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F Wientjes

Publications and source records attributed to F Wientjes.

9 recordsLinked to original sources
PubMed2001

Structure-function relationship in the interaction of mastoparan analogs with neutrophil NADPH oxidase.

Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown to block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxidase activation was now demonstrated in the semirecombinant NADPH oxidase system. The structural basis of the inhibitory effect of MP on oxidase assembly was explored employing a variety of truncated and specifically substituted synthetic peptide analogs. The data indicated that an alpha helical fold, positive net charge, hydrophobicity and amphiphilicity were essential for the inhibitory potency and that peptide analogs below eleven residues were inactive. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rac1 in conjunction with immobilized MP or soluble (125)I-tyr-MP, respectively. The data implicated p67-phox as the main MP-binding component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-phox and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a low affinity towards MP.

Cell-Free System↗
PubMed1997

Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.

Human neutrophils contain a complex of proteins similar to the actin-related protein 2/3 (Arp2/3) complex of Acanthamoeba. We have obtained peptide sequence information for each member of the putative seven-protein complex previously described for Acanthamoeba and human platelets. From the peptide sequences we have identified cDNA species encoding three novel proteins in this complex. We find that in addition to Arp2 and Arp3, this complex contains a relative of the human (Suppressor of Profilin) SOP2Hs protein and four previously unknown proteins. These proteins localize in the cytoplasm of fibroblasts that lack lamellipodia, but are enriched in lamellipodia on stimulation with serum or platelet-derived growth factor. We propose a conserved and dynamic role for this complex in the organization of the actin cytoskeleton.

3T3 Cells↗
PubMed1997

Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils.

The NADPH oxidase generates microbicidal superoxide in phagocytes, and when defective it leads to chronic granulomatous disease (CGD). Oxidase specific proteins in the cytosol, p47phox and p67phox, as well as the small GTP binding protein p21rac are important for activation of superoxide production. Because the activity of this oxidase is normally tightly restricted to the phagocytic vacuole, and its temporal and spatial organisation might be regulated by cytoskeletal proteins, we examined the cytosolic phox proteins for interactions with cytoskeletal elements. p67phox copurified with a 57 kDa protein, identified as coronin, an actin binding protein that is important for movement and phagocytosis in Dictyostelium. Binding studies revealed that coronin attaches to the C-terminal half of p40phox, a binding partner of p67phox. The phox proteins and coronin had a similar distribution in the cell, and both accumulated around the phagocytic vacuole. PMA activation of adherent neutrophils resulted in a major rearrangement of these proteins, and of actin, which were lost from the periphery of the cell and condensed around the nucleus. The rearrangement of F-actin and coronin in adherent cells, were absent, or markedly diminished, in cells from patients lacking p47phox or p67phox in which an abnormally large proportion of the coronin was present as part of a large complex. The cytosolic phox proteins might play a regulatory role in the reorganisation of the cytoskeleton accompanying superoxide generation.

Actins↗
PubMed1996

Tyrosine phosphorylation and subcellular redistribution of p125 ras guanosine triphosphatase-activating protein in human neutrophils stimulated with FMLP.

In this paper, we show that the p125 ras guanosine triphosphatase-activating protein (p125 GAP) is present in the cytosol of human neutrophils and is transiently tyrosine phosphorylated and translocated to the membranes upon cell activation with formyl-methionyl-leucyl-phenylalanine (FMLP). When concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), which both induced a long-lasting respiratory burst, were used as stimuli, tyrosine phosphorylation and translocation of p125 GAP did not occur.

Cell Membrane↗
PubMed1996

Translocation of p190rho guanosine triphosphatase-activating protein from cytosol to the membrane in human neutrophils stimulated with different agonists.

In this paper, we investigated the subcellular distribution of p190rho guanosine triphosphatase-activating protein (p190 GAP) in human neutrophils stimulated with different agonists. The results show that in neutrophils treated with formyl-methionyl-leucyl-phenylalanine (FMLP) (1) p190 GAP was translocated from the cytosol to the membranes; (2) the translocation of p190 GAP took place only at doses of FMLP that induced the translocation of rac 1 and rac 2 and the activation of the NADPH oxidase; and (3) the kinetic of translocation of p190 GAP paralleled that of rac 1 and rac 2. However, when the agonist was concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), rac 1 and rac 2, but not the p190 GAP, were translocated.

Cell Membrane↗
PubMed1995

Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b558.

The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell-free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane-bound and octyl-glucoside-extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91-phox cytochrome b558, two were selected, both of which were found to bind to the beta subunit of cytochrome b558 and to inhibit superoxide formation in the oxidase reconstituted cell-free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X-linked chronic granulomatous disease patients did not produce O2- in the reconstituted system and did not bind to the antibodies.

Animals↗
PubMed1992

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.

Amino Acid Sequence↗