney type II cells released much higher amounts of AA and prostaglandin E2. In both stationary and non-confluent cells, AA was released by type IV cPLA2 (cytosolic phospholipase A2), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA2 activation was not due to a difference in expression, or in phosphorylation of the enzyme, or in the amount of its substrate. To find out the mechanism by which cPLA2 activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA2, p11, a natural inhibitor of the enzyme, and annexin II, the natural ligand of p11. These three proteins were expressed at a constant level, regardless of the cell confluence. In contrast, whereas annexin II and cPLA2 interacted at a constant rate, p11 and cPLA2 interacted more strongly in stationary cells, thus indicating that cPLA2 activation is regulated by its accessibility to p11, independent of their expression level. Our results indicate that, in epithelial cells, the cell confluence, i.e. the establishment of cell-cell contacts, rather than cell proliferation directly controls cPLA2 activation by changing the stoichiometry of p11/cPLA2 interaction