Assembly of phagocyte NADPH oxidase: A concerted binding process?

Background

The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b₅₅₈ and four cytosolic proteins (p67^phox, p47^phox, p40^phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.

Methods

A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47^phox–p67^phox complex.

Results

The data presented here are consistent with the absence of a catalytic role of the p47^phox–p67^phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b₅₅₈ allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b₅₅₈. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core.

Conclusion and general significance

The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.     

A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis

The assembly of cytosolic p47^phox and p67^phox with flavocytochrome b₅₅₈ at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47^phox and p67^phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67^phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67^phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47^phox PRR. This fragment was recruited in a flavocytochrome b₅₅₈-dependent, p67^phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67^phox SH3b domain and suggest that the p47^phox/p67^phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67^phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.     

Conformational changes in p47 upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis

The neutrophil NADPH oxidase is an enzymatic complex involved in innate immunity. Phosphorylation of p47^phox promotes its translocation with p67^phox and p40^phox, followed by membrane interaction and assembly with flavocytochrome b₅₅₈ into a functional complex. To characterise p47^phox conformational changes during activation, we used wild-type and the S303/304/328E triple mutant mimicking the phosphorylated state. Hydrogen/deuterium exchange and limited proteolysis coupled to mass spectrometry were used to discriminate between the various structural models. An increase in deuteration confirmed that p47^phox adopts an open and more flexible conformation after activation. Limited proteolysis correlated this change with increased auto-inhibitory region (AIR) accessibility. These results establish a structural link between the AIR release and the exposure of the Phox homology (PX) domain.     

Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O₂⁻), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b₅₅₈ (cyt b₅₅₈) and four cytosolic components: p40^phox, p47^phox, p67^phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b₅₅₈ to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47^phox. We also used a fused chimera consisting of p47^phox(aa 1–286), p67^phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47^phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b₅₅₈. They also have an impact on O₂.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.     

Expression of functional mammal flavocytochrome b558 in yeast: Comparison with improved insect cell system

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b₅₅₈ (Cytb₅₅₈) which consists of a heterodimer of the gp91^phox and p22^phox subunits. The Cytb₅₅₈ is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb₅₅₈ (rCytb₅₅₈). We expressed the gp91^phox and p22^phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91^phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb₅₅₈ displayed comparable spectral properties to neutrophil Cytb₅₅₈. In contrast to rCytb₅₅₈ produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.     

p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

Conformational changes in truncated p47phox proteins monitored by fluorescent labeling

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O^– ₂ at the expense of NADPH. During activation, the cytosolic oxidase components p47^phox and p67^phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47^phox and p67^phox associate with cytochrome b₅₅₈, a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47^phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N-di-methyl-N(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47^phox-(SH3)₂-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47^phox. However, the fluorescence of C-terminal-truncated p47^phox-N-(SH3)₂ and that of both C-terminal and N-terminal truncated p47^phox-(SH3)₂ were not altered by the activators. These results indicate that the C-terminal region of p47^phox is a primary target of the conformational change during the activation of NADPH oxidase.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O₂^–). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b₅₅₈ (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91⁰, X91^– or X91⁺) according to their cytochrome b₅₅₈ expression and NADPH oxidase activity. Nine patients had X91⁰ CGD, one had X91^– CGD and one had X91⁺ CGD. Six mutations in CYBB were novel. Of the four new X91⁰ CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91⁰ CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91^– CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91⁺ CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.     

Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi

NADPH oxidases (Nox) are membrane complexes that produce O₂^?. Researches in mammals, plants and fungi highlight the involvement of Nox‐generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91^phox/Nox2 is associated with p22^phox forming the flavocytochrome b₅₅₈ ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22^phox gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22^phox homologue as being mutated in the Podospora anserina mutant IDC⁵⁰⁹. Functional studies show that the fungal p22^phox, PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91^phox homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co‐localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.     

NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47^phox, p67^phox and p40^phox. p67^phox, due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67^phox is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67^phox. Our data reveals that p67^phox behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67^phox. We suggest that the dual character of p67^phox conformation in solution is central to ensure the numerous interactions to be accommodated.     

Invariant local conformation in p22phox p.Y72H polymorphisms suggested by mass spectral analysis of crosslinked human neutrophil flavocytochrome b

The NADPH oxidase of phagocytic leukocytes generates superoxide that plays a critical role in innate immunity and inflammatory responses. The integral membrane protein flavocytochrome b (Cyt b, a.k.a. cytochrome b_558/559) is the catalytic core of the complex and serves as a prototype for homologs important in regulating signaling networks in a wide variety of animal and plant cells. Our analysis identifies a naturally-occurring Tyr72/His72 polymorphism (p.Y72H) in the p22^phox subunit of Cyt b at the protein level that has been recognized at the nucleotide level (c.214T > C, formerly C242T) and implicated in cardiovascular disease. In the present study, Cyt b was isolated from human neutrophils and reacted with chemical crosslinkers for subsequent structure analysis by MALDI mass spectrometry. Following mild chemical modification of Cyt b with two pairs of isotopically-differentiated lysine crosslinkers: BS²G-d₀/d₄ and BS³-d₀/d₄, the reaction mixtures were digested with trypsin and purified on C₁₈ZipTips to generate samples for mass analysis. MALDI analysis of tryptic digests from each of the above reactions revealed a series of masses that could be assigned to p22^phox residues 68-85, assuming an intra-molecular crosslink between Lys71 and Lys78. In addition to the 30 ppm mass accuracy obtained with internal mass calibration, increased confidence in the assignment of the crosslinks was provided by the presence of the diagnostic mass patterns resulting from the isotopically-differentiated crosslinking reagent pairs and the Tyr72/His72 p22^phox polymorphisms in the crosslinked peptides. This work identifies a novel, low-resolution distance constraint in p22^phox and suggests that the medically-relevant p.Y72H polymorphism has an invariant structural motif in this region. Because position 72 in p22^phox lies outside regions identified as interactive with other oxidase components, the structural invariance also provides additional support for maturational differences as the source of the wide variation in observed reactive oxygen species production by cells expressing p.Y72H.     

