Cytosolic NADPH Research Articles

The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A

Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.

Pre-B Cell Colony-Enhancing Factor (PBEF/Nampt/Visfatin) Primes Neutrophils for Augmented Respiratory Burst Activity through Partial Assembly of the NADPH Oxidase

Pre-B cell colony-enhancing factor ([PBEF] also known as Nampt/visfatin) is a pleiotropic 52-kDa cytokine-like molecule whose activity has been implicated in multiple inflammatory disease states. PBEF promotes polymorphonuclear neutrophil (PMN) proinflammatory function by inhibiting constitutive PMN apoptosis. We investigated whether PBEF activates or primes for PMN respiratory burst. We found that although PBEF did not activate respiratory burst on its own, it primed for increased reactive oxygen species generation through the NADPH oxidase. PBEF promoted membrane translocation of cytosolic NADPH oxidase subunits p40 and p47, but not p67, induced p40 phosphorylation on Thr(154), and activated the small GTPase Rac. Priming, translocation, and phosphorylation were dependent on activation of p38 and ERK MAPKs, but not of PI3K. Priming by PBEF occurred independent of its NAD-generating capacity because neither nicotinamide mononucleotide or NAD could recapitulate the effects, and a specific inhibitor of PBEF, APO-866, could not inhibit priming. Taken together, these results demonstrate that PBEF can prime for PMN respiratory burst activity by promoting p40 and p47 translocation to the membrane, and this occurs in a MAPK-dependent fashion.

Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells.

Abstract 4072: Insulin and NADPH-generating enzymes as molecular targets to repress colon cancer cell proliferation

Abstract An increasing body of evidence implicates obesity and the attendant hyperinsulinemia as major risk factors for human colorectal cancers. The lipogenic pathway supports growth and survival of colon cancer cells. In particular, inhibition of Fatty Acid Synthase (FASN) or Acetyl-CoA Carboxylase α (ACCα) induces apoptosis of colon cancer cells. The relative involvement in colon cancer initiation, progression and pathogenesis of cytosolic NADPH that is used in lipogenesis remains less clear. In the present study, we have determined the effects of insulin and the involvement of the major NADPH-generating cytosolic enzymes on proliferation of human colon cancer cell lines. Insulin (1-10 nM; to mimic normo- to hyper-insulinemic states) induced a significant increase (7-15%, p < 0.05) in proliferation of HT-29 and HCT-116 colon cancer cell lines in vitro (assayed by MTT assay). Addition of insulin augmented (by 5-10%) proliferation of both cell lines upon concurrent treatment with cancer chemotherapeutic agents (5-fluorouracil, oxaliplatin, irinotecan; each added at a dose < EC50). The insulin-mediated increase in HT-29 cell growth was associated with a 2-3 fold elevation in FASN gene expression and a 50% decrease in expression of the FASN-targeting micro RNAs, miR-103 and miR-107. However, insulin did not induce mRNA abundance for the cytoplasmic NADPH-generating enzymes [Malic Enzyme 1 (ME1), Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6PGD), or Isocitrate Dehydrogenase-1 (IDH1]. siRNA-mediated knockdown of FASN or of the individual major cytosolic NADPH-generating enzymes caused decreases in proliferation of HT-29 and HCT-116 cells (15-30%, p < 0.05) compared to the control scrambled siRNA. Data highlight pro-proliferative roles for insulin, FASN and cytosolic NADPH-generating enzymes in colorectal cancer cell growth. Implementation of pharmacologic and/or nutritional means to reduce serum insulin levels, enhance FASN miR expression and inhibit tumor cell NADPH-generation may serve as useful adjuncts for future cancer treatment and prevention. Supported by NIH grant RO1 CA136493 and the Arkansas Biosciences Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2011-4072

Organoid Culture of Bovine Coronary Arteries Reverses the Response to Hypoxia from Relaxation to Contraction Associated with Alterations in Mechanisms Controlling Protein Kinase G

