Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

LE CYTOCHROME B 5 5 8 CUR MEMBRANAIRE REDOX DE LA NADPH OXYDASE

LE CYTOCHROME B 5 5 8 DU COMPLEXE DE LA NADPH OXYDASE GENERE DES IONS SUPEROXYDE GRACE A UN TRANSFERT D'ELECTRONS DU NADPH VERS L'OXYGENE. LA NADPH OXYDASE DORMANTE ET DISSOCIEE DANS LES CELLULES AU REPOS DEPEND POUR FONCTIONNER DE L'ASSEMBLAGE DE PROTEINES CYTOSOLIQUES (P47-PHOX, P67-PHOX, P40-PHOX ET RAC) SUR LE CONSTITUANT MEMBRANAIRE REDOX, LE FLAVOCYTOCHROME B 5 5 8, HETERODIMERE FORME DE DEUX SOUS-UNITES GP91-PHOX GLYCOSYLEE ET P22-PHOX. ACTUELLEMENT, LES MODALITES DE TRANSFERT DES ELECTRONS AU NIVEAU DU CYTOCHROME B 5 5 8 AINSI QUE LES MECANISMES D'ASSEMBLAGE DES FACTEURS CYTOSOLIQUES RESTENT MECONNUS. POUR ABORDER CES ASPECTS NOUS AVONS UTILISE DEUX MODELES : LE LYMPHOCYTE B-EBV QUI EXPRIME TOUS LES COMPOSANTS DE LA NADPH OXYDASE MAIS DONT L'ACTIVITE OXYDASE EST REDUITE, ET LE NEUTROPHILE QUI SERT DE REFERENCE. 1) LE CYTOCHROME B 5 5 8 A ETE ISOLE ET PURIFIE A PARTIR DES MEMBRANES DE LB-EBV. IL EST LE FACTEUR LIMITANT L'ACTIVITE OXYDASE DANS CES CELLULES LORSQU'IL EST INSERE DANS LES MEMBRANES, MAIS UNE FOIS PURIFIE, SES PROPRIETES BIOCHIMIQUES SONT SEMBLABLES A CELLES DU NEUTROPHILE 2) SA FORTE GLYCOSYLATION DANS LES LB-EBV N'INFLUENCE PAS L'ACTIVITE OXYDASE 3) L'ETUDE DES MECANISMES D'ASSEMBLAGE DES PROTEINES DU COMPLEXE A ETE REALISEE PAR DEUX APPROCHES : LA RECONSTITUTION D'UNE ACTIVITE OXYDASE EN MILIEU ACELLULAIRE METTANT EN CONTACT LE CYTOCHROME B 5 5 8 PURIFIE ET LES FACTEURS CYTOSOLIQUES RECOMBINANTS, ET L'ETUDE PAR MICROSCOPIE A FORCE ATOMIQUE DE LA TOPOGRAPHIE DE LIPOSOMES CONTENANT LE CYTOCHROME B 5 5 8 APRES ASSEMBLAGE DE LA NADPH OXYDASE. LES RESULTATS MONTRENT UNE AUGMENTATION DE LA TAILLE DES LIPOSOMES APRES ASSEMBLAGE DES FACTEURS CYTOSOLIQUES SUGGERANT QUE IN VITRO, LA TRANSITION DE L'ETAT INACTIF VERS L'ETAT ACTIF POURRAIT FAIRE INTERVENIR UN PROCESSUS DE REGULATION CONFORMATIONNELLE DANS LEQUEL P67-PHOX JOUERAIT UN ROLE CENTRAL. CE TRAVAIL SE POURSUIT AVEC UN NOUVEAU MODELE, DICTYOSTELIUM DISCOIDEUM CHEZ QUI NOUS AVONS CARACTERISE UNE ACTIVITE NADPH OXYDASE.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases.

International audienceInteraction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint

Participation of Rac GTPase activating proteins in the deactivation of the phagocytic NADPH oxidase.

International audienceThe aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase

Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.

International audienceActivation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states

An Absence of Non-specific Immune Response towards para -Sulphonato-calix[ n ]arenes

International audienceThe effects of para-Sulphonato-calix[4]arene, para-Sulphonato-calix[6]arene and para-Sulphonato-calix[8]arene on the activation of NADPH oxidase in neutrophils has been studied. All three molecules do not induce NADPH oxidase activation, and hence do not stimulate neutrophils. Measurement of cell viability demonstrates that these three water-soluble calix[n]arene derivatives are not cytotoxic

Glucocerebroside inhibits NADPH oxidase activation in cell-free system.

International audienceWe reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O(2)(-)) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients' phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O(2)(-). We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47(phox) and p67(phox). In contrast, O(2)(-) generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/xanthine oxidase system, GC had no effect on the generation of O(2)(-). These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC

Protein kinase C as an effector of lipid-derived second messengers.

International audienceMembers of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these immune cells. Since neutrophils provide a ready source of human primary tissue, these methods are also useful for pharmacological studies on the protein kinase C system and for evaluation of protein kinase C activators and inhibitors in the context of human primary cells. Furthermore, since protein kinase C activity is determined by a number of lipid-generating signaling systems, the methods described here can also be employed to study the pharmacology of these "upstream" signaling systems