Endothelial NOS

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

In human eNOS is predominantly formed in endothelial cells, which express the innermost layer of cells in blood and lymph vessels. There eNOS and nitric oxide play a central role in the regulation of blood pressure and for the function of blood vessels. Decreased activity or malfunction of eNOS contribute to the development of vascular diseases such as atherosclerosis. Of particular importance is the phenomenon of eNOS uncoupling. Decoupled eNOS produces superoxide instead of nitric oxide, thereby promoting oxidative stress in the endothelium and so harms blood vessels more than profit them. Because of this dual function, eNOS is also known as a Janus-like enzyme.

  • 3.1 Chromosomal localization and protein structure
  • 3.2 Catalytic mechanism and role of cofactors
  • 5.1 Regulation of eNOS gene transcription 5.1.1 Promoter structure
  • 5.1.2 transcription
  • 5.1.3 Epigenetic mechanisms
  • 5.1.4 mRNA stability
  • 5.2.1 lipid anchor
  • 5.2.2 phosphorylations
  • 5.2.3 S -nitrosylation
  • 5.2.4 acetylation
  • 5.2.5 glycosylation
  • 5.2.6 Protein-Protein Interactions
  • 5.2.7 Substrate Availability

Discovery of eNOS

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

→ nitric # History

Physiological significance

Effects of nitric oxide in blood vessels

→ nitric # Physiological significance

Electricity produced by eNOS messenger nitric oxide ( NO) exerts numerous beneficial effects in blood vessels and is therefore regarded as vascular protective factor. The main physiological effects of nitric oxide in blood vessels are:

  • Nitric oxide then activates the enzyme soluble guanylate cyclase, which initiates through the formation of the messenger cyclic guanosine monophosphate ( cGMP), the relaxation of vascular smooth muscle cells. In this way, eNOS is involved in the regulation of blood pressure.
  • Nitric oxide inhibits the production of adhesion molecules on the surface of endothelial cells. This inhibits the adhesion of leukocytes, one of the first steps in the development of atherosclerosis.
  • Nitric oxide inhibits the oxidation of low density lipoprotein (LDL). The uptake of oxidized LDL by macrophages also contributes to the development of atherosclerosis.
  • Nitric oxide inhibits the aggregation of platelets ( platelet aggregation) and thus the formation of blood clots that can block blood vessels (see also: heart attack).
  • Nitric oxide suppresses the abnormal cell division of vascular smooth muscle cells, a process that contributes to the narrowing of blood vessels ( see also: restenosis).

The many vascular -protective properties, nitric oxide make it clear that a reduction in nitric oxide production by eNOS promotes the development of vascular disease.

ENOS uncoupling

The term eNOS uncoupling describes a condition in which superoxide eNOS produced in place of nitric oxide. In this case, the enzymatic reduction of oxygen by the catalytic reaction with L-arginine is decoupled. The condition occurs primarily when too little of the cofactor tetrahydrobiopterin ( BH4) is present in the endothelium (see also: catalytic mechanism of eNOS).

The superoxide formed by uncoupled eNOS is very responsive with nitric oxide to peroxynitrite. Peroxynitrite in turn builds from BH4, which eNOS is increasingly decoupled 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 conditions, eNOS converts from one vessel to a vascular damaging Protective enzyme. One speaks in this context of endothelial dysfunction.

The phenomenon of eNOS uncoupling is also the reason that the increase in eNOS expression in endothelial this does not necessarily mean that more nitric oxide is formed in the vessel. This is only the case if the appropriate amount of BH4 is available.

A BH4 deficiency in the endothelium may in principle be addressed in two ways: You can try to increase the formation of BH4 or BH4 to protect them from degradation. Drugs from the group of statins may increase, for example, the expression of the BH4 - producing enzyme GTP cyclohydrolase I ( GTPCH I). Thus they increase the BH4 concentration in endothelial cells and improve eNOS function. Ascorbic acid ( vitamin C) BH4 stabilized chemically and inhibits its degradation by oxidative stress. Various antihypertensive drugs such as ACE inhibitors and angiotensin receptor blockers increase the BH4 concentration in the endothelium presumably by reducing oxidative stress in blood vessels.

Structure and function of the eNOS

Chromosomal localization and protein structure

In the human genome, the gene is NOS3 encoding eNOS on chromosome 7 ( 7q35 -36). It comprises 26 exons and extends over a length of about 21 kilobases of DNA. The eNOS protein has 1203 amino acids with a molecular mass of 133 kDa. Structurally, eNOS divided into two protein domains with different catalytic activity: At the C -terminal end is the reductase domain. In it are binding sites for the cofactors nicotinamide 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 ( BH4). Both sub-units are connected to each other via a binding motif for the protein calmodulin. To be catalytically active, two eNOS proteins must assemble to form a homodimer. It forms the oxygenase domain of a monomer having the reductase domain of the other functional unit. To stabilize the protein complex is a zinc ion, which each monomer is complexed tetrahedral with two cysteine ​​residues is used. Calmodulin is usually also bound to the homodimer, so that such an eNOS protein complex - strictly speaking - is a tetramer.

