On the Mechanism of Inhibition of the Neutrophil Respiratory Burst Oxidase by a Peptide from the C-Terminus of the Large Subunit of Cytochrome b558

Dj, Uhlinger; Tyagi,; Jd, Lambeth

doi:10.1021/bi00002a017

Cited by 15 publications

(9 citation statements)

References 19 publications

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“…Domain I comprises residues 559-565, which correspond to yet another region reported to participate in the interaction of Nox2 with p47 phox . This proposal was based on mapping by peptide-phage display libraries [18] and on inhibition of oxidase activation by a Nox2 peptide containing these residues [51], but careful kinetic analysis did not support the proposed mechanism of the peptide competing with Nox2 for binding of p47 phox [52].…”

Section: Location Of Domains Deduced From Clusters Of Oxidase Inhibitmentioning

confidence: 99%

Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study

Dahan

Molshanski-Mor

Pick

2011

Journal of Leukocyte Biology

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In this study, the "peptide walking" approach was applied to the DH region of Nox2 (residues 288-570) with the purpose of identifying domains of functional importance in the assembly and/or catalytic function of the NADPH oxidase complex of phagocytes. Ninety-one overlapping 15-mer peptides were synthesized to cover the full length of the Nox2 DH region, and these were tested for the ability to interfere with the activation of the oxidase in vitro in two semi-recombinant cell-free systems. The first consisted of phagocyte membranes p47(phox), p67(phox), and Rac1 and an amphiphile; the second was p47(phox)- and amphiphile-free and contained prenylated Rac1. We identified 10 clusters of inhibitory peptides with IC(50) values of 10 μM, all of which were inhibitory, also in the absence of p47(phox). Based on the identification of residues shared by peptides in a particular cluster, we defined 10 functional domains in the Nox2 DH region. One domain corresponded to one FAD-binding subdomain, and four domains overlapped parts of three NADPH-binding subdomains. As expected, most inhibitory peptides acted only when added prior to the completion of oxidase assembly, but peptides associated with two NADPH-binding subdomains were also active after assembly. Kinetic analysis demonstrated that inhibition by peptides was not explained by competition for substrates (FAD, NADPH) but was of a more complex nature: noncompetitive with respect to FAD and uncompetitive with respect to NADPH. We conclude that oxidase-inhibitory peptides, in five out of 10 clusters identified, act by interfering with FAD- and NADPH-related redox reactions.

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Section: Location Of Domains Deduced From Clusters Of Oxidase Inhibitmentioning

confidence: 99%

Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study

Dahan

Molshanski-Mor

Pick

2011

Journal of Leukocyte Biology

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“…An antibody directed against the carboxyl-terminal 13 amino acids of gp91 phox inhibits superoxide formation in the cell-free assay (29). Synthetic peptides derived from this region inhibit NADPH oxidase activity in permeabilized neutrophils (29,34) and in the cell-free oxidase assay (29,31,57 ) have been identified by alanine scanning as important for the function of this domain, based on the relative ability of alanine-substituted peptides to inhibit oxidase activity in a cell-free assay (33). However, we found that comparable alanine substitutions in the gp91 phox polypeptide resulted in either no or only a modest reduction in NADPH oxidase activity when the mutant gp91 phox was expressed in whole cells.…”

Section: Mutagenesis Of Phagocyte Flavocytochrome B 558 Gp91mentioning

confidence: 99%

“…A kinetic analysis of oxidase inhibition by 559 RGVHFIF 565 has suggested that this peptide acts as a non-competitive inhibitor (57), which might explain why many of the mutations in the gp91 phox polypeptide did not have a profound effect. Alternatively, additional sites of interaction between p47 phox and gp91 phox may obviate the requirement for an intact 559 RGVHFIF 565 domain for oxidase assembly in intact cells.…”

Section: Mutagenesis Of Phagocyte Flavocytochrome B 558 Gp91mentioning

confidence: 99%

Probing the Role of the Carboxyl Terminus of the gp91 Subunit of Neutrophil Flavocytochromeb 558 using Site-directed Mutagenesis

