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.     

Functional analysis of two-amino acid substitutions in gp91<Emphasis Type="Italic">phox</Emphasis> in a patient with X-linked flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>558</Subscript>-positive chronic granulomatous disease by means of transgenic PLB-985 cells

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b₅₅₈, which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91⁺ CGD. We have previously reported an X⁺ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X⁺ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism.Clara Bionda and Xing Jun Li contributed equally to this work     

Expression of components of the superoxide generating NADPH oxidase by human leucocytes and other cells

Summary NADPH oxidase of phagocytic leucocytes contains a membrane cytochromeb with two subunits, gp91 ^phox and p22 ^phox , together with three cytosolic proteins, p47 ^phox , p67 ^phox and p2 ^rac . The presence of some of these components has been sought in non-phagocytes, using Western blot analysis for protein expression and PCR to amplify and detect mRNA. All components were detected in EBV-transformed B lymphocytes and peripheral blood B lymphocytes. Fibroblasts and human kidney mesangial cells contained mRNA for p67 ^phox , p47 ^phox , and p22 ^phox but not gp91 ^phox . Levels of expression varied with growth conditions, but it appears possible than an isozyme of cytochromeb which lacks gp9 ^phox is present in these cells. Proteins of p47 ^phox and p67 ^phox were expressed, in low concentrations, in these two cell types. Expression of mRNA for p47 ^phox and p67 ^phox was found to be widespread in many cell types.Abbreviations IL-1 interleukin 1 - PMA phorbol myristate acetate - CGD chronic granulomatous disease - EBV-BL Epstein-Barr virus transformed B-lymphocytes - PBBL peripheral blood B lymphocytes     

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.     

Phage display epitope mapping of human neutrophil flavocytochrome b558. Identification of two juxtaposed extracellular domains

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.     

Adenosine A(2A) receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91^phox deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A_2A receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A_2A receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91^phox deficient mice is attenuated by CGS21680. These data suggest that treatment with A_2AR agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91^phox deficiency.     

Proton conduction through full-length gp91phox requires histidine 115

Summary.  The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 ^phox , one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 ^phox has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 ^phox has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 ^phox . Stable CHO cell lines were established which expressed full-length gp91 ^phox in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 ^phox and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 ^phox . The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 ^phox . Therefore, histidine 115 is required for proton conduction by both full-length gp91 ^phox and the N-terminal 230-amino-acid fragment of gp91 ^phox . Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Bristol Institute for Transfusion Sciences, Bristol, United Kingdom. RID="*" ID="*" Correspondence and reprints: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.     

Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox

Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the approximately 58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.     

NADPH oxidase subunit gp91phox: A proton pathway

Summary The generation of superoxide by the NADPH oxidase is an electrogenic process resulting in a rapid depolarisation of the membrane potential of the cell. The efflux of H⁺ ions through an arachidonate-activatable, Zn²⁺-inhibitable H⁺ pathway accompanies the efflux of electrons and provides the necessary charge compensation. Inhibition of H⁺ flux leads to inhibition of superoxide generation. The protein gp91^phox, a transmembrane component of the NADPH oxidase, was demonstrated to be capable of acting as the NADPH oxidase-associated H⁺ channel in a stable CHO cell line, CHO91. The N-terminal 230 amino acids contain all that is required for the protein to form an H⁺ channel and specifically histidine 115 is important to the ability of gp91^phox to conduct H⁺ ions. The recording of outward currents from CHO91 cells, in the whole-cell configuration, demonstrated that gp91^phox is also capable of functioning as a voltage-gated H⁺ conductance pathway. The similarity in properties between voltage-elicited outward currents, from both wild type and the mutations, and the arachidonate-activated H⁺ flux strongly suggests that these H⁺ pathways are one in the same. Among the recently identified homologues of gp91^phox only NOH-1S has so far been demonstrated to also act as an H⁺ conductance pathway.Abbreviation CGD chronic granulomatous disease     

Bidirectional interactions between NOX2‐type NADPH oxidase and the F‐actin cytoskeleton in neuronal growth cones

NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)‐type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F‐actin content, retrograde F‐actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91^phox localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40^phox. p40^phox itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91^phox and p40^phox with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40^phox with NOX2/gp91^phox at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth.

     

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47 phox -deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47 phox gene

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47 ^phox , a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47 ^phox . The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47 ^phox -deficient CGD patients, as an in vitro model of gene therapy for p47 ^phox -deficient CGD. The transduced cells expressed both P-glycoprotein and p47 ^phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47 ^phox -deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases. Received: 8 June 1998 / Accepted: 5 August 1998     

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     

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.     

NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

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.     

Melatonin alleviates lipopolysaccharide‐compromised integrity of blood–brain barrier through activating AMP‐activated protein kinase in old mice

Blood–brain barrier (BBB) dysfunction is considered to be an early event in the pathogenesis of a variety of neurological diseases in old patients, and this could occur in old people even when facing common stress. However, the mechanism remains to be defined. In this study, we tested the hypothesis that decreased melatonin levels may account for the BBB disruption in old mice challenged with lipopolysaccharide (LPS), which mimicked the common stress of sepsis. Mice (24–28 months of age) received melatonin (10 mg kg^?1 day^?1, intraperitoneally, i.p.) or saline for one week before exposing to LPS (1 mg kg^?1, i.p.). Evan's blue dye (EB) and immunoglobulin G (IgG) leakage were used to assess BBB permeability. Immunostaining and Western blot were used to detect protein expression and distribution. Our results showed that LPS significantly increased BBB permeability in old mice accompanied by the degradation of tight junction proteins occludin and claudin‐5, suppressed AMP‐activated protein kinase (AMPK) activation, and elevated gp91^phox protein expression. Interestingly, administration of melatonin for one week significantly decreased LPS‐induced BBB disruption, AMPK suppression, and gp91^phox upregualtion. Moreover, activation of AMPK with metformin significantly inhibited LPS‐induced gp91^phox upregualtion in endothelial cells. Taken together, our findings demonstrate that melatonin alleviates LPS‐induced BBB disruption through activating AMPK and inhibiting gp91^phox upregulation in old mice.     

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.     

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.     

NADPH-oxidase activation and cognition in Alzheimer disease progression

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91^phox, or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91^phox, p67^phox, p47^phox, p40^phox, and p22^phox). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67^phox, p47^phox, and p40^phox ) were significantly elevated with disease progression; the membrane-bound subunits (gp91^phox and p22^phox) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.