Endothelial nitric oxide synthase

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endothelial nitric oxide synthase
Properties of human protein
Mass / length primary structure 1203 amino acids
Secondary to quaternary structure Homodimer
Cofactor Heme, FAD, FMN, tetrahydrobiopterin
Identifier
Gene name NOS3
External IDs
Enzyme classification
EC, category 1.14.13.39 ,  dioxygenase
Response type oxidation
Substrate L- arginine + n NADPH + m O 2
Products L- citrulline + NO + n NADP + + m H 2 O
Occurrence
Homology family eNOS
Parent taxon Jaws

The protein endothelial nitric oxide synthase ( eNOS ) belongs to the enzyme family of NO synthases . It catalyzes the formation of nitric oxide from the amino acid L - arginine .

In humans, eNOS is mainly formed in endothelial cells , which are the innermost cell layer in blood and lymph vessels. There eNOS and nitric oxide play a central role in regulating blood pressure and the function of blood vessels. Reduced activity or a malfunction of the eNOS favor the development of vascular diseases such as atherosclerosis . The phenomenon of eNOS decoupling is particularly important. Decoupled eNOS produces superoxide instead of nitric oxide, thereby promoting oxidative stress in the endothelium and thus damaging blood vessels more than they are useful. Because of this dual function, eNOS is also known as a Janus-headed enzyme.

Discovery of the eNOS

In 1980 Robert Francis Furchgott discovered that the well-known vasodilator acetylcholine only relaxes blood vessels if the endothelial cell layer is intact. He concluded that acetylcholine causes the release of an unknown substance in endothelial cells, which is responsible for this effect. This substance was identified as nitric oxide in 1987. Two years later (1989) Robert Palmer and Salvador Moncada discovered the enzyme responsible for this. The term “endothelial NOSynthase” (eNOS) serves to distinguish it from the other isoforms iNOS (inducible NO synthase) and nNOS (neuronal NO synthase).

→ nitric oxide # story

Physiological importance

Effects of nitric oxide in blood vessels

→ Nitric oxide # Physiological significance

The messenger substance nitric oxide (NO) produced by eNOS has numerous positive effects in blood vessels and is therefore considered a vascular protective factor. The main physiological effects of nitric oxide in blood vessels are:

The many vascular protective properties of nitric oxide make it clear that a reduction in nitric oxide production by eNOS favors the development of vascular diseases.

eNOS decoupling

The term eNOS decoupling describes a condition in which eNOS produces superoxide instead of nitric oxide. In this case, the enzymatic reduction of oxygen is decoupled from the catalytic reaction with L- arginine. This condition occurs above all when too little of the cofactor tetrahydrobiopterin (BH 4 ) is present in the endothelium (see also: catalytic mechanism of eNOS).

The superoxide formed by decoupled eNOS reacts very easily with nitrogen monoxide to form peroxynitrite . Peroxynitrite, in turn, breaks down BH 4 , which decouples eNOS more and more in a vicious circle. This increases the oxidative stress in blood vessels and forms an important basis for the development of high blood pressure, atherosclerosis and other cardiovascular diseases. Under these circumstances, eNOS transforms from a vascular protective enzyme to a vascular damaging enzyme. In this context, one speaks of an endothelial dysfunction.

The phenomenon of eNOS decoupling is also the reason why the increase in endothelial eNOS expression does not necessarily lead to more nitrogen monoxide being formed in the vessel. This is only the case if the corresponding amount of BH 4 is available.

A BH 4 deficiency in the endothelium can basically be countered in two ways: Either one tries to increase BH 4 formation or to protect BH 4 from degradation. Drugs from the group of statins can, for example, increase the expression of the BH 4 -producing enzyme GTP-Cyclohydrolase I (GTPCH I). This increases the BH 4 concentration in endothelial cells and improves eNOS function. Ascorbic acid (vitamin C) chemically stabilizes BH 4 and inhibits its breakdown through oxidative stress . Various drugs that lower blood pressure, such as ACE inhibitors and angiotensin receptor blockers, increase the BH 4 concentration in the endothelium, presumably by reducing the oxidative stress in blood vessels.

