Biosynthesis of the Phagocyte NADPH Oxidase Cytochromeb 558

Self Cite

151

149

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b 558 (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91 phox and p22 phox to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91 phox precursor, gp65, was processed to gp91phox within 4 -8 h of chase, unassembled gp65 and p22 phox monomers were degraded by the cytosolic proteasome. gp65 associated with p22 phox post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22 phox coprecipitated with unglycosylated gp91 phox primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22 phox was unaffected. In succinyl acetone-treated cells, p22 phox and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22 phox heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22 phox , a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.

Section: Glycosylation and Heterodimer Formation-because The Binding supporting

confidence: 81%

Section: Glycosylation and Heterodimer Formation-because The Binding mentioning

confidence: 99%

Processing and Maturation of Flavocytochromeb 558 Include Incorporation of Heme as a Prerequisite for Heterodimer Assembly

DeLeo¹,

Burritt²,

Yu³

et al. 2000

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151

149

“…These are lineage-specific to eosinophils, B-lymphocytes, and monocytes, respectively. Surface gp91 phox of the cells was stained as described above with fluorescein isothiocyanateconjugated 7D5 (22). 3 These cells were washed twice with phosphatebuffered saline containing 0.5% bovine serum albumin, and their fluorescence intensities were analyzed on a FACScan (Becton Dickinson, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

Eosinophil-specific Regulation of gp91 Gene Expression by Transcription Factors GATA-1 and GATA-2

Yang

Suzuki

et al. 2000

The glycoprotein gp91phox is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91 phox gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91 phox promoter in various types of gp91 phox -expressing cells. A 10-base pair (bp) region from bp ؊105 to ؊96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp ؊101 to ؊96 (؊98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the ؊98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the ؊105/؉12 construct in a ؊98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91 phox gene in human eosinophils.

“…In human leukocytes, Nox2 is synthesized as a 65-kDa precursor in the Golgi apparatus, whereas mature Nox2 has a molecular mass of 91 kDa because of heavy glycosylation of the protein. The glycosylation pattern, however, has no impact on the function of the protein (3,4).…”

mentioning

confidence: 99%

Direct Interaction of the Novel Nox Proteins with p22phox Is Required for the Formation of a Functionally Active NADPH Oxidase

Ambasta

Kumar

Griendling

et al. 2004

477

376

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan-and yellowfluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.