Disease relevance of NCF2

• Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking [1].
• Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia [2].
• This system is characterized by the partial reconstitution of O-(2) production in an Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell line from a patient with p67-phox-deficient CGD by transfection with an expression plasmid containing the 67-phox cDNA in the sense orientation [3].
• To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds) [2].
• Expression of p67-phox was upregulated by rhinovirus challenge [4].

Psychiatry related information on NCF2

• The present study is the first to show that superoxide (O(-)(2)) forming NADPH oxidase is activated in Alzheimer's disease (AD) brains by demonstrating the marked translocation of the cytosolic factors p47-phox and p67-phox to the membrane [5].

High impact information on NCF2

• Rac1 interacted directly with p67phox in a GTP-dependent manner [6].
• Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity [6].
• Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558 [7].
• A 65-kilodalton factor, NCF-2, restored activity to defective neutrophil cytosol from one patient with autosomal CGD [8].
• NCF-1, NCF-2, and a third cytosol fraction, NCF-3, were inactive alone or in pairs, but together replaced unfractionated cytosol in cell-free O2.- generation [8].

Chemical compound and disease context of NCF2

• The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease [9].
• Molecular characterization of autosomal recessive chronic granulomatous disease caused by a defect of the nicotinamide adenine dinucleotide phosphate (reduced form) oxidase component p67-phox [10].
• Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils [11].
• Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease [12].

Biological context of NCF2

• p67(phox) and gp91(phox) are components of the phagocyte-specific respiratory burst oxidase that are encoded by the NCF2 and CYBB genes, respectively [13].
• In contrast, RNA decay assays demonstrated that there was no significant difference between stability of NCF2 intron 1 transcripts and the exon 1 5'-UTR variant in HL-60, MonoMac 6, and U937 cells [14].
• Haplotype analyses and homozygosity mapping with microsatellite markers around known CGD genes assigned the genetic defect to NCF1 in four patients, to NCF2 in four patients and to CYBA in two patients [15].
• Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation [16].
• Approximately 5% of CGD patients have an autosomal recessive form of disease caused by a severe deficiency of p67-phox, a 526-amino acid subunit of the oxidase that appears to regulate electron transport within the enzyme [10].

Anatomical context of NCF2

• The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25 [17].
• The CYBB and NCF2 genes encode the phagocyte respiratory burst oxidase proteins, gp91PHOX and p67PHOX [18].
• Therefore, decreased HoxA10 repression contributes to increased CYBB and NCF2 transcription in differentiating myeloid cells [19].
• NCF2 5'-UTR variant transcripts were differentially expressed in various cell lines and human tissues [14].
• The NCF2 gene encodes p67(phox), an essential component of the multi-protein NADPH oxidase enzyme in phagocytic leukocytes, as well as in certain non-phagocytic cells [14].

Associations of NCF2 with chemical compounds

• These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox [1].
• However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases [20].
• We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production [2].
• These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH [21].
• However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated [21].

Physical interactions of NCF2

• The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs [22].
• We identify a sequence in the NCF2 promoter that is homologous to the HoxA10-binding CYBB cis element [13].
• We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus [23].
• Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox [24].

Regulatory relationships of NCF2

• Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation [25].
• Because IFN-gamma induces simultaneous expression of p67phox and gp91phox, these investigations identify a molecular event that coordinates oxidase gene transcription during the inflammatory response [26].
• Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase [23].
• A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity [27].
• Consistent with this observation, we also demonstrate that NCF2 mRNA and p67phox protein are up-regulated by TNF-alpha in various myeloid cell lines as well as in human monocytes [28].

Other interactions of NCF2

• In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558 [29].
• We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox [30].
• Consonant with this finding, the protein content of three essential components of phagocyte oxidase (p22-phox, p67-phox, and p47-phox) was also eosinophils > neutrophils > monocytes = macrophages [31].
• Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils [1].
• Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases [32].

Analytical, diagnostic and therapeutic context of NCF2

• Western blot analysis showed that both cell lines expressed the phagocyte oxidase (phox) cytosolic proteins Rac1, p40phox, and p67phox [33].
• P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy [34].
• Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox [35].
• That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient [36].
• Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox [37].

References