pathway [173, 176]. To determine whether rotenone- induced autophagy was Nox2 dependent, Pal and col- leagues used the human neuroblastoma SH-SY5Y cell line. They observed that short exposure to rotenone (0.5 μM; 6 h) results in a ~2-fold increase in reactive oxygen species generation, impairs autophagic flux and promotes protein accumulation. Pre-incubation with the Nox2 docking se- quence (Nox2ds)-tat significantly attenuates the levels of LC3 and p62 proteins, indicating that the effect on autoph- agy is mediated by Nox-2 dependent reactive oxygen spe- cies. When SH-SY5Y dopaminergic cells are exposed to higher doses of rotenone (10 μM; 24 h), a ~3.5 increase in reactive oxygen species generation is observed compared to untreated cells. Nox2-ds abolish Nox2-generated reactive oxygen species generation (measured using the Nox2- specific redox sensor p47-roGFP) while total intracellular reactive oxygen species (measured using DCF-DA) is par- tially, but not completely, inhibited . This suggests that 10 μM rotenone for 24 h stimulates reactive oxygen species generation not only through Nox2, but also possibly from mitochondria. Importantly, preincubation with Nox2-ds par- tially attenuates LC3 and p62 protein levels and protects against rotenone-dependent upregulation in apoptotic sig- naling . These data highlight a novel mechanism by which Nox2-dependent oxidative stress could promote the pathogenesis of PD. Noteworthy, Nox4 was also found to promote autophagy and survival in cancer cells  and in cardiomyocytes in response to nutrient deprivation and is- chemia , while no studies have been performed yet in the neuronal context. Thus, this emerging evidence indicates the importance of NADPHoxidases in the regulation of au- tophagy. Future studies are warranted to delineate the asso- ciation between NADPHoxidases-dependent impaired autophagy, mitochondria dysfunction and cell death in PD.
Animal models of NOX-DUOX2 deficiency do not recapitulate human IBD susceptibility-
Altogether the above studies suggest that NOX2, NOX1 and DUOX2 loss-of-function is a susceptibility factor for IBD development in humans. However, although NOX2- and DUOXA- deficient mice recapitulate human CGD and congenital hypothyroidism, respectively (56, 131), these mice and NOX1-deficient mice do not develop spontaneous intestinal inflammation. Even under conditions where intestinal inflammation is induced, such as in dextran sulfate sodium (DSS) and trinitrobenzenesulfonic acid (TNBS) models, the consequences of the lack of NADPHoxidases are still unclear, as it can be a worsening factor in some models, but be protective in others (7). Therefore, the role of the NADPHoxidases in IBD might be context- specific and a second hit related to genetics, environment or microbiota may be required to observe significant clinical consequences. In contrast, loss of function mutations of IL-10 in patients with VEO-IBD (86) can be recapitulated in IL-10-deficient mice who develop spontaneous general enterocolitis under conventional breeding (83, 89).
NADPHoxidases are one of the main enzymatic sources of ROS in cells and the relative expression of NOX 1 and NOX 4 was linked to melanoma cell survival [10, 26]. To identify which isoform(s) contributed to ROS levels in A375 cells, we tested the kinetics of eNOS, NOX1, NOX2, NOX4 expression as a function of time by western blots (Figure 1C). After 1 hour in the presence of NS1, a transient NOX1 increase was observed, which decreased after 24 hours and beyond. NOX2 and NOX4 strongly decreased, reaching a very low level after 72 hours. In agreement with a cross-talk between eNOS and NOX4, eNOS level increased . NOX1 and NOX4 also decreased in SK-Mel28 and 1205 Lu cell lines as in A375 but smaller effects were observed in 1205 Lu. Only small changes in eNOS levels were observed in these cells as compared to the large increase observed in A375 cells (Figure 1C–1E). Since NOX4 is a self-sufficient enzyme, a decrease of NOX4 level directly reflects the ROS levels. Previous studies also showed that NS1 inhibits e/nNOS and NOX4 [19, 24]. The observed changes in NOX1 and NOX2 levels could not be directly linked with their ROS production since both proteins require additional proteins for full activation. To further confirm a direct inhibition of NOX by NS1, we used RAW 264.7 macrophage cells treated with PMA. PMA- activated NOX2- released superoxide anion radicals that were trapped by the cyclic nitrone DEPMPO. The nitroxide DEPMPO-OOH spin- adduct was detected by EPR spectroscopy. The addition of increasing amounts of NS1 to the PMA-stimulated RAW cells led to half-inhibition of the rates of DEPMPO- OOH spin-adduct formation with an IC 50 = 120 + 25 μM (Figure 2A). These results show NS1 inhibition of NOX (NOX2) activity at the cell membrane [27, 28].
et al., 2005). However, the precise mechanisms by which
celastrol achieves these benefits is not fully known.
The NOX enzymes are a family of ROS-generating nicoti- namide adenine dinucleotide phosphate (NADPH) oxidases (NOX) comprising seven members (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2). All NOX isoforms func- tion as electron transporters and catalyse the reduction of molecular oxygen to generate the superoxide anion. Whereas NOX enzymes are expressed throughout the body, each isoform possesses a unique pattern of tissue and subcellular distribution and there is little redundancy among NOX iso- forms. The role of NOX enzymes in oxidative stress-related pathologies is increasingly recognized, in particular for car- diovascular and neurodegenerative diseases, and they repre- sent a promising pharmacological target (Lambeth et al., 2008).
NADPHoxidases, Nox: new isoenzymes family
NADPHoxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acqui- red from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophy- siological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the func- tion of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes. ‡ REMERCIEMENTS
2 O 2 ), by means of the single-electron reduction of molecular oxygen using NADPH as the electron donor (1). This family has been deﬁned based on the structural homol- ogy of their members with gp91 PHOX (now renamed NOX2), the catalytic subunit of the phagocyte NADPH oxidase. In phagocytes, such as neutrophils and mono- cytes/macrophages, this enzyme system plays a critical role in antimicrobial host defenses through the robust production of highly cytotoxic ROS in a phenomenon known as the “respiratory burst” (2). The enzymatic activity of the phagocyte NADPH oxidase has been studied for .30 yr, and its structure and regulation have been studied extensively over the past decade (3, 4). The phagocyte NADPH oxidase is a multicompo- nent enzyme complex constituted of several cytosolic and membrane-bound components: in the cytosol, p47 PHOX (phox for phagocyte oxidase), p67 PHOX , p40 PHOX , and a small GTPase Rac1 or Rac2; and in the membrane, gp91 PHOX and p22 PHOX , which together comprise cytochrome b558. The catalytic core of the enzyme gp91 PHOX contains both ﬂavin and heme groups, which are necessary for electron transfer from NADPH to oxygen. It is only recently that several homologs of gp91 PHOX have been described in many nonphagocytic cells within various tissues. This new family of NADPHoxidases (NOX) encompasses 7 members: NOX1, NOX2 (gp91 PHOX ), NOX3, NOX4, NOX5, DUOX1, and DUOX2 (5–9). They have been shown to mediate various biological functions, includ- ing innate immunity, angiogenesis, cell growth and
Our group has long studied NADPHoxidases (NOXes), a family of complex multidomain, and sometimes multicom- ponent, enzymes ( 12–14 ). NOXes are transmembrane flavo- cytochromes that catalyze the production of reactive oxygen species associated with diverse downstream physiological outcomes ranging from hormone synthesis, balance, cardio- vascular tone regulation, and innate immunity. The range of pathologies impacted by NOX make this family a drug target of high value for pharmaceutical companies. We are particularly interested in the neutrophilic NOX complex essential to innate immunity in humans. In the phagocytic cell, NOX2 is activated to generate the ‘‘respiratory burst,’’ producing superoxide anion and initiating a cascade of reac- tions leading to the production of microbicidal hypochlorite. Its activation includes the assembly of several cytosolic components—p67 phox , p47 phox , p40 phox , and Rac—onto the membranous flavocytochome b 558 . The latter is composed of the catalytic subunit NOX2 and an essential accessory transmembrane subunit p22 phox ( Fig. 1 ). Struc- tural information exists for domains or entire proteins of the cytosolic factors ( 15–22 ), along with some data regarding association between these cytosolic factors ( 23 ). For the transmembrane component of this complex, howev- er, structural data are scarce, and very little is known about how assembly leads to enzyme activity. The SANS signal
part of p22 phox , reflecting an intermediate conformation
between the autoinhibited and activated forms.
NADPHoxidases (Nox) and Dual oxidases (Duox) are mul- tienzymatic complexes found in many cell types (1) that play a wide range of physiological roles (2). The seven different iso- forms of these enzymatic complexes differ in their membrane redox components: Nox1 to Nox5, Duox1 and Duox2. Despite this molecular heterogeneity, they all share the common fea- ture of reactive oxygen species production, which is either con- stitutive (Nox4) or inducible by cytosolic factors (Nox1, Nox2, and Nox3) or Ca 2⫹ (Nox5, Duox1, and Duox2).