We provided evidence that bovine coronary arteries (BCA) relax to hypoxia (PO2 ~10 torr) by oxidizing cytosolic NADPH and that hypoxia contracts pulmonary arteries by removing a peroxide/protein kinase G (PKG)-mediated relaxation. Hypoxia-elicited relaxation of 20mM KCl contracted BCA caused increased thiol oxidation-dimerization of PKG and increased PKG activity (VASP phosphorylation). BCA were organoid cultured for 48 hours in the absence (48h Control) and presence of 10μM ODQ to deplete soluble guanylate cyclase (by ~75%, chronic ODQ). Contraction to hypoxia was observed in 48h organoid cultured BCA, associated with decreased dimerization of PKG. Chronic ODQ treatment enhanced KCl contraction (vs. 48h Control BCA) and attenuated contraction to hypoxia and relaxation to an NO donor. While acute treatments with 10μM ODQ and 0.1mM ebselen (a peroxide & peroxynitrite scavenger) did not alter BCA relaxation to hypoxia, these agents attenuated the contraction to hypoxia in organoid cultured BCA. The data suggest that organoid culture changes the mechanisms regulating PKG in a manner which seems to participate in converting the response to hypoxia from relaxation to contraction. Supported HL31069, HL43023 and HL66331

A Novel Motif in the Crohn's Disease Susceptibility Protein, NOD2, Allows TRAF4 to Down-regulate Innate Immune Responses

The Crohn's disease and early onset sarcoidosis susceptibility protein, NOD2, coordinates innate immune signaling pathways. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis. In this work, we identify the atypical tumor necrosis factor-associated factor (TRAF) family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-κB activation and directly binds to NOD2 to inhibit NOD2-induced bacterial killing. We find that two consecutive glutamate residues in NOD2 are required for interaction with TRAF4 and inhibition of NOD2 signaling because mutation of these residues abrogated both TRAF4 binding and inhibition of NOD2. This work identifies a novel negative regulator of NOD2 signaling. Additionally, it defines a TRAF4 binding motif within NOD2 involved in termination of innate immune signaling responses.

Redox regulation of guanylate cyclase and protein kinase G in vascular responses to hypoxia.

The production of cGMP by the soluble form of guanylate cyclase (sGC) in bovine pulmonary arteries (BPA) is controlled by cytosolic NADPH maintaining reduced thiol and heme sites on sGC needed for activation by NO, and the levels of Nox oxidase-derived superoxide and peroxide that influence pathways regulating sGC activity. Our recent studies in BPA suggest that the activities of peroxide metabolizing pathways in vascular smooth muscle potentially determine the balance between sGC stimulation by peroxide and a cGMP-independent activation of cGMP-dependent protein kinase (PKG) by a disulfide-mediated subunit dimerization. Cytosolic NADPH oxidation also appears to function in BPA through its influence on protein thiol redox control as an additional mechanism promoting vascular relaxation through PKG activation. These processes regulating PKG may participate in decreases in peroxide and increases in NADPH associated with contraction of BPA to hypoxia and in cytosolic NADPH oxidation potentially mediating bovine coronary artery relaxation to hypoxia.

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol.

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD + dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate.

Redox regulation of responses to hypoxia and NO‐cGMP signaling in pulmonary vascular pathophysiology

Pulmonary vascular responses elicited by hypoxia and NO-cGMP signaling are potentially influenced by ROS and redox mechanisms that change during the progression of disease processes. Our studies in endothelium-rubbed bovine pulmonary arteries suggest increased glucose-6-phosphate dehydrogenase levels (compared to coronary arteries) seem to maintain a tonic peroxide-mediated relaxation removed by hypoxia through NADPH fueling superoxide generation from Nox oxidase. The activities of glucose-6-phosphate dehydrogenase, oxidases (i.e., Nox4), and systems metabolizing superoxide and peroxide markedly influence hypoxic pulmonary vasoconstriction (HPV). Activation of soluble guanylate cyclase and cGMP protein kinase seems to participate in peroxide-elicited relaxation. Endogenous NO helps maintain low pulmonary arterial pressure and suppresses HPV. Multiple redox processes potentially occurring during the progression of pulmonary hypertension may also attenuate NO-mediated relaxation beyond its scavenging by superoxide, including oxidation of guanylate cyclase heme and thiols normally maintained by cytosolic NADPH redox control.

