Abstract
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 yellow-fluorescent 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.
Highlights
The phagocyte NADPH oxidase generates superoxide anions (O2Ϫ) by reducing molecular oxygen by a transmembrane heterodimer, which carries two hemes and forms a cytochrome b558 complex [1, 2]
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
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
Summary
The phagocyte NADPH oxidase generates superoxide anions (O2Ϫ) by reducing molecular oxygen by a transmembrane heterodimer, which carries two hemes and forms a cytochrome b558 complex [1, 2]. 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. Interaction of p22phox and Nox antibodies directed against the GFP part of the constructs are available and allow the detection of the fusion proteins by Western blot analysis and even immunoprecipitation.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have