Structure and function of the eNOS

Chromosomal location and protein structure

In the human genome , the gene NOS3 , which codes for eNOS, is located on chromosome 7 (7q35–36). It comprises 26 exons and extends over a length of approx. 21 kilobases on the DNA . The eNOS protein has 1203 amino acids with a molecular mass of 133  kDa . Structurally, eNOS is divided into two protein domains with different catalytic activity: The reductase domain is located at the C-terminal end. This contains binding sites for the cofactors nicotinic acid amide-adenine-dinucleotide-phosphate (NADP), flavin-adenine-dinucleotide (FAD) and flavin mononucleotide (FMN). The oxygenase domain at the N-terminus contains a heme group as well as binding sites for oxygen and the cofactor tetrahydrobiopterin (BH 4 ). Both subunits are linked to one another via a binding motif for the protein calmodulin . In order to be catalytically active, two eNOS proteins have to combine to form a homodimer . The oxygenase domain of one monomer forms a functional unit with the reductase domain of the other. A zinc ion , which is tetrahedrally complexed with two cysteine residues of each monomer, serves to stabilize the protein complex . Usually calmodulin is also bound to the homodimer, so that such an eNOS protein complex - strictly speaking - represents a tetramer .

Catalytic mechanism and role of cofactors

In the reaction catalyzed by eNOS, the guanidine group of the amino acid L -arginine is oxidized in the presence of oxygen. The two products nitrogen monoxide (NO) and L - citrulline are produced in equal quantities.

In the first reaction step, an electron is split off from NADPH at the reductase domain. This electron is then passed on to the oxygenase domain with the help of the cofactors FAD , FMN and calmodulin. In the catalytic center, molecular oxygen is available as an electron receptor through binding to the iron atom of the heme group. As a result, oxygen is reduced and reacts with the carbon atom of the guanidine group in the substrate L- arginine. The intermediate product Nώ-hydroxy- L- arginine is thus initially formed . In a second pass, Nώ-hydroxy- L- arginine serves as a substrate and is converted into L- citrulline. A nitrogen atom from the guanidine group is split off and released as NO.

The stoichiometry of the reaction is:

2 L -arginine + 3 NADPH + 4 O 2 → 2 L -citrulline + 3 NADP + 4 H 2 O + 2 NO

BH 4 also plays an important role as a cofactor. A deficiency in BH 4 means that the enzymatic reduction of oxygen is decoupled from the catalytic reaction with L- arginine. In this case, the oxygen-heme complex breaks down and a superoxide radical is created instead of NO. This eNOS decoupling plays a central role in the development of cardiovascular diseases.

Subcellular localization

The distribution of eNOS within the cell is mainly determined by the fatty acids attached to the enzyme, so-called lipid anchors . As a result, eNOS is transported to specific points on the cell membrane, the caveolae . Caveolae are indentations in the cell membrane with a typical composition of lipids and proteins. Often there are collections of functionally linked proteins. Various proteins are found in caveolae that can influence eNOS activity. These include caveolin-1 , protein kinase B (AKT), the heat shock protein Hsp90 , G-protein-coupled receptors (see: eNOS regulation through protein-protein interactions ).

In addition, eNOS activation can lead to their redistribution within the cell. The eNOS agonists acetylcholine , bradykinin or vascular endothelial growth factor (VEGF) remove fatty acid residues and move eNOS protein into the Golgi apparatus . Furthermore, the binding of nitric oxide to certain eNOS cysteine ​​residues, so-called S-nitrosylations, influences the location of the protein.

In general, the cell membrane is the site of the highest eNOS activity. In other cell compartments, NO production is significantly lower. Among other things, eNOS was also detected in the cell nucleus and in mitochondria . The existence of a mitochondrial NO synthase (mtNOS) and its possible physiological significance is currently being discussed intensively, but has not yet been conclusively clarified (as of 2008). In the cytoskeleton, which is involved in eNOS protein transport, eNOS appears to be inactive.

Regulation of the eNOS

Regulation of eNOS gene transcription

Promoter structure

The eNOS promoter lacks a TATA box , as is found in many permanently expressed proteins. However, there are two important positive regulatory domains (PRD) that lie between base pairs −104 and −95 (PRD I) and −144 to −115 (PRD II). Various activating transcription factors bind there. An enhancer element is located approximately 4.5 kilobases in front of the start of transcription . Even further away, at about −5.3 kilobases, there is a hypoxia- sensitive binding element ( hypoxia responsive element ). Inside the eNOS intron 4 there is a 27 nucleotide long, repetitive sequence that serves as a binding site for β-actin and represents the site of origin of a regulatory microRNA .

transcription

Although eNOS is regarded as a permanently expressed enzyme, there are numerous physiological and pathophysiological stimuli or messenger substances that can influence eNOS expression.

