The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Gross, Aya Federman; Rafalowski, Meirav; Pick, Edgar

doi:10.3389/fchem.2015.00003

Cited by 21 publications

(45 citation statements)

References 82 publications

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“…Mechanistic evaluation in the cell-free NOX2 semirecombinant assay using WST-1 revealed a competitive inhibition of GSK2795039 toward NADPH and excluded a potential action on intracellular cysteines, a known inhibitory mode of action specific for NOX2 (6). This finding was confirmed when NADPH depletion was used as readout.…”

mentioning

confidence: 63%

“…Compounds able to oxidize dithiols to form a disulfide complex such as phenylarsine oxide (PAO) (39) and gliotoxin (67) in the NOX2 sequence, which is absent in NOX1, NOX3, NOX4, and NOX5 (33) and is necessary for NOX2 function, probably by mediating the binding of the p67 phox subunit (6). When the NOX2 complex is not assembled, the cysteine thiol groups are exposed and gp91 phox is more sensitive to thioloxidizing compounds.…”

Section: Enzyme Mode Of Action Studiesmentioning

confidence: 99%

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Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

Hirano¹,

Chen²,

Chueng³

et al. 2015

Antioxidants & Redox Signaling

155

141

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Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cellbased NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358-374.

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mentioning

confidence: 63%

Section: Enzyme Mode Of Action Studiesmentioning

confidence: 99%

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

Hirano¹,

Chen²,

Chueng³

et al. 2015

Antioxidants & Redox Signaling

155

141

View full text Add to dashboard Cite

show abstract

“…The N-terminal portion of this region is adjacent to the FAD binding site. Interestingly, peptides derived from the homologous region of Nox2 bind p67phox [56] suggesting an evolutionary conservation of regulatory protein:protein interactions in this area. The region between aa 572–600 contains a loop connecting helices in the NADPH-binding domain.…”

Section: Discussionmentioning

confidence: 99%

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Chen

Haigh

et al. 2015

Free Radical Biology and Medicine

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Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490–550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.

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“…Wild‐type p67 phox (1–526) was constructed by subcloning of the Bam HI‐ Eco RI fragment from plasmid pGEX‐2T‐p67 phox (1–526) into a modified plasmid pET‐30a‐His 6 , as described before, resulting in the generation of N‐terminally His 6 ‐tagged protein. p67 phox (1–242) was constructed by PCR on pET‐30a‐His 6 ‐p67 phox (1–526) with pT7 and 3′–p67 phox (242) ‐Eco RI primers.…”

Section: Methodsmentioning

confidence: 99%

“…The chimeric protein (p67 phox (1–212)‐Rac1Q61L(1–192)) and the fusion proteins NusA–Nox2(357–570) and NusA–Nox2(372–570) were constructed and expressed as described before . The bacteria were induced with 0.4 mM IPTG and cultured for 14–16 h at 18°C.…”

Section: Methodsmentioning

confidence: 99%

p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond—Canaan sighted?

Bechor

Zahavi

Amichay

et al. 2020

Journal of Leukocyte Biology

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Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67phox and there is evidence for a change in the configuration of p67phox being required for binding to Nox2. To study this, we measured binding of p67phox to a library of Nox2 peptides and binding of NusA–Nox2 fusion proteins to p67phox. We found, serendipitously, that deletion of residues 259–279 in p67phox (p67phoxΔ(259–279)), endowed it with the ability to bind selectively to Nox2 peptide 369–383 (peptide 28). There was no binding to scrambled Nox2 peptide 28 and to Nox4 peptide 28. Binding was cysteine independent and resistant to reducing and alkylating agents. Truncations of peptide 28 revealed that the actual binding site consisted of residues 375–383. Binding of p67phoxΔ(259–279) to peptide 28 was mimicked by that of a (p67phox‐RacGTP) chimera. Both p67phoxΔ(259–279) and the (p67pho–RacGTP) chimera bound a NusA–Nox2 fusion protein, comprising residues 375–383. Specific single residue deletion mutants, within the p67phox sequence 259–279, were also bound to Nox2 peptide 28. Peptides synthesized to correspond to the 259–279 sequence in p67phox, were found to autobind p67phox, suggesting that an intramolecular bond exists in p67phox, one pole of which was located within residues 259–279. We conclude that “resting” p67phox exists in a “closed” conformation, generated by an intramolecular bond. Deletion of specific residues within the 259–279 sequence, in vitro, or interaction with RacGTP, in vivo, causes “opening” of the bond and results in binding of p67phox to a specific, previously unknown, site in Nox2.

show abstract

The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

Cited by 21 publications

References 82 publications

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

p67phox binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond—Canaan sighted?

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