RESUMEN
Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and ß-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.
Asunto(s)
Toxinas Bacterianas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Neutrófilos/patología , Fagocitosis/fisiología , Anticuerpos Antiidiotipos/inmunología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/uso terapéutico , Muerte Celular/fisiología , Células Cultivadas , Humanos , Neutrófilos/fisiologíaRESUMEN
Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the alpha-subunit of Mac-1, a leukocyte beta2 integrin required for innate immunity to invading microbes. The GAS Mac-1-like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcgammaRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.
Asunto(s)
Proteínas Bacterianas , Integrinas/metabolismo , Antígeno de Macrófago-1/farmacología , Proteínas Opsoninas , Fagocitosis/efectos de los fármacos , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/patogenicidad , Enfermedad Aguda , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/sangre , Sitios de Unión , Convalecencia , Integrinas/genética , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/metabolismo , Modelos Inmunológicos , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Faringitis/inmunología , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/metabolismo , Fiebre Reumática/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Myeloperoxidase (MPO) deficiency is a common inherited disorder linked to increased susceptibility to infection and malignancy. We identified a novel missense mutation in the MPO gene at codon 173 whereby tyrosine is replaced with cysteine (Y173C) that is associated with MPO deficiency and assessed its impact on MPO processing and targeting in transfectants expressing normal or mutant proteins. Although the precursor synthesized by cells expressing the Y173C mutation (MPOY173C) was glycosylated, associated with the molecular chaperones calreticulin and calnexin, and acquired heme, it was neither proteolytically processed to mature MPO subunits nor secreted. After prolonged association with calreticulin and calnexin in the endoplasmic reticulum, MPOY173C was degraded. Furthermore, the 20S proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl inhibited its degradation, suggesting that the proteasome mediates proteolysis of MPOY173C and, thus, participates in quality control in this novel form of hereditary MPO deficiency.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Ligamiento Genético , Complejos Multienzimáticos/metabolismo , Mutación , Peroxidasa/deficiencia , Peroxidasa/genética , Secuencia de Aminoácidos , Transporte Biológico/genética , Humanos , Datos de Secuencia Molecular , Complejo de la Endopetidasa ProteasomalRESUMEN
Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.
Asunto(s)
Lipopolisacáridos/farmacología , NADPH Oxidasas/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Membrana Celular/enzimología , Grupo Citocromo b/metabolismo , Citosol/enzimología , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/química , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Estallido Respiratorio/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Phagocytes such as neutrophils play a key role in the body's innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi-protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane-bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process.
Asunto(s)
NADPH Oxidasas/química , Fagocitos/enzimología , Proteínas Bacterianas/química , Grupo Citocromo b/química , Activación Enzimática , Proteínas Activadoras de GTPasa , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/química , Modelos Biológicos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas/química , Fosfoproteínas/fisiología , Unión Proteica , Proteínas/química , Dominios Homologos srcRESUMEN
The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.
Asunto(s)
Electroporación , Neutrófilos/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/inmunología , Grupo Citocromo b/farmacología , Difusión , Citometría de Flujo , Fluoresceína , Fluoresceínas , Humanos , Datos de Secuencia Molecular , NADPH Oxidasas/antagonistas & inhibidoresRESUMEN
Phagocytic leukocytes such as neutrophils and macrophages are essential for the innate immune response against invading bacteria. Binding and ingestion of bacteria by these host cells triggers potent anti-microbial activity, including production of reactive oxygen species. Although phagocytes are highly adept at destroying bacteria, modulation of leukocyte apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Whereas induction of macrophage apoptosis by pathogens may adversely affect the host immune response to infection, acceleration of neutrophil apoptosis following phagocytic interaction with bacteria appears essential for the resolution of infection. This idea is supported by the finding that some bacterial pathogens alter normal phagocytosis-induced neutrophil apoptosis to survive and cause disease. This review summarizes what is currently known about modulation of phagocyte apoptosis by bacteria and describes a paradigm whereby bacteria-induced neutrophil apoptosis plays a role in the resolution of infection.
Asunto(s)
Apoptosis/fisiología , Bacterias/crecimiento & desarrollo , Fagocitos/fisiología , Animales , Humanos , Infecciones/fisiopatología , Macrófagos/microbiología , Macrófagos/fisiología , Modelos Biológicos , Neutrófilos/microbiología , Neutrófilos/fisiología , Fagocitos/microbiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Human PMNs (polymorphonuclear leucocytes or neutrophils) are essential to the innate immune response against bacterial pathogens and are a key part of the acute inflammatory response. Although progress has been made, the molecular basis for termination of inflammation during bacterial infection in humans is largely undefined. To that end, we used genomics strategies to gain new insight into processes that facilitate resolution of neutrophil-mediated inflammation and bacterial infection. On the basis of a series of recent studies, we propose that global changes in PMN gene expression after phagocytosis comprise an apoptosis differentiation programme, which represents the final stage of transcription-regulated PMN maturation. Our studies indicate that the apoptosis differentiation programme regulates multiple post-phagocytic processes in human neutrophils, such as cell fate and proinflammatory activity, and is modulated by PMN-derived reactive oxygen species. Collectively, these studies establish a global model of host cell-pathogen interaction, which provides fundamental insight into the resolution of infection in humans.
Asunto(s)
Apoptosis , Leucocitos Mononucleares/patología , Diferenciación Celular , Regulación hacia Abajo , Humanos , Infecciones , Inflamación , Leucocitos Mononucleares/microbiología , Neutrófilos/patología , Fagocitosis , Especies Reactivas de Oxígeno , Transcripción GenéticaRESUMEN
Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.
