Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding.

Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA).

Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat.

Objective: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation.

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C.

NADPH oxidase.

NADPH oxidase is an enzyme that catalyzes the production of superoxide from oxygen and NADPH. It is a complex enzyme consisting of two membrane-bound components and three components in the cytosol, plus rac 1 or rac 2. Activation of the oxidase involves the phosphorylation of one of the cytosolic components.

Evidence for ozone formation in human atherosclerotic arteries.

Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro.

Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O(2)(-) at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47(PHOX) occurs during the activation of the enzyme in intact cells. p47(PHOX) carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47(PHOX) is required for the activation of the oxidase.

Investigating antibody-catalyzed ozone generation by human neutrophils.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H(2)O(3)) and ozone (O(3)) from singlet oxygen ((1)O(2)(*)) and water. Given that neutrophils have the potential both to produce (1)O(2)(*) and to bind antibodies, we considered that these cells could be a biological source of O(3). We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O(3).

The leukocyte NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O2- (superoxide) at the expense of NADPH. The O2- then dismutes to H2O2, which serves to oxidize Cl- to HOCl, a potent microbicidal agent that is used by leukocytes to kill invading microorganisms. This oxidation is catalyzed by myeloperoxidase.

Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing.

The C2A domain of JFC1 binds to 3'-phosphorylated phosphoinositides and directs plasma membrane association in living cells.

Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3'-phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3'-phosphoinositides in vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3'-phosphoinositides in vivo.

JFC1 is transcriptionally activated by nuclear factor-kappaB and up-regulated by tumour necrosis factor alpha in prostate carcinoma cells.

The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549 bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide -321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-kappaB (NF-kappaB).

Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing.

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair.

The activity of leukocyte NADPH oxidase: regulation by p47PHOX cysteine and serine residues.

The leukocyte NADPH oxidase is regulated chiefly by phosphorylation of the serines of p47(PHOX), one of its cytosolic subunits. Its activity is also regulated, however, by the four cysteines of the same subunit, as indicated by the replacement of those cysteines by alanines.

Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558 II.

Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac with the membrane-bound cytochrome b558. We recently established a direct interaction between p67PHOX and cytochrome b558. In the present study, we show that removal of the C-terminal domain of p67PHOX increased its binding to cytochrome b558.