Certain growth factors ( VEGF , TGF-β1 ) and hormones ( insulin , estrogen ) increase eNOS expression in endothelial cells. A particularly effective stimulus for eNOS expression are also laminar shear forces, which are exerted on endothelial cells as the blood flows past (see also: laminar flow). In addition, some oxygen radicals , especially hydrogen peroxide , as well as hypoxia and certain drugs ( statins ) increase eNOS expression. Increased eNOS transcription can also represent a counter-regulation that pursues the goal of compensating for an eNOS protein deficiency due to reduced eNOS mRNA stability (see also: mRNA stability). The transcription factor Krüppel-like factor 2 (Klf-2), which also controls endothelial inflammatory processes, seems to be of particular importance. Both statins and laminar shear forces mediate their effect on eNOS expression at least partially via Klf-2.

Epigenetic Mechanisms

Epigenetic mechanisms play an important role in the endothelium-specific eNOS expression .

The PRD I and PRD II domains of the eNOS promoter are highly methylated in non-endothelial cells. As a rule, such methylation of DNA bases is a characteristic of transcriptionally inactive promoters. Accordingly, the eNOS promoter is hardly methylated in endothelial cells. In addition, nucleosomes of active eNOS promoters are more frequently acetylated on the histone proteins H3 and H4. This epigenetic marking is a characteristic of transcriptionally active promoters. Similarly, demethylating substances and histone deacetylase inhibitors can force eNOS expression in non-endothelial cell types.

mRNA stability

Since the half-life of the mRNA coding for eNOS is normally between 10 and 35 hours (REF), eNOS protein is still formed from the mRNA present for some time after the transcription has ended immediately. Modulating the mRNA stability is therefore a faster and more efficient way to influence protein expression. So far, three such mechanisms have been described (as of 2008):

  • Polyadenylation at the 3 'end stabilizes the mRNA. Increased eNOS mRNA polyadenylation is observed during treatment with drugs from the group of statins or laminar shear forces, which are exerted on endothelial cells by blood flowing past.
  • The binding of various proteins to the 3'-UTR region destabilizes the mRNA. Among other things, tumor necrosis factor-α (TNF-α), bacterial lipopolysaccharides , oxidized low density lipoprotein (oxLDL) or thrombin cause such an effect.
  • An mRNA transcript called sONE, which comes from the non-codogenic strand of the eNOS gene, reduces the cellular eNOS mRNA concentration. Since this antisense RNA (aRNA) is mainly found in non-endothelial cells, it probably helps to restrict eNOS expression to the endothelium to a large extent.

Post-translational modifications

Lipid anchor

The anchoring of eNOS in the cell membrane is mainly determined by fatty acids that are attached to the eNOS protein as part of post-translational modifications . One therefore speaks of so-called lipid anchors. Linking them to eNOS takes place in two steps. First, eNOS is irreversibly myristoylated at Gly-2. Then, in the Golgi apparatus, Cys-15 and Cys-26 are palmitoylated , which are the cause of the eNOS translocation in caveolae. Activation of eNOS, for example by phosphorylation, can lead to the cleavage of palmitic acid , whereby eNOS is transported back into the Golgi apparatus .

Phosphorylations

eNOS phosphorylations play a major role in regulating eNOS activity. So far, six phosphorylation sites in the eNOS protein are known (as of 2008). eNOS Ser-1177 is generally considered to be the most important phosphorylation site. Almost all eNOS activators lead to phosphorylation of Ser-1177 by various kinases , such as protein kinase A (PKA), protein kinase B (PKB, AKT), protein kinase C (PKC), AMP-activated protein kinase and Ca 2+ / calmodulin-dependent Kinase. Phosphorylation at Ser-1177 accelerates the flow of electrons within the reductase domain and promotes the binding of calmodulin to eNOS. Protein phosphatase 2A can reverse phosphorylation at Ser-1177 and return eNOS to its basic state after activation.

Ser-633 is another important activating phosphorylation site. It is located within the FMN binding site and is under the control of PKA. Many PKA-activating eNOS agonists (e.g. laminar shear forces, bradykinin, VEGF) also lead to phosphorylation at Ser-633. Since the phosphorylation there is slightly delayed compared to Ser-1177, it is assumed that Ser-633 phosphorylation causes longer-lasting NO production. Phosphorylation at Ser-615, which is also in the FMN binding site, is mediated by AKT. This presumably facilitates the binding of calmodulin. However, this has not yet been conclusively clarified (as of 2008). Thr-495 is the major eNOS negative phosphorylation site. It is normally constantly phosphorylated, which is primarily due to protein kinase C (PKC). Thr-495 phosphorylation reduces eNOS activity because it disrupts the binding of calmodulin to eNOS. Protein Phosphatase 1, Protein Phosphatase 2A (PP2A) and Protein Phosphatase 2B can dephosphorylate eNOS Thr-495. Above all, a rapid increase in the intracellular calcium concentration leads to Thr-495 dephosphorylation. This removes the negative influence on the calmodulin binding and increases the eNOS activity.

