Literature summary for 1.6.3.1 extracted from

Lassegue, B.; San Martin, A.; Griendling, K.K.
Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system (2012), Circ. Res., 110, 1364-1390.

Search for more literature for 1.6.3.1

Activating Compound

Activating Compound	Comment	Organism	Structure
angiotensin II	stimulates Nox1	Homo sapiens
betaPix	a Rac1 guanine nucleotide exchange factor, appears to be constitutively bound to Nox1 and essential for its activity	Homo sapiens
H2O2	Nox5 can be upregulated and activated by minute concentrations of hydrogen peroxide	Homo sapiens
heat shock protein 90	binding of heat shock protein 90 to the C-terminus of Nox5 appears to stabilize the protein and enhance expression and activity	Homo sapiens
interleukin-1beta	stimulates Nox1	Homo sapiens
additional information	agonists appear to stimulate Nox1 in specific locations, thus determining where superoxide is produced: extracellularly by muscarinic agonists and thrombin, in endosomes by IL-1beta and TNF-alpha, both inside and outside cells by angiotensin II. Nox activators comprise p67phox and the structurally similar Noxa1. In colon the cytosolic subunits p47phox and p67phox are not expressed and are replaced by Noxo1 and Noxa1. Besides p47phox, other possible organizers include Tks4 and Tks5, two Src substrates with a PX domain and multiple SH3 domains capable of binding p22phox and Noxa1, but not p67phox. Cdc42 cannot activate Nox1	Homo sapiens
NOXA1	in contrast to its Noxo1 partner, Noxa1 activity appears to be tightly regulated. Noxa1 contains four Rac-binding TPR motifs, a Nox activation domain and an SH3 domain that interacts with the prolinerich region of an organizer subunit, But the p40phox-binding PB1 domain is not well conserved and the SH3 domain in the middle of the molecule is missing. Phosphorylation of Noxa1 by protein kinase A favors binding to 14-3-3 and dissociation from Nox1, whereas other kinases appear to decrease Noxa1 affinity for Rac1 and Nox1. In contrast, phosphorylation of Noxa1 by Src on tyrosine 110 increases Nox1 activity	Homo sapiens
NOXO1	In contrast to its Noxo1 partner, Noxa1 activity appears to be tightly regulated. Unlike p47phox, because Noxo1 lacks an autoinhibitory domain, it is thought to constitutively bind the cytochrome, but similar to p47phox, Noxo1 facilitates oxidase assembly by binding both an activator subunit and p22phox. The proline-rich region of Noxo1 binds to an SH3 domain of the activator, whereas the tandem SH3 domains of Noxo1 bind to the proline-rich region of p22phox. Noxo1 also binds to the dehydrogenase domain of Nox1. The PX domain of Noxo1 provides an essential affinity for membrane phosphoinositides	Homo sapiens
p67phox	activation domain of p67phox triggers FAD reduction by Nox2. P40phox appears to increase oxidase activity in cooperation with p47phox not by inducing translocation to the membrane, but by retaining the oxidase at the phagosome	Homo sapiens
Poldip2	reactive oxygen species production is enhanced by the multifunctional Poldip2, which also interacts with p22phox, presumably at the beginning of the cytosolic C-terminus, upstream of the region dispensable for Nox4 activity	Homo sapiens
Rac1	in addition to cytosolic organizers and activators, Nox1 also requires Rac1 for activity. Rac1 interacts directly with the C-terminus of Nox1, even in the absence of Noxa1. Nox1 is stimulated by constitutively active Rac1 and inhibited by Rac1 knockdown. Rac1 provides a crucial mechanism for activation by agonists, particularly in cells that exclusively express Nox1/Noxo1/Noxal. Rac1 does not activate Nox4 in transfected cells. Rac1 may participate in Nox5 activation	Homo sapiens
thrombin	stimulates Nox1 extracellularly	Homo sapiens
TNF-alpha	stimulates Nox1	Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
additional information	Nox5 is unaffected by expression or knockdown p22phox intransfected cells. The cytosolic N-terminal segment, containing 4 calcium binding EF-hands is missing in Nox5S, a short calcium-insensitive variant, which is the dominant isoform in carcinoma cells, and expressed together with the long Nox5L in endothelial cells. Replacing the first transmembrane domain of Nox4 by that of Nox1, or altering the last extracellular loop of Nox4, makes it produce superoxide, rather than peroxide. Deletion of the NADPH binding domain produces a dominant-negative Nox4. Nox1-Nox4 and Nox2-Nox4 chimeras are active without transfection of cytosolic subunits, whereas the opposite Nox4-Nox2 chimera requires activation. Mutation of the proline-rich domain of p22phox required for docking organizers does not affect Nox4 activity	Homo sapiens
P437H	the mutation in the canonical NADPH binding motif of Nox4, analogous to the Nox2 mutation of a CGD patient, abolishes activity	Homo sapiens
R96E	Nox4 is inhibited by an R96E mutation in the cytosolic B loop, a region of the amino-terminal domain that interacts with the NADPH binding site	Homo sapiens

