RESUMEN
The mammalian gut microbiota is essential for normal intestinal development, renewal, and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host-derived and bacterial peptides and small molecules. Here we report that specific members of the gut microbiota stimulate FPR1 on intestinal epithelial cells to generate reactive oxygen species via enterocyte NADPH oxidase 1 (NOX1), causing rapid phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase mitogen-activated protein kinase. These events stimulate migration and proliferation of enterocytes adjacent to colonic wounds. Taken together, these findings identify a novel role of FPR1 as pattern recognition receptors for perceiving the enteric microbiota that promotes repair of mucosal wounds via generation of reactive oxygen species from the enterocyte NOX1.
Asunto(s)
Homeostasis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Oxidación-Reducción , Receptores de Formil Péptido/metabolismo , Transducción de Señal , Animales , Bacterias , Colon/inmunología , Colon/metabolismo , Colon/microbiología , Colon/patología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Modelos Biológicos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , Especies Reactivas de Oxígeno/metabolismo , Cicatrización de HeridasRESUMEN
The NADPH oxidase of phagocytes catalyzes the conversion of oxygen to O2(-). This multicomponent enzyme complex contains five essential protein components, two in the membrane and three in the cytosol. Unassembled and inactive in resting phagocytes, the oxidase becomes active after translocation of cytosolic components to the membrane to assemble a functional oxidase. Multiple factors regulate its assembly and activity, thus serving to maintain this highly reactive system under spatial and temporal control until recruited for antimicrobial or proinflammatory events. The recent identification of homologs of one of the membrane components in nonphagocytic cells will expand understanding of the biological contexts in which this system may function.
Asunto(s)
NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Grupo Citocromo b/metabolismo , Regulación Enzimológica de la Expresión Génica , Fosfoproteínas/metabolismoRESUMEN
High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.
Asunto(s)
Matriz Extracelular/metabolismo , Flavoproteínas , NADPH Oxidasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/anatomía & histología , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Clonación Molecular , ADN Complementario/genética , Oxidasas Duales , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Fagocitos/enzimología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Emerging evidence indicates that reactive oxygen species are important regulators of vascular function. Although NAD(P)H oxidases have been implicated as major sources of superoxide in the vessel wall, the molecular identity of these proteins remains unclear. We recently cloned nox1 (formerly mox-1), a member of a new family of gp91(phox) homologues, and showed that it is expressed in proliferating vascular smooth muscle cells (VSMCs). In this study, we examined the expression of three nox family members, nox1, nox4, and gp91(phox), in VSMCs, their regulation by angiotensin II (Ang II), and their role in redox-sensitive signaling. We found that both nox1 and nox4 are expressed to a much higher degree than gp91(phox) in VSMCS: Although serum, platelet-derived growth factor (PDGF), and Ang II downregulated nox4, they markedly upregulated nox1, suggesting that this enzyme may account for the delayed phase of superoxide production in these cells. Furthermore, an adenovirus expressing antisense nox1 mRNA completely inhibited the early phase of superoxide production induced by Ang II or PDGF and significantly decreased activation of the redox-sensitive signaling molecules p38 mitogen-activated protein kinase and Akt by Ang II. In contrast, redox-independent pathways induced by PDGF or Ang II were unaffected. These data support a role for nox1 in redox signaling in VSMCs and provide insight into the molecular identity of the VSMC NAD(P)H oxidase and its potentially critical role in vascular disease.
Asunto(s)
Glicoproteínas de Membrana/genética , Músculo Liso Vascular/metabolismo , Animales , Northern Blotting , Línea Celular , Células Cultivadas , ADN sin Sentido/genética , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Análisis de Secuencia de ADN , Transducción de Señal , Superóxidos/metabolismo , Factores de TiempoRESUMEN
Nox1, a homologue of gp91phox, the catalytic moiety of the superoxide (O(2)(-))-generating NADPH oxidase of phagocytes, causes increased O(2)(-) generation, increased mitotic rate, cell transformation, and tumorigenicity when expressed in NIH 3T3 fibroblasts. This study explores the role of reactive oxygen species (ROS) in regulating cell growth and transformation by Nox1. H(2)O(2) concentration increased approximately 10-fold in Nox1-expressing cells, compared with <2-fold increase in O(2)(-). When human catalase was expressed in Nox1-expressing cells, H(2)O(2) concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. A large number of genes, including many related to cell cycle, growth, and cancer (but unrelated to oxidative stress), were expressed in Nox1-expressing cells, and more than 60% of these returned to normal levels on coexpression of catalase. Thus, H(2)O(2) in low concentrations functions as an intracellular signal that triggers a genetic program related to cell growth.
Asunto(s)
División Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , NADH NADPH Oxidorreductasas/fisiología , Animales , Secuencia de Bases , Catalasa/metabolismo , División Celular/fisiología , Línea Celular , Cartilla de ADN , Ratones , Ratones Desnudos , NADPH Oxidasa 1 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
gp91phox is the catalytic subunit of the respiratory burst oxidase, an NADPH-dependent, superoxide generating enzyme present in phagocytes. In phagocytes, the enzyme functions in host defense, but reactive oxygen generation has also been described in a variety of non-phagocytic cells, including cancer cells. We previously reported the cloning of Nox1 (NADPH oxidase1), a homolog of gp91phox, its expression in colon and vascular smooth muscle, and its oncogenic properties when overexpressed [Suh et al. (1999). Nature 401, 79-82]. Herein, we report the cloning and tissue expression of three additional homologs of gp91phox, termed Nox3, Nox4 and Nox5, members of a growing family of gp91phox homologs. All are predicted to encode proteins of around 65 kDa, and like gp91phox, all show 5-6 conserved predicted transmembrane alpha-helices containing putative heme binding regions as well as a flavoprotein homology domain containing predicted binding sites for both FAD and NADPH. Nox3 is expressed primarily in fetal tissues, and Nox4 is expressed in not only fetal tissues, but also kidney, placenta and glioblastoma cells. Nox5 is expressed in a variety of fetal tissues as well as in adult spleen and uterus. Nox isoforms are aberrantly expressed in several cells derived from human cancers, with Nox4 being the isoform most frequently expressed in the tumor cells investigated. Thus, expression of Nox family members is likely to account for some of the reactive oxygen generation seen in non-phagocytic cells.
Asunto(s)
Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , NADPH Oxidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CACO-2 , Bovinos , Clonación Molecular , ADN Complementario , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasa 5 , ARN Mensajero , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
A series of truncated forms of gp91phox were expressed in Escherichia coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin. TRX-gp91phox (306-569), which contains the putative FAD and NADPH binding sites, showed weak NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91phox (304-423) and TRX-gp91phox (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91phox (306-569), and showed the same K(m) for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. In the presence of Rac1, the cytosolic regulatory protein p67phox stimulated the NBT reductase activity, but p47phox had no effect. Truncated p67phox containing the activation domain (residues 199-210) [C.-H. Han, J.R. Freeman, T. Lee, S.A. Motalebi, and J.D. Lambeth (1998) J. Biol. Chem. 273, 16663-16668] stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: (i) TRX-gp91phox (306-569) contains binding sites for both pyridine and flavin nucleotides; (ii) this flavoprotein domain shows weak diaphorase activity; and (iii) the flavin-binding domain of gp91phox is the target of regulation by the activation domain of p67phox.
Asunto(s)
Flavoproteínas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Sitios de Unión , Factores Biológicos/farmacología , Clonación Molecular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavina-Adenina Dinucleótido/metabolismo , Flavina-Adenina Dinucleótido/farmacología , Cinética , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , NADP/metabolismo , NADP/farmacología , NADPH Deshidrogenasa/antagonistas & inhibidores , NADPH Deshidrogenasa/genética , NADPH Oxidasa 2 , Compuestos Onio/farmacología , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Superóxido Dismutasa/metabolismo , Termodinámica , Proteína de Unión al GTP rac1/metabolismoAsunto(s)
Glicoproteínas de Membrana/genética , NADH NADPH Oxidorreductasas/metabolismo , Animales , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Peso Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasas/química , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal-regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.
Asunto(s)
Ciclofilina A/metabolismo , Músculo Liso Vascular/metabolismo , NADPH Oxidasas , Estrés Oxidativo , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Aminoquinolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Traumatismos de las Arterias Carótidas/metabolismo , División Celular/efectos de los fármacos , Ciclofilina A/análisis , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Nitroprusiato , Compuestos Onio/farmacología , Isomerasa de Peptidilprolil , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Enfermedades Vasculares/etiologíaRESUMEN
The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.
