scholarly journals Consequences of the constitutive NOX2 activity in living cells: cytosol acidification, apoptosis, and localized lipid peroxidation

2021 ◽  
Author(s):  
Hana Valenta ◽  
Sophie Dupré-Crochet ◽  
Tania Bizouarn ◽  
Laura Baciou ◽  
Oliver Nüsse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Living Cells ◽  
Small Gtpase ◽  
Activation Mechanism ◽  
Cell Physiology ◽  
List Type ◽  
Catalytic Core ◽  
Cytosolic Proteins ◽  
Phagocyte Nadph Oxidase

ABSTRACTThe phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•−), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.HighlightsTrimera is a tool to trigger a continuous ROS production in living cellsContinuous NOX2 activity causes cytosol acidification and apoptosisROS overproduction leads to localized oxidation of the membrane lipidsTrimera tends to clusterize in the plasma membrane of COSNOX and COS-7 cells

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase

2009 ◽  
Vol 419 (2) ◽  
pp. 329-338 ◽  
Author(s):  
Masahiko Taura ◽  
Kei Miyano ◽  
Reiko Minakami ◽  
Sachiko Kamakura ◽  
Ryu Takeya ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Crucial Role ◽  
Sh3 Domain ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Wild Type ◽  
Catalytic Core ◽  
Membrane Recruitment ◽  
Phagocyte Nadph Oxidase ◽  
Isoleucine Residue

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox–p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

scholarly journals Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Jérémy Joly ◽  
Elodie Hudik ◽  
Sandrine Lecart ◽  
Dirk Roos ◽  
Paul Verkuijlen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Proteolytic Enzymes ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Human Neutrophils ◽  
Number Of Clusters ◽  
Phagocyte Nadph Oxidase ◽  
Phagosomal Membrane

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.

Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells

2003 ◽  
Vol 285 (2) ◽  
pp. F219-F229 ◽  
Author(s):  
Yves Gorin ◽  
Jill M. Ricono ◽  
Nam-Ho Kim ◽  
Basant Bhandari ◽  
Goutam Ghosh Choudhury ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Mesangial Cells ◽  
Protein Kinase B ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Ros Generation ◽  
Ang Ii ◽  
Phagocyte Nadph Oxidase

ANG II induces protein synthesis through the serine-threonine kinase Akt/protein kinase B (PKB) in mesangial cells (MCs). The mechanism(s) of activation of Akt/PKB particularly by G protein-coupled receptors, however, is not well characterized. We explored the role of the small GTPase Rac1, a component of the phagocyte NADPH oxidase, and the gp91 phox homologue Nox4/Renox in this signaling pathway. ANG II causes rapid activation of Rac1, an effect abrogated by phospholipase A2 inhibition and mimicked by arachidonic acid (AA). Northern blot analysis revealed high levels of Nox4 transcript in MCs and transfection with antisense (AS) oligonucleotides for Nox4 markedly decreased NADPH-dependent reactive oxygen species (ROS)-producing activity. Dominant negative Rac1 (N17Rac1) as well as AS Nox4 inhibited ROS generation in response to ANG II and AA, whereas constitutively active Rac1 stimulated ROS formation. Moreover, N17Rac1 blocked stimulation of NADPH oxidase activity by AA. N17Rac1 or AS Nox4 abolished ANG II- or AA-induced activation of the hypertrophic kinase Akt/PKB. In addition, AS Nox4 inhibited ANG II-induced protein synthesis. These data provide the first evidence that activation by AA of a Rac1-regulated, Nox4-based NAD(P)H oxidase and subsequent generation of ROS mediate the effect of ANG II on Akt/PKB activation and protein synthesis in MCs.

scholarly journals Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent α-helix of p22phox

2006 ◽  
Vol 396 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Ikuo Nobuhisa ◽  
Ryu Takeya ◽  
Kenji Ogura ◽  
Noriko Ueno ◽  
Daisuke Kohda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Regulatory Protein ◽  
Sh3 Domain ◽  
Alanine Scanning Mutagenesis ◽  
Catalytic Core ◽  
Intact Cells ◽  
Sh3 Domains ◽  
Phagocyte Nadph Oxidase ◽  
Phagocyte Oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151–160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161–164), adopting an α-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

scholarly journals RAB11-Mediated Trafficking and Human Cancers: An Updated Review

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 26
Author(s):  
Elsi Ferro ◽  
Carla Bosia ◽  
Carlo C. Campa
Keyword(s):  
Plasma Membrane ◽  
Normal Cell ◽  
Vesicular Trafficking ◽  
Small Gtpase ◽  
Cell Physiology ◽  
Biological Mechanisms ◽  
Cellular Processes ◽  
Human Cancers ◽  
Toxic Materials ◽  
Dependent Pathway

Many disorders block and subvert basic cellular processes in order to boost their progression. One protein family that is prone to be altered in human cancers is the small GTPase RAB11 family, the master regulator of vesicular trafficking. RAB11 isoforms function as membrane organizers connecting the transport of cargoes towards the plasma membrane with the assembly of autophagic precursors and the generation of cellular protrusions. These processes dramatically impact normal cell physiology and their alteration significantly affects the survival, progression and metastatization as well as the accumulation of toxic materials of cancer cells. In this review, we discuss biological mechanisms ensuring cargo recognition and sorting through a RAB11-dependent pathway, a prerequisite to understand the effect of RAB11 alterations in human cancers.