General Stability

General Stability	Organism
association with p22phox is required for Nox4 activity, the 2 proteins stabilize each other	Homo sapiens
Nox1 activity is dependent on chaperones Hsp90 and PDI, which appear to be necessary not only for protein folding after synthesis, but also to maintain enzyme stability	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
apocynin	inhibition of NOX1 and NOX2	Homo sapiens
Cdc42	a small monomeric GTPase, competitive inhibitor of Nox2, might also be a competitive inhibitor of Nox1	Homo sapiens
GK-136901	inhibition of NOX1 and NOX4	Homo sapiens
ML171	inhibition NOX1	Homo sapiens
additional information	V204A mutant is a competitive inhibitor of wild-type p67phox	Homo sapiens
Nox2ds-tat	inhibition of NAOX1 and NOX2	Homo sapiens
Plumbagin	inhibition of NOX4	Homo sapiens
VAS2870	inhibition of NOX2 and NOX4	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
caveola	NOX1	Homo sapiens	5901	-
cytoplasm	NOX4	Homo sapiens	5737	-
endoplasmic reticulum	NOX4	Homo sapiens	5783	-
endosome	-	Homo sapiens	5768	-
endosome	NOX1, Nox1 stimulation in endosomes is dependent on ClC-3, where this ion exchanger is required to balance the electrogenic activity of the enzyme	Homo sapiens	5768	-
focal adhesion	NOX4	Homo sapiens	5925	-
membrane	Nox1, like Nox2, associates with p22phox to form a membrane-bound cytochrome	Homo sapiens	16020	-
mitochondrion	NOX4	Homo sapiens	5739	-
additional information	Nox4 location varies according to cell type, different Nox4 isoforms may be present in specific subcellular locations	Homo sapiens	-	-
nuclear pore complex	NOX2	Homo sapiens	-	-
nucleus	NOX4	Homo sapiens	5634	-
plasma membrane	NOX1, NOX5, NOX2, and NOX4. The signal peptide at the N-terminus of Nox1 is important for localization at the plasma membrane, as it is prevented by replacement with the N-terminus of Nox4. A polybasic region in Nox5 with affinity for phosphoinositides may favor translocation to the plasma membrane and extracellular superoxide release	Homo sapiens	5886	-
stress fiber	NOX4	Homo sapiens	1725	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	an increase in cytosolic calcium concentration triggers high superoxide production by Nox5. Calcium induces binding of the N-terminal domain of Nox5 to the dehydrogenase domain, thus relieving autoinhibition. Two mechanisms may increase Nox5 sensitivity, allowing activation by resting calcium concentrations: (1) calcium-dependent binding of calmodulin to another site in the dehydrogenase domain157 and (2) phosphorylation of serine and threonine residues by protein kinase C and calcium/calmodulin-dependent kinase II	Homo sapiens
Fe2+	heme iron	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Homo sapiens	Nox4 can produce a higher hydrogen peroxide to superoxide ratio than Nox1 and Nox2. NOX2-produced superoxide can be released outside the cell when Nox2 is located at the plasma membrane, thus allowing it to intercept, e.g., with endothelial-derived nitric oxide before it reaches the adjacent smooth muscle cell layer in vessels	?	-	?
NADH + H+ + O2	Homo sapiens	2 electrons are transferred from cytosolic NADPH to FAD and in succession across the membrane, via redox changes in heme irons. Finally, each electron reduces a molecule of oxygen to a superoxide radical, which is subsequently released outside the cell or in a topologically equivalent compartment, such as a vesicle lumen	NAD+ + H2O2	-	?
NADPH + H+ + O2	Homo sapiens	-	NADP+ + H2O2	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	3 loops of Nox2 are extracellular and include consensus asparagine glycosylation sites. Phosphorylation of p67phox, mediated by PKCdelta, ERK2 and p38 MAPK, increases superoxide production by Nox2	Homo sapiens
phosphoprotein	phosphorylation of serine 461 by protein kinase A, serine 282 by ERK1/2 or p38 MAPK, or serine 172 by PKA or PKC, decreases ROS production by Nox1. Phosphorylation of p40phox on serine and threonine by PKC is increased during stimulation, particularly at threonine 154	Homo sapiens

