• Home
  • Search Results
  • Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369 Cys-Gly-Cys371 triad in Nox2 and in p67phox.

Binding of p67phox to Nox2 is stabilized by disulfide bonds between cysteines in the 369 Cys-Gly-Cys371 triad in Nox2 and in p67phox.

Journal of leukocyte biology (2018-07-17)
Tanya Fradin, Edna Bechor, Yevgeny Berdichevsky, Iris Dahan, Edgar Pick
ABSTRACT

A central event in the activation of the phagocyte NADPH oxidase involves binding of p67phox to the dehydrogenase region of Nox2. The identity of the binding site in Nox2 is unknown. By measuring binding of p67phox to synthetic Nox2 peptides, we previously identified a sequence corresponding to Nox2 residues 357-383, as a potential binding site. A key role was attributed to a 369 Cys-Gly-Cys371 triad, shared by peptides 357-371 (peptide 24) and 369-383 (peptide 28). In this study, we show that (1) oxidation of cysteines in peptides 24 and 28 by a variety of oxidants markedly enhances the binding of p67phox ; (2) replacing cysteines by arginine abolishes the response to oxidants and the enhanced binding of p67phox ; (3) oxidants act by generating an intramolecular disulfide bond linking cysteines 369 and 371, generating such bond during peptide synthesis reproduces the effect of oxidants; (4) for the disulfide bond to lead to enhanced binding, cysteines must be separated by an intervening residue; bonds joining adjacent cysteines, or cysteines located on two peptides, do not enhance binding; (5) dissociating disulfide bonds by reducing agents abolishes enhanced binding; (6) treating p67phox with the alkylating agent N-ethylmaleimide suppresses binding; and (7) mutating all nine cysteines in p67phox to serines abolishes binding and diminishes the ability of p67phox to support NADPH oxidase activation in vitro. Results show that the primary interaction of p67phox with Nox2 is followed by a stabilizing step, based on the establishment of disulfide bonds between cysteine(s) in the 369 Cys-Gly-Cys371 triad and cysteine(s) in p67phox .

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
TWEEN® 20, viscous liquid
Sigma-Aldrich
2-Mercaptoethanol, for molecular biology, suitable for electrophoresis, suitable for cell culture, BioReagent, 99% (GC/titration)
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
DL-Dithiothreitol, for molecular biology, ≥98% (HPLC), ≥99% (titration)
Sigma-Aldrich
Aprotinin from bovine lung, lyophilized powder, 3-8 TIU/mg solid
Sigma-Aldrich
Vitamin B12, ≥98%
Sigma-Aldrich
Catalase from bovine liver, lyophilized powder, 2,000-5,000 units/mg protein
Sigma-Aldrich
Casein sodium salt from bovine milk
Sigma-Aldrich
N-Ethylmaleimide, crystalline, ≥98% (HPLC)
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, ≥93% (HPLC)
Sigma-Aldrich
Glycine, ACS reagent, ≥98.5%
Sigma-Aldrich
(−)-Riboflavin, from Eremothecium ashbyii, ≥98%
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate
Sigma-Aldrich
Flavin adenine dinucleotide disodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Anti-polyHistidine−Peroxidase antibody, Mouse monoclonal antibody produced in mouse, clone HIS-1, purified from hybridoma cell culture
Sigma-Aldrich
Diamide
Sigma-Aldrich
Riboflavin 5′-monophosphate sodium salt hydrate, synthetic, ≥70% (HPLC)
Sigma-Aldrich
Lithium dodecyl sulfate, ≥98.5% (GC)
Sigma-Aldrich
2,3-Dimercapto-1-propanol, ≥98% (iodometric)
Sigma-Aldrich
L-Glutathione oxidized, BioXtra, ≥98%
Sigma-Aldrich
Imidazole, ≥99% (titration), crystalline
Sigma-Aldrich
Phenylarsine oxide, ≥97%, powder
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate, ~98%, pkg of 100 mg
Sigma-Aldrich
ECO TWEEN® 20, viscous liquid