Phosphorylation at Ser-114, the only phosphorylation site in the eNOS oxygenase domain to date, may inhibit eNOS activity. However, the function is controversial (as of 2008). eNOS can also be phosphorylated at various tyrosine residues, for example by laminar shear forces. The regulation and function of eNOS tyrosine phosphorylation is currently only incompletely understood (as of 2008).

S-nitrosylation

S-nitrosylation is understood to be the reversible binding of nitrogen monoxide, or radical compounds derived therefrom such as peroxynitrite, to thiol groups . In proteins, the thiol groups usually come from the amino acid cysteine. In the resting state, the catalytic activity of the eNOS is inhibited by nitrosylation at Cys-93 and Cyst-98. It is unclear whether this affects the stability of the homodimer or whether this rather inhibits the flow of electrons between the two monomers (as of 2008). After eNOS activation, there is a rapid, temporary denitrosylation. This acts as a signal to move the eNOS protein into the cytosol. While the eNOS activity declines there, the protein is increasingly nitrosylated again and finally transported back into the caveolae.

Acetylation

There are initial indications that acetylation can also influence eNOS activity. Apparently, eNOS is permanently acetylated at Lys-496 and Lys-506 (based on the amino acid sequence of bovine eNOS). Both lysine residues are in the calmodulin binding region and could therefore have a negative influence on calmodulin binding similar to that of phosphorylation at Thr-495. SIRT1, a class III histone deacetylase, can reverse this acetylation.

Glycosylation

Cell proteins can be altered by the attachment of N- acetyl-glucosamine (GlcNAc) to serine or threonine residues. Elevated blood sugar levels increase the concentration of GlcNAc in endothelial cells. This favors the binding of GlcNAc to eNOS Ser-1177 and has the consequence that less Ser-1177 can be phosphorylated. This has a lasting effect on eNOS activity. This eNOS glycosylation may contribute to the accompanying vascular diseases typical of diabetes mellitus.

Protein-protein interactions

Numerous proteins are known that interact with eNOS and thus influence endothelial NO production. These include protein kinases, phosphatases, membrane proteins, as well as adapter and cytoskeletal proteins. Calmodulin was the first protein to be shown to interact with eNOS. Calmodulin binding is a prerequisite for maximum catalytic eNOS activity. At low intracellular calcium concentrations, calmodulin binding is impaired by an auto-inhibitory loop in the FMN binding domain. If the intracellular calcium concentration increases, calmodulin shifts this loop, binds to eNOS and also displaces caveolin-1, an important negative regulator of eNOS. All of this accelerates the flow of electrons between the reductase and oxygenase domains (see also: catalytic mechanism of eNOS). Since many signaling pathways lead to a rapid increase in intracellular calcium, calmodulin binding is a very common mechanism for rapidly increasing eNOS activity. The heat shock protein 90 (Hsp90) facilitates the binding of calmodulin to eNOS. It also acts as a protein scaffold that brings eNOS together with protein kinase B, among other things. Protein kinases and phosphatases generally have a great influence on eNOS activity (see also: eNOS phosphorylation). In caveolae, other proteins accumulate in the immediate vicinity of the eNOS. G-protein-coupled receptors, for example for bradykinin (B 2 receptor), acetylcholine (M 2 receptor) or histamine , transmit the eNOS-activating effects of their extracellular ligands into the cell. The cationic amino acid transporter CAT-1 ( cationic amino acid transporter ) supplies eNOS directly with the substrate L- arginine due to its spatial proximity . Possibly this is an explanation for the "arginine paradox". NOSTRIN ( eNOS traffic inducer ), NOSIP ( NOS interacting protein ), actin filaments of the cytoskeleton and the motor protein Dynamin-2 are involved in the intracellular transport of eNOS.

Substrate availability

The substrate L- arginine must be available in sufficient quantities for maximum cellular NO production . For the Michaelis constant of the eNOS, values ​​of 3 to 30 μM L -arginine were measured. Although arginine occurs in significantly higher concentrations in the cell (up to 2 mM), an additional administration of arginine, for example by means of an infusion, can increase endothelial NO production. Currently, three hypotheses are being discussed that should provide an explanation for this so-called "arginine paradox" (as of 2008):

  • eNOS is permanently inhibited by asymmetric dimethylarginine (ADMA), because this competes with L -arginine for the binding site in the catalytic center. ADMA can promote eNOS decoupling and is considered a risk factor for atherosclerosis.
  • L- arginine is unevenly distributed within the cell. Caveolae form a cellular compartment in which the arginine concentration differs from that of the cytosol due to the spatial proximity of eNOS and the arginine transporter CAT-1.
  • High local arginine concentrations, such as those found in infusions, activate eNOS by binding to α 2 -adrenoceceptors. This also applies to significantly lower concentrations of the arginine breakdown product agmatine.