Reaction

Reaction	Comment	Organism	Reaction ID
NAD(P)H + H+ + O2 = NAD(P)+ + H2O2	a cytosolic C-terminal dehydrogenase domain includes an FAD cofactor and an NADPH substrate binding site. On activation, electrons are transferred from NADPH to FAD and across the membrane, via heme irons, to molecular oxygen, thus producing superoxide anion, which can be dismutated into hydrogen peroxide	Homo sapiens	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
blood vessel	-	Homo sapiens	-
carcinoma cell	the cytosolic N-terminal segment, containing 4 calcium binding EF-hands is missing in Nox5S, a short calcium-insensitive variant, which is the dominant isoform in carcinoma cells, and expressed together with the long Nox5L in endothelial cells. Nox5S may be constitutively active or be a competitive inhibitor of calcium-dependent activation when present in the same tetrameric complex as Nox5L	Homo sapiens	-
cardiomyocyte	in ischemic cardiomyocytes, Nox2 is upregulated in the cytosol and targeted to the nuclear pore complex	Homo sapiens	-
cardiovascular system	-	Homo sapiens	-
colon	Nox1 is most highly expressed in colon epithelium. In colon the cytosolic subunits p47phox and p67phox are not expressed and are replaced by Noxo1 and Noxa1	Homo sapiens	-
endothelial cell	-	Homo sapiens	-
epithelium	-	Homo sapiens	-
fibroblast	adventitial and cardial	Homo sapiens	-
lung	five splice variants of Nox4, named Nox4A through E, are found in lung epithelial cells	Homo sapiens	-
additional information	Nox5 expression is restricted to fewer tissues. Nox4 activity is constitutive, addition of cytosol to membrane fractions in transfected cells does not increase Nox4 activity, also transfection of organizer and activator subunits does not increase reactive oxygen species production	Homo sapiens	-
phagocyte	high expression level of Nox2	Homo sapiens	-
vascular smooth muscle cell	-	Homo sapiens	-
vascular wall	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	Nox4 can produce a higher hydrogen peroxide to superoxide ratio than Nox1 and Nox2. NOX2-produced superoxide can be released outside the cell when Nox2 is located at the plasma membrane, thus allowing it to intercept, e.g., with endothelial-derived nitric oxide before it reaches the adjacent smooth muscle cell layer in vessels	Homo sapiens	?	-	?
NADH + H+ + O2	-	Homo sapiens	NAD+ + H2O2	-	?
NADH + H+ + O2	2 electrons are transferred from cytosolic NADPH to FAD and in succession across the membrane, via redox changes in heme irons. Finally, each electron reduces a molecule of oxygen to a superoxide radical, which is subsequently released outside the cell or in a topologically equivalent compartment, such as a vesicle lumen	Homo sapiens	NAD+ + H2O2	-	?
NADPH + H+ + O2	-	Homo sapiens	NADP+ + H2O2	-	?