A Prenylated p47phox-p67phox-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator: MEMBRANE ASSOCIATION AND FUNCTIONAL CAPACITY*

The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b₅₅₉, a membrane-associated heterodimer composed of two subunits (Nox2 and p22^phox), and four cytosolic proteins (p47^phox, p67^phox, Rac, and p40^phox). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b₅₅₉ and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47^phox, p67^phox, and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122–22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47^phox-p67^phox-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47^phox phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp¹⁹³ in p47^phox persists. Prenylated GFP-p47^phox-p67^phox-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.     

Role for the first SH3 domain of p67 in activation of superoxide-producing NADPH oxidases

The membrane-bound NADPH oxidase in phagocytes, gp91^phox (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91^phox/Nox2 requires assembly with the cytosolic proteins p67^phox and p47^phox, each containing two SH3 domains. Although the C-terminal SH3 domain of p67^phox is responsible for binding to p47^phox, little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67^phox-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91^phox/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67^phox in cells, suggesting that SH3(N) primarily increases the affinity of p67^phox for the oxidase complex. On the other hand, p67^phox-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91^phox/Nox2. Thus p67^phox-SH3(N) specifically functions in gp91^phox/Nox2 activation probably via facilitating oxidase assembly.     

Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production

The phagocyte NADPH oxidase Nox2, heterodimerized with p22^phox in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47^phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22^phox and p67^phox, leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47^phox conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47^phox and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67^phox, AA induces the direct interaction of Rac-GTP-bound p67^phox with the C-terminal cytosolic region of Nox2. p67^phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67^phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67^phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.     

Conservation of fungal and animal nicotinamide adenine dinucleotide phosphate oxidase complexes

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) are a group of eukaryotic flavoenzymes that catalyse the reduction of dioxygen to the superoxide anion using electrons provided by NADPH. An integral membrane flavocytochrome b558 heterodimer, composed of the catalytic subunit gp91^phox and the adaptor protein p22^phox, is essential for catalytic activity of the mammalian Nox2 complex. Two homologues of the mammalian gp91^phox, NoxA and NoxB, have been identified in fungi and shown to be crucial for distinct fungal cell differentiation and developmental processes, but to date, no homologue of the p22^phox adaptor protein has been identified. Isolation of a mutant from Podospora anserina with a phenotype identical to a previously characterised PaNox1 mutant, combined with phylogenetic analysis, identified a fungal homologue of p22^phox called PaNoxD. The same adaptor protein was shown to be a component of the Botrytis cinerea NoxA complex as supported by the identical phenotypes of the bcnoxA and bcnoxD mutants and direct physical interaction between BcNoxA and BcNoxD. These results suggest that NoxA/NoxD is the fungal equivalent of the mammalian gp91^phox/p22^phox flavocytochrome complex. Tetraspanin (Pls1) mutants of P. anserina and B. cinerea have identical phenotypes to noxB mutants, suggesting that Pls1 is the corresponding integral membrane adaptor for assembly of the NoxB complex.     

Identification of allele-specific p22-phox mutations in a compound heterozygous patient with chronic granulomatous disease by mismatch PCR and restriction enzyme analysis

A rare subgroup (approx. 5%) of all chronic granulomatous disease (CGD) patients suffers from mutations in the gene encoding the small p22-phox subunit of the flavocytochrome b ₅₅₈ heterodimer, the terminal redox component of the phagocyte NADPH oxidase. A male CGD patient with neutrophil granulocytes devoid of any spectrometrically detectable cytochrome b ₅₅₈ owing to autosomally inherited p22-phox deficiency (phenotype, A22^–) is reported. The patient was identified as being compound heterozygous for two independent mutations of his p22-phox alleles. On the maternal allele a single base substitution (A186 to T) was found that predicts a nonconservative replacement of Glu 53 by Val. On his paternal p22-phox allele a G was found to be added to a G stretch between nucleotides G195 and G199 in the cDNA sequence. The resulting frame shift predicts an aberrant open reading frame, 16 amino acids longer than the normal p22-phox polypeptide. Genomic DNA was tested for the presence of the mutant allele by mismatch PCR (polymerase chain reaction). For this purpose, a single base mismatch was introduced at nucleotide position 189, leading to digestion of the normal allele by the restriction enzyme HinfI. The maternal allele was found to be present in 50% of the patient's DNA and in 50% of the DNA from his mother. The same mismatch PCR analysis with control DNA from 35 healthy individuals ruled out the possibility that the single base substitution (A186 to T) represents a common polymorphism. Inheritance of the second allelic mutation (G insertion) was verified by restriction enzyme analysis using BslI [CC(N)₇GG] to digest PCR-amplified genomic DNA at the mutation site. PCR in combination with restriction enzyme analysis proved to be a powerful tool for verification of point mutations in the compound heterozygous CGD patient analyzed and may be used for prenatal diagnosis in this family.Abbreviations phox phagocyte oxidase - CGD chronic granulomatous disease - PCR polymerase chain reaction     

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Khz (Fusion of Ganoderma lucidum and Polyporus umbellatus Mycelia) Induces Apoptosis by Increasing Intracellular Calcium Levels and Activating JNK and NADPH Oxidase-Dependent Generation of Reactive Oxygen Species

Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47^phox and p67^phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca²⁺]_i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47^phox and p67^phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca²⁺]_i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.