Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst.
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ABSTRACT:
SUBMITTER: Ramsey IS
PROVIDER: S-EPMC2669790 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.
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Project description:Key pointsLRRC8A-containing anion channels associate with NADPH oxidase 1 (Nox1) and regulate superoxide production and tumour necrosis factor-α (TNFα) signalling. Here we show that LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. LRRC8C knockdown inhibited TNFα-induced O2 •- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited LRRC8C currents but potently (∼80%) inhibited LRRC8D currents. Substitution of the extracellular loop (EL1, EL2) domains of 8D into 8C conferred significantly stronger (69%) ChlorT-dependent inhibition. ChlorT exposure impaired subsequent current block by DCPIB, which occurs through interaction with EL1, further implicating external oxidation sites. LRRC8A/C channels most effectively sustain Nox1 activity at the plasma membrane. This may result from their ability to remain active in an oxidized microenvironment.AbstractTumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2 •- ) required for subsequent signalling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. VRAC currents are modulated by oxidants, suggesting that channel oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2 •- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and colocalized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric LRRC8C and LRRC8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited 8C, but potently inhibited 8D currents. ChlorT exposure also impaired subsequent current block by the VRAC blocker DCPIB, implicating external sites of oxidation. Substitution of the 8D extracellular loop domains (EL1, EL2) into 8C conferred significantly stronger ChlorT-mediated inhibition of 8C currents. Our results suggest that LRRC8A/C channel activity can be effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.
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Project description:ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1? generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91(phox)-deficient or p47(phox)-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1? levels compared with mice transplanted with wild-type bone marrow. IL-1? receptor blockade abrogated aggravated NCGN in gp91(phox)-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1? generation in gp91(phox)-deficient and p47(phox)-deficient monocytes compared with wild-type monocytes. This enhanced IL-1? generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1? generation were inversely related in human monocytes. Furthermore, transplantation of gp91(phox)/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91(phox) bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants.
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Project description:SignificanceVoltage-gated proton channels are increasingly implicated in cellular proton homeostasis. Proton currents were originally identified in snail neurons less than 40 years ago, and subsequently shown to play an important auxiliary role in the functioning of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Molecular identification of voltage-gated proton channels was achieved less than 10 years ago. Interestingly, so far, only one gene coding for voltage-gated proton channels has been identified, namely hydrogen voltage-gated channel 1 (HVCN1), which codes for the HV1 proton channel protein. Over the last years, the first picture of putative physiological functions of HV1 has been emerging.Recent advancesThe best-studied role remains charge and pH compensation during the respiratory burst of the phagocyte NADPH oxidase (NOX). Strong evidence for a role of HV1 is also emerging in sperm biology, but the relationship with the sperm NOX5 remains unclear. Probably in many instances, HV1 functions independently of NOX: for example in snail neurons, basophils, osteoclasts, and cancer cells.Critical issuesGenerally, ion channels are good drug targets; however, this feature has so far not been exploited for HV1, and hitherto no inhibitors compatible with clinical use exist. However, there are emerging indications for HV1 inhibitors, ranging from diseases with a strong activation of the phagocyte NOX (e.g., stroke) to infertility, osteoporosis, and cancer.Future directionsClinically useful HV1-active drugs should be developed and might become interesting drugs of the future.
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Project description:It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase ? abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase ? mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.
| S-EPMC3743482 | biostudies-literature
Project description:Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ?1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (K d) of ?1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.
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Project description:AimsHere, we develop a novel cancer treatment modality using mitochondria-targeting, high-fluence, low-power laser irradiation (HF-LPLI) in mouse tumor models and explore the mechanism of mitochondrial injury by HF-LPLI.ResultsWe demonstrated that the initial reaction after photon absorption was photosensitization of cytochrome c oxidase (COX), to inhibit enzymatic activity of COX in situ and cause respiratory chain superoxide anion (O2(-•)) burst. We also found that HF-LPLI exerted its main tumor killing effect through mitochondrial O2(-•) burst via electron transport chain (ETC). These phenomena were completely absent in the respiration-deficient cells and COX knockdown cells. With a carefully selected irradiation protocol, HF-LPLI could efficaciously destroy tumors. The inhibition of enzymatic activity of COX and generation of O2(-•) by HF-LPLI in vivo were also detected.InnovationIt is the first time that the mechanism involved in the interaction between light and its photoacceptor under HF-LPLI treatment is clarified. Our results clearly indicate that HF-LPLI initiates its effects via targeted COX photoinactivation and that the tumor-killing efficacy is dependent of the subsequent mitochondrial O2(-•) burst via ETC.ConclusionBased on both in vitro and in vivo results, we conclude that HF-LPLI can selectively photoinactivate respiratory chain oxidase to trigger a fatal mitochondrial O2(-•) burst, producing oxidative damage on cancer cells. This study opens up the possibilities of applications of HF-LPLI as a mitochondria-targeting cancer phototherapy.
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Project description:Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws of Ncf1(?/?) mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficient Ncf1(?/?) mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst.
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Project description:The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. We here show that insolubility of NADPH oxidase subunits in nonionic detergents TX-100, Brij-58, and Brij-98 is a consequence of inclusion into cholesterol-enriched membrane microdomains (lipid rafts). Thus, flavocytochrome b558, in a cholesterol-dependent manner, segregated to the bouyant low-density detergent-resistant membrane (DRM) fraction, and the cytosolic NADPH oxidase factors associated dynamically with low-density DRM. Further, superoxide production following cholesterol depletion was severely compromised in intact cells or in a cell-free reconstituted system, correlating with a reduced translocation of cytosolic phox subunits to the membrane. In analogy with the widely accepted role of lipid rafts as signaling platforms, our data indicate that cholesterol-enriched microdomains act to recruit and/or organize the cytosolic NADPH oxidase factors in the assembly of the active NADPH oxidase.
| S-EPMC380990 | biostudies-literature
Project description:Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.
| S-EPMC3253173 | biostudies-literature