Subunits

Subunits	Comment	Organism
More	Nox2 structure homology modeling, and structure and activation mechanism of NOX proteins, overview. Nox2 is composed of two main domains of equal sizes with very different properties. The amino-terminal moiety includes six transmembrane alpha-helices I-VI connected by 5 loops A-E. The cytosolic carboxy-terminal moiety of Nox2 constitutes a dehydrogenase domain that includes consensus binding sites for its NADPH substrate and FAD cofactor. NOX2 p40phox is composed of an N-terminal PX domain, a central SH3 domain and a C-terminal PB1 domain. The C-terminal PB1 domain of p40phox interacts with the PB1 domain of p67phox	Homo sapiens

Synonyms

Synonyms	Comment	Organism
gp91phox	-	Homo sapiens
NADPH oxidase	-	Homo sapiens
NOX1	-	Homo sapiens
Nox2	-	Homo sapiens
Nox4	-	Homo sapiens
NOX5	-	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
FAD	a dehydrogenase domain is binding FAD and NADPH, Nox1, Nox2, and Nox4 requires FAD and NADPH. Nox5 is similar to other Nox enzymes, with 6 transmembrane helices expected to bind 2 hemes and a cytosolic dehydrogenase domain including FAD and NADPH binding sites	Homo sapiens
heme	4 conserved histidine residues in transmembrane helices III and V, thought to coordinate 2 heme molecules, mutation of these histidines abolishes binding to p22phox	Homo sapiens
NADH	-	Homo sapiens
NADPH	a dehydrogenase domain is binding FAD and NADPH, Nox1, Nox2, and Nox4 requires FAD and NADPH. Nox5 is similar to other Nox enzymes, with 6 transmembrane helices expected to bind 2 hemes and a cytosolic dehydrogenase domain including FAD and NADPH binding sites	Homo sapiens

Expression

Organism	Comment	Expression
Homo sapiens	NOX1 is upregulated by angiotensin II, PDGF, PGF, LDL, TNF-alpha, oscillatory shear stress BMP4, aldosterone plus salt, IFN-gamma, ET-1, T3, urokinase8, oxidized LDL, and vascular injury. NOX2 is upregulated by angiotensin II, ET-1, TGF-beta, IFN-gamma, oxidized LDL oscillatory shear stress, aldosterone plus salt, Ischemia, and vascular injury.NOX4 is upregulated by TGF-beta, thromboxane, TNF-alpha. IFN-gamma, urotensin, urokinase, oscillatory shear stress, hypoxia, hyperoxia vascular injury. NOX5 iss upregulated by angiotensin II, ET-1, thromboxane A2, TNF-alpha, atherosclerosis. Nox5 can be upregulated and activated by minute concentrations of hydrogen peroxide. In ischemic cardiomyocytes, Nox2 is upregulated in the cytosol and targeted to the nuclear pore complex	up