Zhen¹,

Yu²,

Dinauer³

1998

Journal of Biological Chemistry

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Site-directed mutagenesis was used to generate a series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91 phox , the 91-kDa subunit of the phagocyte NADPH oxidase flavocytochrome b 558 . This region encompasses 559 RGVHFIF 565 , implicated as a contact point for the cytosolic oxidase subunit p47 phox during oxidase activation, and a carboxyl-terminal phenylalanine (Phe 570 ), which corresponds in position to a highly conserved aromatic residue that interacts with the flavin group in the ferredoxin-NADP ؉ reductase flavoenzyme family, of which gp91 phox is a member. Mutant proteins were expressed in human myeloid leukemia cells which lack expression of endogenous gp91 phox due to targeted disruption of the X-linked gp91 phox gene. Although specific residues within 559 RGVHFIF 565 had previously been identified by alanine scanning as essential for peptide inhibition of oxidase activity in a cellfree assay, comparable substitutions in the gp91 phox polypeptide had either no or only a modest effect on oxidase activity in whole cells. Replacement of nonpolar with polar or charged residues had greater effects on oxidase activity, but were also associated with decreased gp91 phox expression, suggesting that overall protein structure was perturbed. No stable gp91 phox protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal Phe 570 in gp91 phox resulted in a 2-fold reduction in superoxide production. This contrasts with a Ϸ300 -800-fold reduction reported for comparable mutations in pea ferredoxin-NADP ؉ reductase, which suggests that structural or functional differences exist between the carboxyl terminus of gp91 phox and other ferredoxin-NADP ؉ reductases.Neutrophils and other phagocytic leukocytes possess an NADPH oxidase (respiratory burst oxidase) that generates large quantities of superoxide during the respiratory burst (1). Upon phagocyte activation by opsonized bacteria or other inflammatory stimuli, the active NADPH oxidase complex is rapidly assembled at the plasma membrane from cytosolic and membrane components to catalyze the transfer of electrons from NADPH to molecular oxygen. Oxidase subunits include two polypeptides, gp91 phox and p22 phox

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“…However, the first sign that the a priori assumption of a competitive mechanism is risky came from the kinetic analysis of Nox2 peptide 559-565, found to inhibit oxidase activation in vitro and proposed to act by competing with Nox2 for binding of p47 phox [27,49]. Contrary to expectations, an excess of p47 phox did not suppress the effect of the peptide and kinetic analysis did not yield a competitive but a mixed pattern of inhibition [50]. A more scathing analysis of a seemingly competitive mechanism of oxidase inhibition by Nox2 peptides overlapping NADPH binding sites is offered in [37].…”

Section: Kinetic Analysis Of Inhibition By Peptidesmentioning

confidence: 99%

“…In both Nox2 and Nox4, this region binds to the cytosolic dehydrogenase region, a link that is critical for electron flow from the cytosolic (NADPH, FAD) to the membranal (hemes) redox centers. It is remarkable that Nox2 peptide 86-94, which is one of the most intensively studied and tested in several in vivo situations, and which has a potential for clinical applications, has never been subjected to a proper kinetic analysis in relation to its role as a binding site for p47 phox , such as the one applied to Nox2 peptide 559-565 [50]. It has been suggested that p47 phox might not bind directly to the B loop in Nox2 and that, in fact, the B loop-dehydrogenase region interaction affects the binding of p47 phox to the dehydrogenase region [55].…”

Section: Inhibition Of Oxidase Activity By Peptides Versus Binding Tomentioning

confidence: 99%

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”

Dahan

Pick

2012

Cell. Mol. Life Sci.

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Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.

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On the Mechanism of Inhibition of the Neutrophil Respiratory Burst Oxidase by a Peptide from the C-Terminus of the Large Subunit of Cytochrome b558

Cited by 15 publications

References 19 publications

Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study

Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A “peptide walking” study

Probing the Role of the Carboxyl Terminus of the gp91 Subunit of Neutrophil Flavocytochromeb 558 using Site-directed Mutagenesis

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”

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