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Carbonyl compounds methylglyoxal and glyoxal affect interleukin‐8 secretion in intestinal cells by superoxide anion generation and activation of MAPK p38

The carbonyl compounds methylglyoxal (MG) and glyoxal (GL) are reactive intermediates of glucose degradation pathways and capable of inducing cellular damage. Although immune-stimulating activity has been investigated in endothelial cells, little is known about the signaling pathways of cytokine induction of these compounds in the intestine. Hence, we investigated the impact of mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) on IL-8 production by human intestinal cells (Caco-2 and HT-29) after stimulation by MG and GL. Both compounds induced a dose-dependent enhancement of IL-8 secretion in human intestinal cells. MAPK p38 and extracellular signal-regulated kinase (ERK) were phosphorylated in these cells after having been stimulated by MG and GL. Furthermore, inhibitors of MAPK p38 (SB 203580 and 239063), ERK1/2 (PD 98059) and NF-κB activation (SM-7368 and SC-514) reduced IL-8 secretion. The most important mechanism by which MG and GL induced IL-8 secretion was the generation of superoxide anions which was confirmed by the inhibition of the cytosolic NADPH oxidase with diphenyl iodonium (DPI) or by application of superoxide dismutase (SOD). Our data suggest that multiple pathways were simultaneously activated; however, superoxide dependent MAPK p38 activation seems to be the most dominant pathway for IL-8 secretion in intestinal cells.

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

Potential Role of NADPH Redox in Regulating Thiol Oxidation‐Elicited Subunit Dimerization Activation of Protein Kinase G in the Relaxation of Bovine Pulmonary Arteries to Pentose Phosphate Pathway Inhibitors

We have previously provided evidence that inhibition of glucose‐6‐phosphate dehydrogenase (G6PD) in the pentose phosphate pathway promotes oxidation of NADPH, and elicits vascular relaxation through coordinating multiple processes that lower intracellular calcium. Since it was reported that H2O2 induces coronary relaxation associated with a NO/cGMP‐independent thiol oxidation/subunit dimerization activation of protein kinase G (PKG), we investigated if inhibition of G6PD activates PKG through disulfide‐mediated subunit dimerization under conditions where relaxation of isolated endothelium‐removed bovine pulmonary arteries (BPA) is observed. BPA precontracted with 20mM KCl under hypoxic conditions were exposed to either 1mM 6‐aminonicotinamide (6‐AN) or 0.5mM epiandrosterone (Epi) to inhibit G6PD. It was observed that both 6‐AN and Epi increased PKG dimerization under conditions where relaxation was observed. These responses were associated with increased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at the serine‐239 site known to be mediated by PKG. Thus, inhibition of G6PD promotes thiol oxidation‐elicited dimerization activation of PKG, suggesting that PKG activation is likely to be a participant in mechanisms through which cytosolic NADPH oxidation promotes vascular relaxation.Supported by NIH grants HL31069, HL43023 and HL66331

Involvement of mitochondria in the control of plant cell NAD(P)H reduction levels

NADPH and NADH mediate reductant flow between cellular processes, linking central carbon and energy metabolism with intermediary metabolism, stress defence and development. Recent investigations have revealed paths of functional interactions, and have suggested that mitochondrial NADPH oxidation, especially together with the oxidative pentose phosphate pathway, is an important regulator of the cytosolic NADPH reduction level. Furthermore, stress-dependent metabolic pathways substantially affect the NADPH reduction level in particular physiological situations. The mitochondrial impact on the NADPH reduction level provides a model example of the physiological significance of the mitochondrial NAD(P)H dehydrogenase set-up, which is more complex in plants than in other organisms.