General Information

General Information	Comment	Organism
malfunction	in pathological circumstances, excess Nox2 can lead to oxidative stress and disease development. NOX2 V204A mutant is a competitive inhibitor of wild-type p67phox. Binding of the PB1 domain of NOX2 to p40phox is abolished by a K355A mutation in NOX2. Depletion or mutation of p40phox impairs reactive oxygen species production in neutrophils and endothelial cells. Upregulation of Nox1 can lead to oxidative stress in the cardiovascular system	Homo sapiens
metabolism	tight regulation, critical to avoid excessive production of deleterious superoxide, is evident from the large number of proteins involved in oxidase assembly. These include Nox2 itself, p22phox, p47phox, p67phox, and p40phox, all essential subunits whose mutations can cause CGD Also crucial is Rac GTPase, which binds p67phox and the dehydrogenase domain of Nox2. In the resting state, Nox2 and p22phox form an inactive membrane complex known as cytochrome b558. Product superoxide is the first reactive oxygen species in a cascade of metabolites including hydrogen peroxide and peroxynitrite	Homo sapiens
additional information	Nox5 is similar to other Nox enzymes, with 6 transmembrane helices expected to bind 2 hemes and a cytosolic dehydrogenase domain including FAD and NADPH binding sites, but neither Nox5 isoform appears to require cytosolic subunits or p22phox. Presence of an additional cytosolic N-terminal segment, containing 4 calcium binding EF-hands in Nox5. Nox4 activity is constitutive, isozyme Nox4D appears to be fully active, although it lacks most of the transmembrane domain, it might retain activity by coupling to electron acceptors, such as cytochrome c in mitochondria, which might also be an alternative route of hydrogen peroxide formation by full-length Nox4. Nox4, Nox1 and Nox2 bind to p22phox, the interaction is abolished by mutation of heme-binding histidine 115. Nox2 is composed of two main domains of equal sizes with very different properties. The amino-terminal moiety includes six transmembrane alpha-helices I-VI connected by 5 loops A-E. Because both N- and C-termini are cytosolic, 3 loops are extracellular and include consensus asparagine glycosylation sites, whereas the other 2 are intracellular and accessible to cytosolic regulators. The cytosolic carboxy-terminal moiety of Nox2 constitutes a dehydrogenase domain that includes consensus binding sites for its NADPH substrate and FAD cofactor, activation domain of p67phox triggers FAD reduction by Nox2. A charge compensation mechanism, required to balance electron transport by Nox2 and sustain its activity, is provided by a voltage-gated proton channel8 and the chloride/proton antiporter ClC-3. The first SH3 domain of NOX2 increases oxidase activity, the NOX2 PB1 domain allows binding to p40phox. The C-terminal SH3 domain of p67phox of NOX2 is responsible for binding the proline-rich region of p47phox and therefore allows p67phox translocation to the membrane after activation. The C-terminal PB1 domain of p40phox interacts with the PB1 domain of p67phox. Nox1, like Nox2, associates with p22phox to form a membrane-bound cytochrome	Homo sapiens
physiological function	NADPH oxidase enzymes are critical mediators of cardiovascular physiology and pathophysiology. They are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels, and proteins that regulate the cytoskeleton. On activation, Nox2 uses NADPH to reduce molecular oxygen to superoxide anion, which, in concert with its metabolites, is used by phagocytes to destroy invading microorganisms. Nox organizers include both p47phox and its homologue, Noxo1, whereas Nox activators comprise p67phox and the structurally similar Noxa1. Because Nox2 and Nox1 are closely related, both enzymes can be activated in transfected cells by various organizer and activator pairs. Nox1 plays a host defensive role in colon epithelium. primary biochemical function of vascular Nox1 is superoxide production, which is then rapidly converted to hydrogen peroxide. The moderate physiological activity of Nox1, compared with the phagocytic Nox2, can be attributed to its low expression as well as specific regulatory subunits and signaling cascades. Nox4 is a constitutively active enzyme mostly regulated by transcription. Role of enzyme complex component p22phox, detailed overview. Nox4 can produce a higher hydrogen peroxide to superoxide ratio than Nox1 and Nox2. Isozyme Nox5S may be constitutively active or be a competitive inhibitor of calcium-dependent activation when present in the same tetrameric complex as Nox5L	Homo sapiens

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Release 2023.1 (January 2023)