Asunto(s)
NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Neutrófilos/inmunología , Fagocitosis/inmunología , Activación Enzimática/inmunología , Humanos , NADPH Oxidasas/antagonistas & inhibidores , Neisseria meningitidis/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Proteínas Opsoninas/inmunología , Proteínas Opsoninas/metabolismo , Fagosomas/enzimología , Fagosomas/inmunología , Fagosomas/metabolismo , Fagosomas/microbiología , Fosfoproteínas/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Zimosan/inmunología , Zimosan/metabolismoRESUMEN
Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (approximately 5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.
Asunto(s)
Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Neutrófilos/enzimología , Fagosomas/enzimología , Fosfoproteínas/sangre , Cromosoma X , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Enfermedad Granulomatosa Crónica/sangre , Humanos , Inmunohistoquímica , Cinética , Microscopía Fluorescente , NADPH Deshidrogenasa/sangre , NADPH Oxidasas , Neutrófilos/patología , Neutrófilos/fisiologíaRESUMEN
The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47phox, p67phox, and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47phox that mediates its interaction with flavocytochrome b. From a random peptide phage display library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47phox binding residues. Using this approach, we identified a region of p47phox from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides 315SRKRLSQDAYRRNS328, 323AYRRNSVRFL332, and 334QRRRQARPGPQSPG347 inhibited superoxide (O2-.) production in the broken cell system with IC50 of 18, 57, and 15 microM, respectively. 323AYRRNSVRFL332 and its derivative peptides inhibited phosphorylation of p47phox. However, the functional importance of this peptide was independent of its effects on phosphorylation, since 323AYRRNAVRFL332 inhibited O2-. production, but had no effect on phosphorylation. None of the peptides blocked O2-. production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47phox in the broken cell translocation assay and O2-. production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47phox interacts with flavocytochrome b.
Asunto(s)
Grupo Citocromo b/sangre , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Secuencia de Aminoácidos , Bacteriófagos , Sitios de Unión , Membrana Celular/metabolismo , Secuencia de Consenso , Grupo Citocromo b/química , Citosol/metabolismo , Bases de Datos Factuales , Electroporación , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , NADPH Deshidrogenasa/química , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Fosfoproteínas/químicaRESUMEN
The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22(phox) was found in subcellular compartments containing the mature 91-kDa form of gp91(phox) and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91(phox) and p22(phox) but had little or no impact on p65. These studies demonstrate (a) gp91(phox) is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.
Asunto(s)
Grupo Citocromo b/biosíntesis , Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Transporte de Membrana , Precursores de Proteínas/metabolismo , Diferenciación Celular , Hemo/metabolismo , Humanos , Peso Molecular , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fagocitos/enzimología , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Células Tumorales CultivadasRESUMEN
The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.
Asunto(s)
Grupo Citocromo b/metabolismo , NADPH Oxidasas , Procesamiento Proteico-Postraduccional , Grupo Citocromo b/química , Dimerización , Hemo/química , Staphylococcus aureusRESUMEN
During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.
Asunto(s)
Grupo Citocromo b/metabolismo , Proteínas de Transporte de Membrana , NADH NADPH Oxidorreductasas/biosíntesis , NADPH Deshidrogenasa/metabolismo , Neutrófilos/enzimología , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Sitios de Unión/genética , Biblioteca de Genes , Humanos , Glicoproteínas de Membrana/metabolismo , NADH NADPH Oxidorreductasas/efectos de los fármacos , NADPH Deshidrogenasa/genética , NADPH Oxidasa 2 , NADPH Oxidasas , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Fosfoproteínas/genética , Unión ProteicaRESUMEN
The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.
Asunto(s)
Grupo Citocromo b/química , Hemo/metabolismo , Histidina/metabolismo , Glicoproteínas de Membrana/química , NADPH Oxidasas , Secuencia de Aminoácidos , Animales , Células COS , Grupo Citocromo b/biosíntesis , Dimerización , Humanos , Datos de Secuencia Molecular , NADPH Oxidasa 2 , Relación Estructura-ActividadRESUMEN
Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that (226)RIVRG(230) and (160)IKNP(163) regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues (226)RIVRG(230) and (160)IKNP(163) are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly(165)-Ile(190) and Ser(200)-Glu(225) are adjacent to each other in the membrane.
Asunto(s)
Bacteriófagos/genética , Grupo Citocromo b/química , Mapeo Epitopo , NADPH Oxidasas , Neutrófilos/enzimología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Grupo Citocromo b/genética , Grupo Citocromo b/inmunología , Dimerización , Ensayo de Inmunoadsorción Enzimática , Humanos , Pruebas de PrecipitinaRESUMEN
Superoxide (O2-) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane. The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics. Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells. To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells. Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium. The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b. Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase. In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.
Asunto(s)
Grupo Citocromo b/metabolismo , FMN Reductasa , Hierro/metabolismo , Glicoproteínas de Membrana/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Linfocitos B/metabolismo , Transporte Biológico Activo , Transformación Celular Viral , Grupo Citocromo b/química , Grupo Citocromo b/deficiencia , Enfermedad Granulomatosa Crónica/metabolismo , Herpesvirus Humano 4 , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/química , NADH NADPH Oxidorreductasas/química , NADPH Oxidasa 2 , Neutrófilos/metabolismo , Superóxidos/metabolismoRESUMEN
During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.