Asunto(s)
Actinas/metabolismo , NADPH Oxidasas/química , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Fosfoproteínas/metabolismo , Sitios de Unión , Humanos , NADPH Oxidasas/genética , Neutrófilos/metabolismo , Fosfoproteínas/genética , Unión Proteica , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia/genética , Resonancia por Plasmón de Superficie , Termodinámica , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Reactive oxygen species (ROS) generated in some non-phagocytic cells are implicated in mitogenic signalling and cancer. Many cancer cells show increased production of ROS, and normal cells exposed to hydrogen peroxide or superoxide show increased proliferation and express growth-related genes. ROS are generated in response to growth factors, and may affect cell growth, for example in vascular smooth-muscle cells. Increased ROS in Ras-transformed fibroblasts correlates with increased mitogenic rate. Here we describe the cloning of mox1, which encodes a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase of phagocytes, gp91phox. mox1 messenger RNA is expressed in colon, prostate, uterus and vascular smooth muscle, but not in peripheral blood leukocytes. In smooth-muscle cells, platelet-derived growth factor induces mox1 mRNA production, while antisense mox1 mRNA decreases superoxide generation and serum-stimulated growth. Overexpression of mox1 in NIH3T3 cells increases superoxide generation and cell growth. Cells expressing mox1 have a transformed appearance, show anchorage-independent growth and produce tumours in athymic mice. These data link ROS production by Mox1 to growth control in non-phagocytic cells.
Asunto(s)
Transformación Celular Neoplásica , NADH NADPH Oxidorreductasas/fisiología , Superóxidos/metabolismo , Células 3T3 , Aconitato Hidratasa/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Línea Celular , Clonación Molecular , Colon/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 2 , NADPH Oxidasas/química , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , TransfecciónRESUMEN
An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O2(-.)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). To determine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus <5%) compared with the enzyme reconstituted with native FAD. Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O2(-.) generation also decreased the steady state reduction of flavin. Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD. This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin. Kinetic deuterium isotope effects along with K(m) values permitted calculation of the K(d) for NADPH. (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on V and V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K(d) for NADPH was 40 microM in the presence of wild type p67(phox) and was approximately 55 microM using the weakly activating p67(phox)(V205A). Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
Asunto(s)
Grupo Citocromo b/metabolismo , Flavinas/metabolismo , NADP/metabolismo , Fosfoproteínas/metabolismo , Deuterio , Electrones , Reactivadores Enzimáticos/farmacología , Cinética , NADPH Oxidasas/metabolismo , Piridinas/farmacología , Superóxidos/metabolismoRESUMEN
Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate-stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 microM in vitro. Intriguingly, phosphatidylinositol 4,5-bisphosphate (PIP2) synergistically stimulates the oleate-dependent PLD2 activity with an optimal concentration of 2.5 microM. These results provide the first evidence that oleate is a PLD2-specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP2.
Asunto(s)
Ácido Oléico/farmacología , Fosfolipasa D/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Células U937RESUMEN
Phospholipase D1 (PLD1) is known to be activated by ADP-ribosylation factor 1 (ARF1). We report here that ARF1 co-immunoprecipitates with PLD1 and that the ARF1-dependent PLD activation is induced by the direct interaction between ARF1 and PLD1. We found that RalA, another member of the small GTP-binding proteins, synergistically enhances the ARF1-dependent PLD activity with an EC50 of about 30 nM. Using in vitro binding assay, we show that ARF1 and RalA directly interact with different sites of PLD1. The results suggest that the independent interactions of RalA and ARF1 with PLD1 are responsible for the synergistic activation.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfolipasa D/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Animales , Bovinos , Activación Enzimática , Fosfatidilinositol 4,5-Difosfato/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión , Proteínas de Unión al GTP ralRESUMEN
Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b558 and the cytosolic proteins p47(phox), p67(phox), and Rac. p47(phox) functions as an adaptor protein; it increases the affinity of p67(phox) and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582), implying that p67(phox) and/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67(phox) that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67(phox) showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199-210. p67(phox) mutated at single amino acid residues within this region showed diminished activity, and p67(phox) V204A was completely inactive. The effects of mutations on activity were independent of p47(phox), and mutations did not affect the binding of p67(phox) to Rac. In the presence of wild-type p67(phox), the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67(phox), but not by p47(phox) or Rac. The V204A mutant competed with native p67(phox) for translocation to neutrophil plasma membrane, indicating that p67(phox) V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b558 by an activation domain in p67(phox).