4 1. Introduction
Inflammation is a normal host defense mechanism which protects the host from infection through pathogen killing. This inflammatory response is well regulated in order to protect the host against excessive damage and is accompanied by an increase in body temperature (hyperthermia). Normal body temperature generally does not exceed 37.9 °C. Many causes of fever can be encountered (tumor, rheumatic disorders, granulomatous diseases …) but infections are the most frequent causes of a brief fever which corresponds to an elevation of the body temperature above 38.2 °C. Neutrophils are one of the first phagocyte cells which are sought to fight pathogens by using its efficient enzymatic and killing machinery, which include the NADPH oxidase complex. In phagocyte cells, the NADPH oxidase complex converts the chemical reducing equivalent of NADPH into superoxide anions (O 2 •- ) in the intraphagosomal space. The superoxide anion is the precursor of the so-called Reactive Oxygen Species (ROS) which serve as microbicide weapons. The physiological importance of the phagocyte NADPH oxidase is evidenced by an immunodeficiency disease called chronic granulomatous disease (CGD), in which defective microorganism killing entails recurrent and severe infections (reviewed in ). Studies over the last decade revealed homologs of phagocyte NADPHoxidases forming the NOX family [2, 3], which enlarged considerably the number of its physiological functions (inflammatory response, post-translational proteins processing, inter- and intra-cellular signaling, regulation of endothelial function, regulation of gene expression and cell differentiation…).
The role of ROS in inflammation is complex, as they play a role in tissue damage as well as a positive role in the resolution of in- flammation ( Nathan and Cunningham-Bussel, 2013 ). Under certain circumstances, ROS may be beneficial in promoting wound healing, and NADPH oxidase defects in patients with chronic granuloma- tous disease ( Segal et al., 2011 ) and in animal models ( Morgenstern et al., 1997 ) lead to aberrant inflammation. Chemical NADPH oxi- dase inhibitors such as apocynin have antiinflammatory effects in arthritis models ( ’t Hart et al., 1990 ) and reduce ethanol-induced liver inflammation in mice ( Gustot et al., 2006 ). Disrupting the gp91phox –p47phox interaction using a peptide inhibits NADPH oxidase ( Rey et al., 2001 ). Likewise, targeting PCNA could be a strategy to dampen this neutrophil contribution to inflammation. In a therapeutic setting, chemical targeting of NADPH oxidase protein interaction potentially allows temporary and dose-dependent re- duction of ROS, favoring the dampening of inflammation compared with the complete loss of NADPH oxidase function. It should be noted that in the current study along with the antiinflammatory effect, short T2AA treatment not only did not interfere with epi- thelial cell proliferation in TNBS-induced colitis, but it significantly enhanced epithelium regeneration. Our data strongly suggest that T2AA in this setting primarily targeted inflammatory neutrophils and did not demonstrate a negative effect on regenerating tissue at the site of inflammation. Here we show that T2AA also reduced neutrophil survival as an added antiinflammatory mechanism to limit neutrophil-mediated tissue damage, as previously demon- strated for roscovitine, a cyclin-dependent kinase inhibitor ( Rossi et al., 2006 ). Down-regulating neutrophil activation and promoting neutrophil apoptosis represent important mechanisms in inflam- mation resolution ( Serhan et al., 2007 ; Jones et al., 2016 ). None- theless, evaluation of the therapeutic value of T2AA as an antiinflammatory drug will require investigation of its effects on other models of inflammation. Notably, T2AA also did not adversely affect neutrophil progenitor survival. As previously mentioned, chemical strategies are reversible, and moderate reduction of NADPH oxidase function may provide antiinflammatory effects without compromising antipathogen function.
245. Pethe K, Bifani P, Jang J, Kang S, Park S, Ahn S, Jiricek J, Jung J, Jeon HK, Cechetto J, Christophe T, Lee H, Kempf M, Jackson M, Lenaerts AJ, Pham H, Jones V, Seo MJ, Kim YM, Seo M, Seo JJ, Park D, Ko Y, Choi I, Kim R, Kim SY, Lim S, Yim SA, Nam J, Kang H, Kwon H, Oh CT, Cho Y, Jang Y, Kim J, Chua A, Tan BH, Nanjun- dappa MB, Rao SP, Barnes WS, Wintjens R, Walker JR, Alonso S, Lee S, Kim J, Oh S, Oh T, Nehrbass U, Han SJ, No Z, Lee J, Brodin P, Cho SN, Nam K, and Kim J. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat Med 19: 1157–1160, 2013. 246. Pils D and Schmetterer G. Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803. FEMS Microbiol Lett 203: 217–222, 2001.
Abstract: Copper amine oxidases (CuAOs) are ubiquitous enzymes which play a vital role in the physiology and pathology of mammals in controlling the metabolism of various primary monoamines, diamines and polyamines of endogenous or xenobiotic origin. CuAOs, which belong to the
quinoproteins family, possess two cofactors: tightly bound CuII and a quinone residue which catalyzes the oxidative deamination of primary amines with concomitant production of aldehyde, ammonia and hydrogen peroxide through a « ping pong » mechanism. Interest in human enzymes of the CuAOs class has increased in recent years driven by the discovery that the human vascular adhesion protein-1 (VAP-1), which regulates leucocyte trafficking and glucose transport, is a CuAO enzyme. The activities of CuAOs are increased in various human disorders, such as diabetes, Alzheimer's disease and many inflammation-associated diseases leading to the overproduction of toxic metabolites, especially hydrogen peroxide and aldehyde compounds. As most consequences are pathological, effective and selective inhibitors of CuAOs should be of great interest as therapeutic agents. Nevertheless, the utilization of CuAOs to generate enzymatic toxic products into cancer cells for selective in situ killing deserves to be considered in cancer therapy. This paper briefly highlights recent progress in the study of physiological, pathological and molecular aspects of CuAOs in mammals. Furthermore, a small molecule that mimics the metabolic activity of CuAOs toward endogenous and exogenous amines is described because it could be used as a surrogate of enzymes for a preliminary screening of potential inhibitors of CuAO enzymes.
and C). Increased PMA-induced ROS production in PCNA- overexpressing cells was confirmed by 29,79-dichlorofluorescin diacetate (DCF-DA) FACS analysis. Notably, this increase in PMA-induced ROS production was abolished when PLB985 cells were transfected with PCNA fused to the SV40 nuclear locali- zation sequence (NLS), thereby excluding from the cytosol (Bouayad et al., 2012; Fig. 3, D and E). Transfection of PCNA siRNA efficiently down-regulated PCNA protein expression (Fig. 3 F) and significantly decreased ROS production measured by luminol chemiluminescence (CL) in response to PMA and OZ (Fig. 3, G and H). Likewise, we used dimethylformamide (DMF)- differentiated PLB985 cells overexpressing the protein p21/waf1 (Fig. 3 I), which can bind PCNA and inhibit its antiapoptotic function (Martin et al., 2016). DMF-differentiated PLB985- p21A45R cells have significantly decreased NADPH oxidase ac- tivity triggered by either PMA or OZ relative to the PLB985 cells expressing an empty plasmid (Fig. 3, J and K). Notably, the effect of inhibiting PCNA on NADPH oxidase function is not merely a consequence of loss of neutrophil viability. The decreased NADPH oxidase function in differentiated PLB985 cells by PCNA inhibition (by either siRNA or ectopic expression of p21/waf1) was evident at time points in the absence of significant cell death (Fig. S2, A and B). Altogether, these results demonstrated that cytosolic PCNA controlled NADPH oxidase –dependent ROS production in intact cells.
Pour cette étude, deux modèles ont été utilisés: des lapins témoins (LT) et des lapins avec une réaction inflammatoire (LRI) afin de diminuer l’activité et l’expression du CYP. Six groupes contenant chacun cinq lapins ont été utilisés: un groupe sans CS et deux groupes qui ont pris oralement dans l’eau approximativement 20.5 mg/kg/jour de CS pendant 20 et 30 jours; les lapins des trois groupes restants ont pris du CS comme décrit plus haut, mais ont reçu 5 ml sous-cutanées de térébenthine afin de produire une réaction inflammatoire aseptique (RIA) deux jours avant leur sacrifice, c’est-à-dire aux jours -2, 18 et 28. Les hépatocytes ont été isolés pour évaluer l’activité et l’expression du CYP3A6, CYP1A2 et NADPH et aussi le ARNm de ces protéines. In vitro, nous avons étudié l’effet de différentes concentrations de CS-disaccharides sulfatés, 4S, 6S, et 4,6S de CS, sur l’activité et l’expression du CYP1A2 et du CYP3A6. Pour documenter la présence de la réaction inflammatoire, nous avons mesure les mucoprotéines, dans le sérum des lapins avec une réaction inflammatoire. Aussi nous avons mesuré la présence de l’oxide nitrique (NO) chez les hépatocytes de lapins contrôles et chez les hépatocytes des lapins avec une réaction inflammatoire. La translocation nucléaire du NF-κB a été etudiée par fluorescence chez les hépatocytes.
The copper amine oxidases are ubiquitous quinoproteins which catalyze the two-
electron oxidative deamination of a primary amine to produce the corresponding aldehyde and ammonia, with subsequent two-electron reduction of dioxygen to hydrogen peroxide (1). To carry out this reaction, these enzymes contain two cofactors, an organic cofactor, topaquinone (TPQ) and a cupric ion. It is well established that TPQ catalyzes the conversion of an amine substrate into an aldehyde through a pyridoxal-like transamination mechanism, which results in the reduction of TPQ to an aminoquinol form. Although there is no question regarding the crucial role of copper in the biogenesis of TPQ, the role of the copper cofactor during amine oxidation is, however, less well understood. Recent intensive biochemical studies are fairly consistent with a passive role of copper in the catalyzed amine oxidation process (2-4). A few years ago, we have shown that electrogenerated 3,4-iminoquinone 1 ox acts as an efficient catalyst
Competing Interests: The authors have declared that no competing interests exist. * E-mail: firstname.lastname@example.org
In the oxygen respiratory systems, electrons of low-redox potential electron donors are transferred through a series of membrane-bound proteins or complexes and finally, the reduction of molecular oxygen to water is catalyzed by enzymes called terminal oxidases. These oxygen reductases are complicated integral membrane multi-subunit complexes grouped into two major superfamilies. Most of them belong to the well-character- ized heme-copper oxidases (HCO) superfamily . HCO have been named cytochrome c oxidases or quinol oxidases, depending on the nature of their electron donor and are able to pump protons across membrane. Additionally, based on biochemical and structural differences in their catalytic subunits and on phyloge- netic analysis, a classification of HCO into three families was proposed : i) type A (mitochondrial-like oxidases or aa 3 -type), ii)
Sagittal sections through the corpus callosum of adult macaque monkeys (n = 7) reveal a subpopulation of neurons positive for NADPH-diaphorase (NADPHd). These are sparsely distributed, with 2–12 neurons scored over the anterior two-thirds of the callosum (about 14 mm). Neurons are densely labeled, type 1; but on the basis of soma and dendritic morphology, these neurons exhibit distinct heterogeneity. In one subpopulation, the cell body is narrowly attenuated (7–10 μm in width). These have bipolar dendrites, extending 300–800 μm from the cell body. One or both of the dendrites is often closely associated with blood vessels and tends to be aligned dorso-ventral, perpendicular to the body of the callosum. Another subpopulation of neurons has a larger soma (typically, 15 μm × 20 μm) and more multipolar dendrites, which are not as obviously associated with blood vessels. White matter neurons positive for NADPHd have previously been observed as a transient population, most numerous during development, in the human corpus callosum, as well as in that of other species. Their persistence in the corpus callosum of adult macaques and their close association with blood vessels has not previously been reported and is suggestive of roles other than axon guidance.
2 and 3 with earlier reported 1,4-benzoxazine derivatives. 
In summary, we have demonstrated for the first time that unactivated primary aliphatic amines can be efficiently oxidized by a synthetic model cofactor of amine oxidases in the absence of metal ion. The catalyst 1ox exhibits the same substrate specificity as the copper amine oxidases themselves, that is, poor reactivity with - branched primary amines and no reactivity toward secondary amines. The reaction displays two features that are most often associated with enzymatic systems: a) the reaction is likely enhanced through the participation of neighboring substituents, as they prevented the competing formation of Michael adducts (in this case, the benzoyl and 2-hydroxy groups); b) the presence of an active hydroxyl proton very probably constitutes an essential component of the catalytic activity (in this case, the 2-hydroxy proton, analogous to that of TPQ).  We are currently developing
plants ( Watillon et al., 1995 ). CCaMKs are also rare
in plants and might be expressed in the tissues of
only a few plants ( Poovaiah et al., 1999 ). Moreover,
their exact roles and functions in plants are not well known. Although this study underlined the importance of PK in NADPH-oxidase-like enzyme activation and revealed a calmodulin-dependent step in lead-induced ROS production, the link between PK and calmodulin is not clear.
the CuAOs enzymes that is, high reactivity with unbranched primary amines and with the primary amino group of diamines and polyamines, whereas poor reactivity with branched amines. Overall, 1 ox mimics not only the metabolic activity of PrAO enzymes, as high
catalytic performances have been observed with primary monoamines (benzylamine, aminoacetone, propylamine and methylamine) and the terminal primary amino group of spermidine, but also that of DAOs as shown by the data obtained with putrescine, and to a lesser extent histamine. Contrary to FAD-dependent amine oxidases, no activity was observed with secondary and tertiary amines (Table 2). Finally, a last question emerges whether known selective CuAOs inhibitors can also prevent the activity of the electrocatalyst 1 ox . This study