The cellular arginine concentration also depends on other arginine-processing enzymes such as arginase. Arginase converts arginine into urea and ornithine. The messenger substances TNF-α and thrombin increase the expression of arginase, thereby increasing arginine consumption in endothelial cells and possibly impairing the eNOS function.

literature

  • WK Alderton, CE Cooper, RG Knowles: Nitric oxide synthases: structure, function and inhibition . In: Biochemical Journal , 357 (Pt 3), 2001, pp. 593-615.
  • DM Dudzinski, J. Igarashi, D. Greif, T. Michel: The regulation and pharmacology of endothelial nitric oxide synthase . In: Annual Review of Pharmacology and Toxicology , 46, 2006, pp. 235-276.
  • U. Förstermann: Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal . In: Biological Chemistry , 387 (12), 2006, pp. 1521-1533.
  • H. Li, T. Wallerath, U. Förstermann: Physiological mechanisms regulating the expression of endothelial-type NO synthase . In: Nitric Oxide , 7 (2), 2002, pp. 132-147.
  • PF Mount, BE Kemp, DA Power: Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation . In: Journal of Molecular and Cellular Cardiology , 42 (2), 2007, pp. 271-279.
  • C. Napoli, F. de Nigris, S. Williams-Ignarro, O. Pignalosa, V. Sica, LJ Ignarro: Nitric oxide and atherosclerosis: an update . In: Nitric Oxide , 15 (4), 2006, pp. 265-279.
  • KM Naseem: The role of nitric oxide in cardiovascular diseases . In: Mol Aspects Med , 26 (1-2), 2005, pp. 33-65.
  • CD Searles: Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression . In: Am J Physiol-Cell Physiol , 291 (5), 2006, pp. C803-C816.

Web links

Individual evidence

  1. ↑ a b c d e f U. Förstermann: Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal . In: Biological Chemistry , 387 (12), 2006, pp. 1521-1533, doi: 10.1515 / BC.2006.190 , PMID 17132097 .
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  13. ^ A b H. Li, T. Wallerath, U. Förstermann: Physiological mechanisms regulating the expression of endothelial-type NO synthase . In: Nitric Oxide , 7 (2), 2002, pp. 132-147, doi: 10.1016 / S1089-8603 (02) 00127-1 .
  14. ↑ Ming-Xiang Zhang, Hesheng Ou, Ying H. Shen, Jing Wang, Jian Wang, Joseph Coselli, Xing Li Wang: Regulation of endothelial nitric oxide synthase by small RNA . In: Proc Natl Acad Sci USA , 102 (47), 2005, pp. 16967-16972, doi: 10.1073 / pnas.0503853102 , PMID 16284254 , PMC 1287968 (free full text).
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  16. ^ A b C. Napoli, F. de Nigris, S. Williams-Ignarro, O. Pignalosa, V. Sica, LJ Ignarro: Nitric oxide and atherosclerosis: an update . In: Nitric Oxide , 15 (4), 2006, pp. 265-279, PMID 16684613 .
  17. ↑ Y. Chan, JE Fish, C. D'Abreo, S. Lin, GB Robb, AM Teichert, F. Karantzoulis-Fegaras, A. Keightley, BM Steer, PA Marsden: The Cell-specific Expression of Endothelial Nitric-oxide Synthase: a role for DNA methylation . In: Journal of Biological Chemistry , 279 (33), 2004, pp. 35087-35100, doi: 10.1074 / jbc.M405063200 , PMID 15180995 .
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  21. ↑ a b c d e P. F. Mount, BE Kemp, DA Power: Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation . In: Journal Mol Cell Cardiol , 42 (2), 2007, pp. 271-279, doi: 10.1016 / j.yjmcc.2006.05.023 .
  22. ^ I. Fleming, B. Fisslthaler, S. Dimmeler, BE Kemp, R. Busse: Phosphorylation of Thr (495) regulates Ca (2 +) / calmodulin-dependent endothelial nitric oxide synthase activity . In: Circ Res , 88 (11), 2001, E68-75, PMID 11397791 .
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  24. ↑ Kandasam Ravi, Lisa A. Brennan, Snezana Levic, Patrick A. Ross, Stephen M. Black: S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity . In: Proc Natl Acad Sci USA , 101 (8), 2004, pp. 2619-2624, doi: 10.1073 / pnas.0300464101 , PMID 14983058 .
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