EROS is required for phagocyte NADPH oxidase function in humans and its deficiency causes Chronic Granulomatous Disease

2018 ◽  
Author(s):  
David C. Thomas ◽  
Louis-Marie Charbonnier ◽  
Andrea Schejtman ◽  
Hasan Aldhekri ◽  
Eve Coomber ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Reactive Oxygen ◽  
Human Cells ◽  
List Type ◽  
Oxygen Species ◽  
Granulomatous Disease ◽  
Phagocyte Nadph Oxidase

AbstractThe phagocyte respiratory burst is mediated by the phagocyte NADPH oxidase, a multi-protein subunit complex that facilitates production of reactive oxygen species and which is essential for host defence. Monogenic deficiency of individual subunits leads to chronic granulomatous disease (CGD), which is characterized by an inability to make reactive oxygen species, leading to severe opportunistic infections and auto-inflammation. However, not all cases of CGD are due to mutations in previously identified subunits. We recently showed that Eros, a novel and highly conserved ER-resident transmembrane protein, is essential for the phagocyte respiratory burst in mice because it is required for expression of gp91phox-p22phox heterodimer, which are the membrane bound components of the phagocyte NADPH oxidase. We now show that the function of EROS is conserved in human cells and describe a case of CGD secondary to a homozygous EROS mutation that abolishes EROS protein expression. This work demonstrates the fundamental importance of EROS in human immunity and describes a novel cause of CGD.Clinical ImplicationsChronic granulomatous disease is caused by an inability to make reactive oxygen species via the phagocyte NADPH oxidase. Mutations in C17ORF62/EROS, which controls gp91phox- p22phox abundance, are a novel cause of chronic granulomatous disease.Key MessagesThe murine gene, Eros, is known to regulate abundance of gp91phox-p22phox heterodimer and Eros deficient mice are susceptible to infectionWe show that the function of EROS is conserved in human cells and that a homozygous mutation in EROS causes chronic granulomatous disease

scholarly journals Tetratricopeptide Repeat (TPR) Motifs of p67phoxParticipate in Interaction with the Small GTPase Rac and Activation of the Phagocyte NADPH Oxidase

1999 ◽  
Vol 274 (35) ◽  
pp. 25051-25060 ◽  
Author(s):  
Hirofumi Koga ◽  
Hiroaki Terasawa ◽  
Hiroyuki Nunoi ◽  
Koichiro Takeshige ◽  
Fuyuhiko Inagaki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Small Gtpase ◽  
Tetratricopeptide Repeat ◽  
Phagocyte Nadph Oxidase ◽  
Gtpase Rac

Genetic Defects of Phagocyte Nadph Oxidase Activity and Activation

1989 ◽  
Vol 2 (2) ◽  
pp. 75-86
Author(s):  
P. Bellavite ◽  
Flavia Bazzoni ◽  
G. Scolaro ◽  
G. Poli ◽  
S. Dusi ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Protein A ◽  
Multicomponent System ◽  
Activation Mechanism ◽  
Molecular Entity ◽  
Genetic Defects ◽  
Phagocyte Nadph Oxidase ◽  
Key Enzyme ◽  
Molecular Nature

NADPH oxidase is the key enzyme of the free radical-generating oxidative matabolism of phagocytes. Work from our and other's laboratories has recently established that the oxidase is not a single molecular entity, but it is a multicomponent system including a NADPH-binding protein, a flavoprotein, a b-type cytochrome and other unidentified factors. A working model of the molecular nature and of the activation mechanism of phagocyte NADPH oxidase is here proposed. This model is suitable for the study and the classification of the molecular pathology of the oxidase system. The various genetic defects of the NADPH oxidase, that are the cause of chronic granulomatous disease, (CGD) are here presented and discussed.

Microtubule Dynamics and Organelle Transport in Reticulomyxa

1990 ◽  
Vol 48 (3) ◽  
pp. 194-195
Author(s):  
Yih-Tai Chen ◽  
Ursula Euteneuer ◽  
Ken B. Johnson ◽  
Michael P. Koonce ◽  
Manfred Schliwa
Keyword(s):  
Plasma Membrane ◽  
Light Microscopy ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Model Systems ◽  
Organelle Transport ◽  
Biochemical Characteristics ◽  
Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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