Asunto(s)
NADPH Oxidasas/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sistema Libre de Células , Grupo Citocromo b/metabolismo , Cartilla de ADN , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Humanos , Datos de Secuencia Molecular , Neutrófilos/enzimología , Neutrófilos/metabolismo , Fosfoproteínas/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP racRESUMEN
Phospholipase D (PLD) has been implicated in a variety of cellular processes including vesicular transport, the respiratory burst, and mitogenesis. PLD1, first cloned from human, is activated by small GTPases such as ADP-ribosylation factor (ARF) and RhoA. Rodent PLD2, which is approximately 50% identical to PLD1 has recently been cloned from mouse embryo (Colley, W., Sung, T., Roll, R., Jenco, J., Hammond, S., Altshuller, Y., Bar-Sagi, D., Morris, A., and Frohman, M. (1997) Curr. Biol. 7, 191-201) and rat brain (Kodaki, T., and Yamashita, S. (1997) J. Biol. Chem. 272, 11408-11413). We describe herein the cloning from a B cell library and expression of human PLD2 (hPLD2). The open reading frame is predicted to encode a 933-amino acid protein (Mr of 105,995); this corresponds to the size of the protein expressed in insect cells using recombinant baculovirus. The deduced amino acid sequence shows 53 and 90% identity to hPLD1 and rodent PLD2, respectively. The mRNA for PLD2 was widely distributed in various tissues including peripheral blood leukocytes, and the distribution was distinctly different from that of hPLD1. hPLD1 and hPLD2 both showed a requirement for phosphatidylinositol 4,5-bisphosphate. Both isoforms showed optimal activity at 10-20 mol % phosphatidylcholine in a mixed lipid vesicle system and showed comparable basal activities in the presence of phosphatidylinositol 4,5-bisphosphate. Unexpectedly, ARF-1 stimulated the activity of hPLD2 expressed in insect cells about 2-fold, compared with a 20-fold stimulation of hPLD1 activity. Thus, not only PLD1 but also hPLD2 activity can be positively regulated by both phosphatidylinositol 4,5-bisphosphate and ARF.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfolipasa D/genética , Factores de Ribosilacion-ADP , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Evolución Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Disabled-2 (Dab2), a mammalian structural homolog of Drosophila Disabled (Dab), is a mitogen-responsive phosphoprotein. It has been speculated to be a negative regulator of growth since its expression is lost in ovarian carcinomas. Dab2 contains a C-terminal proline-rich domain with sequences similar to those found in Sos, a guanine nucleotide exchange factor for Ras. The proline-rich sequences of Sos mediate the interaction of Sos with Grb2, an adaptor protein which coupled tyrosine kinase receptors to Sos. Herein, we have investigated the possibility that Dab2 interacts with Grb2. In experiments of co-immunoprecipitation from BAC1.2F5 macrophage cell lysates, significant quantities of Grb2 were associated with both Sos and Dab2, although Dab2 and Sos were not present in the same complex. Transfection of Dab2 into a Dab2-negative cell line (293 cells) decreased the amount of Grb2 associated with Sos, suggesting that Dab2 competes with Sos for binding to Grb2. Proline-rich peptides corresponding to Dab2 (#661-669) and to Sos (#1146-1161) inhibited the binding of Dab2 to Grb2, but were less effective in disrupting the Grb2-Sos complex. The expressed proline-rich domain of Dab2 (#600-730) bound Grb2, but other regions of Dab2 failed to bind Grb2. Both of the individual SH3 domains of Grb2 bound to Sos (N-terminal SH3 domain >> C-terminal SH3 domain), but binding to Dab2 required the intact Grb2, suggesting cooperative binding using both SH3 domains of Grb2. These data indicate that Dab2 binds to the SH3 domains of Grb2 via its C-terminal proline-rich sequences. Dab2 may modulate growth factor/Ras pathways by competing with Sos for binding to Grb2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Unión Competitiva , Línea Celular , Sistema Libre de Células , Proteína Adaptadora GRB2 , Genes Supresores de Tumor , Factores de Intercambio de Guanina Nucleótido , Macrófagos , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Fosfopéptidos/metabolismo , Fosfoproteínas/aislamiento & purificación , Pruebas de Precipitina , Dominios Proteicos Ricos en Prolina , Unión Proteica , Proteínas/aislamiento & purificación , Proteínas Supresoras de Tumor , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fosfoproteínas/metabolismo , Animales , Unión Competitiva , Bovinos , Cristalografía por Rayos X , Colorantes Fluorescentes/metabolismo , Proteínas de Unión al GTP/genética , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Modelos Moleculares , NADPH Oxidasas/metabolismo , Mapeo Peptídico , Fosfoproteínas/genética , Mutación Puntual , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia , ortoaminobenzoatos/metabolismo , Proteínas de Unión al GTP racRESUMEN
Previously, we identified peptides that stimulate phosphoinositide hydrolysis in several leukocyte cell lines from mixtures of random hexapeptide sequences. Moreover, the peptides activate phospholipase C via a pertussis toxin-sensitive G protein-coupled receptor. We now investigate the structure-activity relationship of the peptides with the goal of improving the activity of the peptides, as well as the biologic function of the peptides. Substitution of the L-methionine at the C terminus of peptides with D-methionine markedly increased the effectiveness of the peptides. The half-maximal effective concentrations of MKYMPm-NH2 and WKYMVm-NH2 for stimulation of phosphoinositide hydrolysis in U266 cells were 30 and 0.5 nM, respectively. By BIAcore analysis we confirmed the existence of a receptor for WKYMVm-NH2. Furthermore, the intracellular calcium concentration increase induced by WKYMVm-NH2 was not inhibited by several chemoattractants (FMLP, IL-8, platelet-activating factor, C5a, granulocyte-macrophage CSF, and granulocyte CSF) suggests that WKYMVm-NH2 has a unique cell surface receptor on leukocytes. WKYMVm-NH2 stimulated the phosphoinositide hydrolysis in U937, HL60, and U266 cells, as well as in human neutrophils. Moreover, WKYMVm-NH2 is more effective than FMLP in the production of superoxide in human neutrophils. The data suggest that WKYMVm-NH2 may have the ability to activate the microbicidal functions of human neutrophils.
Asunto(s)
Neutrófilos/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Fosfatidilinositoles/metabolismo , Receptores de Péptidos/fisiología , Superóxidos/metabolismo , Aminoácidos/química , Técnicas Biosensibles , Calcio/metabolismo , Glicina/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Hidrólisis , Líquido Intracelular/metabolismo , Linfoma de Células B , Neutrófilos/efectos de los fármacos , Oligopéptidos/síntesis química , Receptores de Péptidos/metabolismo , Eliminación de Secuencia , Estereoisomerismo , Células Tumorales CultivadasRESUMEN
p37, the major protein of the extracellular enveloped form of vaccinia virus, is involved in the biogenesis of the viral double membrane and in egress of virus from the cell. p37 was expressed as a glutathione S-transferase fusion protein and was purified to homogeneity by silver staining using glutathione-agarose, Sephacryl S-200, and DEAE-cellulose chromatography. Incubation of p37 with phosphatidylcholine labeled in the fatty acyl side chains resulted in the production of multiple lipid products that were identified by thin layer chromatography and mass spectrometry as diacylglycerol, free fatty acid, monoacylglycerol, and lysophosphatidylcholine. Lipid-metabolizing activities colocalized with p37-containing fractions throughout the chromatographic steps. p37 also metabolized phosphatidylethanolamine efficiently, but it had less activity toward phosphatidylinositol and little or no activity toward phosphatidylserine. The purified enzyme also metabolized triacylglycerol to diacylglycerol but was inactive toward sn-1, 2-diacylglycerol. p37 was also expressed in insect cells as a poly-His fusion protein; cell lysates and partially purified proteins also generated products expected from phospholipase C and A activities. Thus, p37 is a broad specificity lipase with phospholipase C, phospholipase A, and triacylglycerol lipase activities.