The antimicrobial peptide parabutoporin competes with p47<sup>phox</sup> as a PKC‐substrate and inhibits NADPH oxidase in human neutrophils

A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(؊/؊) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(؊/؊) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase C␦, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity.

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“…It has been demonstrated that PKC␦, on one hand, directly interacts with Shc (40 -43); also, PKC␦ is an activator of p47phox (27,39,59,60).…”

Section: Rac Activation Is Similar In P66shc(ϫ/ϫ) Macrophages-mentioning

confidence: 99%

Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice

Tomilov

Bicocca

Schoenfeld

et al. 2010

Journal of Biological Chemistry

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“…It was purified by HPLC and sequenced. Wild-type and mutant 45 aa peptides were chemically synthesized, using standard 9-fluorenylmethyloxycarbonyl programs, as described previously [20].…”

Section: Reagentsmentioning

confidence: 99%

“…Of interest, activation of NADPH oxidase with PMA and fMLP is strongly inhibited by PP at nonlytic concentrations. We have shown recently that PP inhibits the protein kinase C (PKC)-driven phosphorylation of p47 phox and its subsequent translocation to the transmembrane catalytic NADPH oxidase counterparts [20]. Therefore, we hypothesized that incubation with PP would prolong PMN survival, as has been reported for CGD neutrophils [13].…”

Section: Introductionmentioning

confidence: 96%

“…At micromolar concentrations, it was initially shown to have antimicrobial properties, but in the same concentration range, it also appeared to induce pore formation in human cells, including neutrophils [19]. However, at submicromolar concentrations, PP is nonlytic [18,20]. Of interest, activation of NADPH oxidase with PMA and fMLP is strongly inhibited by PP at nonlytic concentrations.…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Remijsen

Berghe

Parthoens

et al. 2008

Journal of Leukocyte Biology

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Neutrophil cell death plays a crucial role in neutrophil homeostasis and the resolution of inflammation. The superoxide-producing NADPH oxidase is involved in pathogen degradation and subsequent activation of cell death programs. Neutrophils from patients with chronic granulomatous disease, who have a deficient NADPH oxidase activity, have been demonstrated previously to have a prolonged lifespan, suggesting that a basal NADPH oxidase activity also regulates spontaneous neutrophil turnover. The NADPH oxidase inhibitor parabutoporin (PP) does delay spontaneous apoptosis, but this effect is completely independent of NADPH oxidase inhibition. Instead, the prosurvival effect of PP depends on activation of protein kinase B/Akt via lipid raft signaling. Disruption of lipid rafts abrogates the prosurvival effect without interfering with NADPH oxidase activity. Furthermore, we cannot detect a different rate of spontaneous apoptosis between normal and NADPH oxidase-deficient neutrophils, arguing against a role of NADPH oxidase in spontaneous neutrophil apoptosis.

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“…This enzyme constitutes the primary source of oxidizing agents used by neutrophils to kill invading microorganisms (Freitas et al 2009a). Neutrophils express only five different PKC isotypes: a, bI, bII, d, and f, which are inhibited by Gö6983 (Remijsen et al 2006). PKC contain zinc finger structures that are important for its enzymatic activity (Beyersmann and Haase 2001).…”

Section: Discussionmentioning

confidence: 99%

Zinc activates neutrophils’ oxidative burst

et al. 2009

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Zinc has been shown to disturb the innate host defense response by interfering in the activation of neutrophils and subsequent oxidative burst, although the exact role of this metal, either as an activator or inhibitor, remains a matter of controversy among research groups. These apparent discrepancies may be due to experimental settings, through modification of zinc availability to neutrophils, or to inaccurate detections of reactive species. Thus, the main objective of the present study was to provide clarification on the role of zinc on the activation of human neutrophils and the subsequent oxidative burst. For that purpose, different detection methods and incubation media were used. The obtained results showed that phosphate buffers (PBS and HBSS) complex with zinc and interfere with the results obtained with this metal. By using Tris-G, it was clearly demonstrated that zinc, at low concentrations (5-12.5 microM), activates NADPH oxidase, mainly via protein kinase C, leading to the formation of superoxide radical (O(2*-). Higher concentrations of zinc results on a rapid dismutation of O2*- to oxygen and hydrogen peroxide, which in turn is used by myeloperoxidase to generate hypochlorous acid (HOCl).

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The antimicrobial peptide parabutoporin competes with p47^phox as a PKC‐substrate and inhibits NADPH oxidase in human neutrophils

Cited by 20 publications

References 26 publications

Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice

Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice

Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Zinc activates neutrophils’ oxidative burst

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The antimicrobial peptide parabutoporin competes with p47phox as a PKC‐substrate and inhibits NADPH oxidase in human neutrophils

Cited by 20 publications

References 26 publications

Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice

Decreased Superoxide Production in Macrophages of Long-lived p66Shc Knock-out Mice

Inhibition of spontaneous neutrophil apoptosis by parabutoporin acts independently of NADPH oxidase inhibition but by lipid raft-dependent stimulation of Akt

Zinc activates neutrophils’ oxidative burst

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The antimicrobial peptide parabutoporin competes with p47^phox as a PKC‐substrate and inhibits NADPH oxidase in human neutrophils