Oxidant-Redox Regulation of Pulmonary Vascular Responses to Hypoxia and Nitric Oxide-cGMP Signaling

Most current theories for the mechanism of hypoxic pulmonary vasoconstriction (HPV) include a role for reactive oxygen species and/or changes in redox regulation, but extreme controversy exists regarding which systems and redox changes mediate the HPV response. Nitric oxide (NO) appears to help to maintain low pulmonary arterial pressure, suppress HPV, and prevent the development of pulmonary hypertension. Our studies have found a key role for glucose-6-phosphate dehydrogenase in bovine pulmonary arterial smooth muscle functioning to maintain elevated levels of cytosolic NADPH which fuels the generation of vasodilator levels of hydrogen peroxide. HPV results from hypoxia removing vasodilation by peroxide. Decreased superoxide generation by Nox4 oxidase and its conversion to peroxide by Cu,Zn-SOD appear to be potential factors in sensing hypoxia, and decreased cGMP-associated vasodilation and removal of redox controlled vasodilator mechanisms by increased cytosolic NADPH may be key coordinators of the HPV response. Oxidant generation associated with vascular disease processes, including the removal of NO by superoxide, and attenuation of its ability to stimulate cGMP production by oxidation of the heme and thiols of soluble guanylate cyclase attenuate potential beneficial actions of NO on pulmonary arterial function. While pulmonary hypertension appears to have multiple poorly understood effects on redox-associated processes, potentially influencing responses to hypoxia and NO-cGMP signaling, much remains to be elucidated regarding how these processes may be important factors in the progression, expression and therapeutic treatment of pulmonary hypertension.

Simultaneous Measurement of Phagosome and Plasma Membrane Potentials in Human Neutrophils By Di-8-Anepps and SEER

Neutrophils are the first line of defense against invading bacteria. Neutrophils engulf the invaders into an internal vacuole, the phagosome. Accompanying phagocytosis is the respiratory burst, in which NADPH oxidase produces reactive oxygen species by transporting electrons from cytosolic NADPH across the membrane to either intra-phagosomal or extracellular oxygen. It is well known that the plasma membrane depolarizes during the respiratory burst but very little is known about the membrane potential of the phagosome. Here we monitor the membrane potential of phagosomes as well as the plasma membrane during the phagocytosis of opsonized zymosan.Neutrophils were isolated from whole blood and plated on glass coverslips. The cells were loaded with 5 μM di-8-ANEPPS for 30 minutes before excess dye was washed away. The cells were stimulated by addition of 2 mg/ml serum opsonized zymozan (OPZ) and were visualized using a Leica SP2 confocal microscope. SEER imaging was performed by simultaneously acquiring two images at 488 nm and 545 nm and collecting at emission ranges 470-560 nm and 570-700 nm respectively.The neutrophil plasma membrane depolarized rapidly coinciding with phagocytosis of the first OPZ particle. The potential generally decreased somewhat, but the plasma membrane potential generally remained positive to 0 mV for many minutes, during which time several phagocytotic events were typically observed. DPI produced repolarization, confirming that the depolarization was due to the electrogenic activity of NADPH oxidase. The membrane potential of each phagosome was highest upon formation and decreased within several minutes, often falling to negative voltages, while the plasma membrane remained depolarized. The phagosome membrane potentials were independent of, and at their peak often exceeded the plasma membrane potential. This study is the first to monitor the phagosome membrane potential in living cells.

Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.

A redox-mediated modulation of stem bolting in transgenic Nicotiana sylvestris differentially expressing the external mitochondrial NADPH dehydrogenase.

Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.

Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii

Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47 phox and p67 phox , to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens.