Nitric oxide (NO), a multifunctional second messenger, is implicated in physiological functions in mammals that range from immune response and potentiation of synaptic transmission to dilation of blood vessels and muscle relaxation. NO is a highly active molecule that diffuses across cell membranes and cannot be stored inside the producing cell. Its signaling capacity must be controlled at the levels of biosynthesis and local availability. Indeed, NO production by NO synthases is under complex and tight control, being regulated at transcriptional and translational levels, through co- and posttranslational modifications, and by subcellular localization. NO is synthesized from L-arginine by a family of nitric oxide synthases (NOS). Three NOS isoforms have been characterized: neuronal NOS (nNOS, NOS1) primarily found in neuronal tissue and skeletal muscle; inducible NOS (iNOS, NOS2) originally isolated from macrophages and later discovered in many other cells types; and endothelial NOS (eNOS, NOS3) present in vascular endothelial cells, cardiac myocytes, and in blood platelets. The enzymatic activity of all three isoforms is dependent on calmodulin, which binds to nNOS and eNOS at elevated intracellular calcium levels, while it is tightly associated with iNOS even at basal calcium levels. As a result, the enzymatic activity of nNOS and eNOS is modulated by changes in intracellular calcium levels, leading to transient NO production, while iNOS continuously releases NO independent of fluctuations in intracellular calcium levels and is mainly regulated at the gene expression level (Pacher et al. 2007).
The NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central calmodulin-binding motif separates an oxygenase (NH2-terminal) domain from a reductase (COOH-terminal) domain. Binding sites for cofactors NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the absence of calmodulin. iNOS does not contain this insert.<p>Because NOS enzymatic activity is modulated by the presence of its substrates and cofactors within the cell, under certain conditions, NOS may generate superoxide instead of NO, a process referred to as uncoupling (uncoupling of NADPH oxidation and NO synthesis).<p>The molecular details of eNOS function are annotated here.
Smagghe BJ, Trent JT, Hargrove MS.; ''NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo.''; PubMedEurope PMCScholia
Venema RC, Ju H, Zou R, Ryan JW, Venema VJ.; ''Subunit interactions of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-oxide synthase isoforms.''; PubMedEurope PMCScholia
Berka V, Yeh HC, Gao D, Kiran F, Tsai AL.; ''Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Gardner PR.; ''Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases.''; PubMedEurope PMCScholia
List BM, Klösch B, Völker C, Gorren AC, Sessa WC, Werner ER, Kukovetz WR, Schmidt K, Mayer B.; ''Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization.''; PubMedEurope PMCScholia
Andjelković M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq JM, Hemmings BA.; ''Role of translocation in the activation and function of protein kinase B.''; PubMedEurope PMCScholia
Michel JB, Feron O, Sacks D, Michel T.; ''Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin.''; PubMedEurope PMCScholia
Zimmermann K, Opitz N, Dedio J, Renne C, Muller-Esterl W, Oess S.; ''NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Klatt P, Schmidt K, Werner ER, Mayer B.; ''Determination of nitric oxide synthase cofactors: heme, FAD, FMN, and tetrahydrobiopterin.''; PubMedEurope PMCScholia
Kone BC, Kuncewicz T, Zhang W, Yu ZY.; ''Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide.''; PubMedEurope PMCScholia
Chen TY, Illing M, Molday LL, Hsu YT, Yau KW, Molday RS.; ''Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca(2+)-calmodulin modulation.''; PubMedEurope PMCScholia
Icking A, Matt S, Opitz N, Wiesenthal A, Müller-Esterl W, Schilling K.; ''NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS.''; PubMedEurope PMCScholia
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM.; ''Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.''; PubMedEurope PMCScholia
Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, Kurzchalia TV.; ''Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.''; PubMedEurope PMCScholia
Halligan KE, Jourd'heuil FL, Jourd'heuil D.; ''Cytoglobin is expressed in the vasculature and regulates cell respiration and proliferation via nitric oxide dioxygenation.''; PubMedEurope PMCScholia
Cillero-Pastor B, Mateos J, Fernández-López C, Oreiro N, Ruiz-Romero C, Blanco FJ.; ''Dimethylarginine dimethylaminohydrolase 2, a newly identified mitochondrial protein modulating nitric oxide synthesis in normal human chondrocytes.''; PubMedEurope PMCScholia
García-Cardeña G, Oh P, Liu J, Schnitzer JE, Sessa WC.; ''Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling.''; PubMedEurope PMCScholia
Reiter CD, Teng RJ, Beckman JS.; ''Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite.''; PubMedEurope PMCScholia
Pacher P, Beckman JS, Liaudet L.; ''Nitric oxide and peroxynitrite in health and disease.''; PubMedEurope PMCScholia
Forbes SP, Druhan LJ, Guzman JE, Parinandi N, Zhang L, Green-Church KB, Cardounel AJ.; ''Mechanism of 4-HNE mediated inhibition of hDDAH-1: implications in no regulation.''; PubMedEurope PMCScholia
Dedio J, König P, Wohlfart P, Schroeder C, Kummer W, Müller-Esterl W.; ''NOSIP, a novel modulator of endothelial nitric oxide synthase activity.''; PubMedEurope PMCScholia
Jourd'heuil D, Jourd'heuil FL, Kutchukian PS, Musah RA, Wink DA, Grisham MB.; ''Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo.''; PubMedEurope PMCScholia
Hamdane D, Kiger L, Dewilde S, Green BN, Pesce A, Uzan J, Burmester T, Hankeln T, Bolognesi M, Moens L, Marden MC.; ''The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin.''; PubMedEurope PMCScholia
Ghosh S, Gachhui R, Crooks C, Wu C, Lisanti MP, Stuehr DJ.; ''Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthase. Consequences for catalysis.''; PubMedEurope PMCScholia
Wang Y, Monzingo AF, Hu S, Schaller TH, Robertus JD, Fast W.; ''Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: toward a targeted polypharmacology to control nitric oxide.''; PubMedEurope PMCScholia
Oess S, Icking A, Fulton D, Govers R, Müller-Esterl W.; ''Subcellular targeting and trafficking of nitric oxide synthases.''; PubMedEurope PMCScholia
Fernández-Hernando C, Fukata M, Bernatchez PN, Fukata Y, Lin MI, Bredt DS, Sessa WC.; ''Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Michel T.; ''Targeting and translocation of endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Burmester T, Ebner B, Weich B, Hankeln T.; ''Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues.''; PubMedEurope PMCScholia
Andjelković M, Maira SM, Cron P, Parker PJ, Hemmings BA.; ''Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase.''; PubMedEurope PMCScholia
Gratton JP, Fontana J, O'Connor DS, Garcia-Cardena G, McCabe TJ, Sessa WC.; ''Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1.''; PubMedEurope PMCScholia
Schilling K, Opitz N, Wiesenthal A, Oess S, Tikkanen R, Müller-Esterl W, Icking A.; ''Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN.''; PubMedEurope PMCScholia
García-Cardeña G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, Sessa WC.; ''Dynamic activation of endothelial nitric oxide synthase by Hsp90.''; PubMedEurope PMCScholia
Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, Tsuruo T, Sessa WC.; ''Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release.''; PubMedEurope PMCScholia
Vásquez-Vivar J, Martásek P, Whitsett J, Joseph J, Kalyanaraman B.; ''The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study.''; PubMedEurope PMCScholia
Tuteja N, Chandra M, Tuteja R, Misra MK.; ''Nitric Oxide as a Unique Bioactive Signaling Messenger in Physiology and Pathophysiology.''; PubMedEurope PMCScholia
Syed NA, Horner KN, Misra V, Khandelwal RL.; ''Different cellular localization, translocation, and insulin-induced phosphorylation of PKBalpha in HepG2 cells and hepatocytes.''; PubMedEurope PMCScholia
Liu J, Sessa WC.; ''Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Takahashi S, Mendelsohn ME.; ''Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex.''; PubMedEurope PMCScholia
Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PR, Kemp BE, Pearson RB.; ''The Akt kinase signals directly to endothelial nitric oxide synthase.''; PubMedEurope PMCScholia
Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T.; ''Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells.''; PubMedEurope PMCScholia
Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC.; ''Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.''; PubMedEurope PMCScholia
García-Cardeña G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, Sessa WC.; ''Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.''; PubMedEurope PMCScholia
Fago A, Hundahl C, Dewilde S, Gilany K, Moens L, Weber RE.; ''Allosteric regulation and temperature dependence of oxygen binding in human neuroglobin and cytoglobin. Molecular mechanisms and physiological significance.''; PubMedEurope PMCScholia
Sawabe K, Yamamoto K, Harada Y, Ohashi A, Sugawara Y, Matsuoka H, Hasegawa H.; ''Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin.''; PubMedEurope PMCScholia
In the second salvage step, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, a process which increases the BH4:BH2 ratio providing BH4 for coupled eNOS production of NO. In mice cell lines, DHFR inhibition or knockdown diminishes the BH4:BH2 ratio and exacerbates eNOS uncoupling (Crabtree et al. 2009).
Peroxynitrite can oxidise BH4 to the BH3 radical, further reducing BH4 availability to couple eNOS activity and compounding the production of superoxide through uncoupled eNOS activity (Kuzkaya et al. 2003).
6-pyruvoyl tetrahydrobiopterin synthase (PTPS) (Takikawa et al. 1986) catalyses the second step in BH4 biosynthesis, the dephosphorylation of DHNTP to 6-pyruvoyl-tetrahydropterin (PTHP). PTPS is believed to function as a homohexamer (Nar et al. 1994, Bürgisser et al. 1994) and has a requirement for Zn2+ (one Zn2+ ion bound per subunit) and Mg2+ ions for activity (Bürgisser et al. 1995). The phosphorylation of Ser-19 is an essential modification for enzyme activity (Scherer-Oppliger et al. 1999).
Superoxide (O2.-) formed from an uncoupled eNOS action, together with nitric oxide (NO) formed from a coupled eNOS action, readily react together to fom peroxynitrite (ONOO-) (Jourd'heuil et al. 2001, Reiter et al. 2000).
The oxidation product of BH4, 7,8-dihydrobiopterin (BH2), can compete with BH4 for binding to eNOS. This can lead to the uncoupling of eNOS and can result in the formation of reactive oxygen species (Vasquez-Vivar et al. 2002).
High levels of the end product, BH4, negatively regulates GCH1. It does this via GTP cyclohydrolase 1 feedback regulatory protein (GCHFR). BH4-dependant GCHFR in the form of a homopentamer complexes with the decameric GCH1 enzyme in the ratio 2:1 to inactivate it. L-phenylalanine reverses this inhibition. These regulatory steps control the biosynthesis of BH4. (Swick & Kapatos 2006, Chavan et al. 2006, Harada et al. 1993).
Heme iron from the oxygenase domain of eNOS can reduce the BH3 radical back to BH4, with itself being oxidised from the ferrous (Fe2+) back to the ferric (Fe3+) form (Berka et al. 2004).
To become active, sepiapterin reductase (SPR) must first be phosphorylated (serine 213 in humans) by Ca2+/calmodulin-dependent protein kinase II (Fujimoto et al. 2002, Katoh et al. 1994).
The first and rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis is GTP cyclohydrolase I (GCH1, GTPCHI). Three different isoforms are produced but only isoform 1 is functionally active (Gütlich et al. 1994). GCH1 is functional as a homodecamer. First, a monomer of GCH1 forms a dimer. Then five dimers arrange into a ring-like structure to form the homodecamer (Nar et al. 1995).
The cofactor tetrahydrobiopterin (BH4) ensures endothelial nitric oxide synthase (eNOS) couples electron transfer to L-arginine oxidation (Berka et al. 2004). During catalysis, electrons derived from NADPH transfer to the flavins FAD and FMN in the reductase domain of eNOS and then on to the ferric heme in the oxygenase domain of eNOS. BH4 can donate an electron to intermediates in this electron transfer and is oxidised in the process, forming the BH3 radical. This radical can be reduced back to BH4 by iron, completing the cycle and forming ferrous iron again. Heme reduction enables O2 binding and L-arginine oxidation to occur within the oxygenase domain (Stuehr et al. 2009).
Ascorbate (vitamin C) can reduce the BH3 radical back to BH4, thereby maintaining BH4 levels (Baker et al. 2001, Patel et al. 2002, Kuzkaya et al. 2003).
In the first of two salvage steps to maintain BH4 levels in the cell, sepiapterin is taken up by the cell and reduced by sepiapterin reductase (SRP) to form BH2 (Sawabe et al. 2008).
BH2 may compete with BH4 to bind eNOS, uncoupling eNOS leading to the formation of superoxide rather than nitric oxide. BH2, the oxidised form of BH4, cannot contribute electrons to heme in the reductase domain of eNOS, thereby uncoupling it from arginine oxidation and producing superoxide from oxygen instead (Vasquez-Vivar et al. 2002).
HSP90 serves as a scaffold to promote productive interaction between AKT1 and eNOS. Due to the proximity of these proteins once complexed with HSP90, AKT1 phosphorylates eNOS at Ser1177. When Ser1177 is phosphorylated, the level of NO production is elevated two- to three-fold above basal level.
eNOS interacts with the SH3 domain of NOSTRIN (positions 434-506). Caveolin-1 also binds directly to NOSTRIN (residues 323-434), thus allowing formation of a ternary complex.
Nitric oxide (NO) is produced from L-arginine by the family of nitric oxide synthases (NOS) enzymes, forming the free radical NO and citrulline as byproduct. The cofactor tetrahydrobiopterin (BH4) is an essential requirement for the delivery of an electron to the intermediate in the catalytic cycle of NOS.
Increases in intracellular calcium and calmodulin stimulate depalmitoylation of eNOS by acyl protein thioesterase 1, which displaces eNOS from the membrane. This might be a mechanism to downregulate NO production following intense stimuli.
Once depalmitoylated, it's proposed that eNOS is displaced from the plasma membrane and redistributed to other intracellular membranes, including the Golgi, where re-palmitoylation occurs. The mechanism of transport from the plasma membrane is still unknown.
Palymitoylated, myristoylated eNOS forms a dimer and is transported from the Golgi to the plasma membrane. Transport is thought to be mediated by intracellular vesicles, but the details remain unknown.
Caveolin-1 is the primary negative regulatory protein for eNOS. Caveolin-1 binding to eNOS compromises its ability to bind Calmodulin (CaM), thereby inhibiting enzyme activity. The major binding region of caveolin-1 for eNOS is within amino acids 60-101 and to a lesser extent, amino acids 135-178.
DHHC-21 is a Golgi-localized acyl transferase that palmitoylates eNOS, which targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.
NOSIP (eNOS interacting protein) binds to the carboxyl-terminal region of the eNOS oxygenase domain. Note that the eNOS binding sites for caveolin and NOSIP overlap.
NOSIP promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis. eNOS appears to be shifted to intracellular sites that colocalize with Golgi and/or cytoskeletal marker proteins.
NOSTRIN translocates eNOS from the plasma membrane to intracellular vesicular structures. NOSTRIN internalization of eNOS is proposed to occur via vesicle fission and caveolar transport through cooperation with dynamin and N-WASP.
The NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central calmodulin-binding motif separates an oxygenase (NH2-terminal) domain from a reductase (COOH-terminal) domain. Binding sites for cofactors NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the absence of calmodulin. iNOS does not contain this insert.<p>Because NOS enzymatic activity is modulated by the presence of its substrates and cofactors within the cell, under certain conditions, NOS may generate superoxide instead of NO, a process referred to as uncoupling (uncoupling of NADPH oxidation and NO synthesis).<p>The molecular details of eNOS function are annotated here.
Original Pathway at Reactome: http://www.reactome.org/PathwayBrowser/#DB=gk_current&FOCUS_SPECIES_ID=48887&FOCUS_PATHWAY_ID=202131
Try the New WikiPathways
View approved pathways at the new wikipathways.org.Quality Tags
Ontology Terms
Bibliography
History
External references
DataNodes
CaM HSP90
p-AKT1CaM
HSP90Caveolin-1 CaM
HSP90Caveolin-1
CaMCaveolin-1 NOSTRIN
Dynamin-2Caveolin-1 NOSTRIN dynamin-2
N-WASPCaveolin-1 NOSTRIN dynamin-2
N-WASPCaveolin-1
NOSTRIN complexCaM HSP90 p-AKT1
BH2CaM HSP90 p-AKT1
BH4CaM HSP90
p-AKT1Annotated Interactions
CaM HSP90
p-AKT1CaM
HSP90CaM
HSP90Caveolin-1 CaM
HSP90Caveolin-1
CaMCaveolin-1 NOSTRIN
Dynamin-2Caveolin-1
NOSTRIN complexCaM HSP90 p-AKT1
BH2CaM HSP90 p-AKT1
BH2CaM HSP90 p-AKT1
BH4CaM HSP90 p-AKT1
BH4CaM HSP90
p-AKT1CaM HSP90
p-AKT1