Catalytic mechanism and role of cofactors

In the catalyzed reaction of eNOS the guanidine group of the amino acid L- arginine is oxidized in the presence of oxygen. The two products, nitric oxide ( NO) and L-citrulline produced in equal amounts.

In the first reaction step an electron from NADPH is cleaved in the reductase domain. Subsequently, this electron is transferred with the help of the cofactors FAD, FMN, and calmodulin to the oxygenase domain. In the catalytic center, molecular oxygen is by binding to the iron atom of the heme group as an electron receiver. As a result, oxygen is reduced and reacts with the carbon atom of the guanidine group in the substrate L-arginine. Thus, the initially formed intermediate N?- Hydroxy-L -arginine. In a second pass -hydroxy- N? L-arginine acts as a substrate and is converted into L- citrulline. In this case, a nitrogen atom of the guanidine group is cleaved and released as NO.

The stoichiometry of the reaction is:

In addition, BH4 plays an important role as a cofactor. BH4 deficiency leads to the enzymatic reduction of oxygen by the catalytic reaction with L- arginine is decoupled. In this case, breaks the oxygen -heme complex and creates a superoxide radical instead of NO. This eNOS uncoupling plays a central role in the pathogenesis of cardiovascular diseases.

Subcellular localization

The distribution of eNOS in the cell is determined primarily by the enzyme attached to the fatty acids, so-called lipid anchor. Thus eNOS is targeted to specific sites of the cell membrane, the caveolae transported. Caveolae are indentations of the cell membrane with a typical composition of lipids and proteins. Often you can find there accumulations functionally linked proteins. In caveolae different proteins occur, which can affect the eNOS activity. These include caveolin -1, protein kinase B (AKT ), the heat shock protein Hsp90, G - protein - coupled receptors (see: eNOS regulation by protein-protein interactions ).

In addition, an eNOS activation leads to their redistribution within the cell. Thus, eNOS agonist acetylcholine, bradykinin or Vascular Endothelial Growth Factor ( VEGF), causing the removal of fatty acid residues and a shift of the eNOS protein in the Golgi apparatus. Furthermore affects the binding of nitric oxide to specific eNOS cysteine ​​residues, so-called S- Nitrosylierungen the whereabouts of the protein.

Generally, the cell membrane with the location of the highest eNOS activity in other cell compartments dar. NO production is much lower. Among other things, eNOS was also detected in the nucleus and in mitochondria. The existence of a mitochondrial NO synthase ( mtNOS ) and its possible physiological significance is currently being intensively discussed, but is not yet fully understood (as of 2008 ). In the cytoskeleton, which is involved in the transport of proteins eNOS, eNOS seem to be inactive.

Regulation of eNOS

Regulation of eNOS gene transcription

Promoter structure

The eNOS promoter lacks a TATA box, such as those found in many permanent expressed proteins. However, there are two major positive regulatory domain (PRD) which are located between base pairs -104 and -95 (PRD I) and -144 to -115 (PRD II). There bind various transcription factors activating. Approximately 4.5 kilobases in front of the transcription start point is an enhancer element. Still farther away, at about -5.3 kilobases is a hypoxia - sensitive binding element ( hypoxia responsive element ). Is within the eNOS intron 4 a 27 nucleotides long, recurring several times sequence that serves as a binding site for β - actin and represents the origin of a regulatory microRNA.

Transcription

Although eNOS is considered to be permanently expressed enzyme, there are numerous physiological and pathophysiological stimuli or messengers that can affect the eNOS expression.

Certain growth factors ( VEGF, TGF- β1 ) and hormones (insulin, estrogen ) increase the eNOS expression in endothelial cells. A particularly effective stimulus of eNOS expression are also laminar shear forces exerted by the passage of the blood flow on endothelial cells (see also: laminar flow ). In addition, some oxygen radicals, especially hydrogen peroxide, as well as hypoxia and certain drugs increase (statins ) eNOS expression. Increased eNOS transcription can also be a counter-regulation, which aims to compensate for eNOS protein deficiency due to reduced eNOS mRNA stability (see also: mRNA stability). Of particular importance, the transcription factor cripple -like factor appears to be 2 ( Klf -2), which also controls endothelial inflammation. Both statins and laminar shear forces mediate their effect on eNOS expression at least partly through Klf -2.

Epigenetic mechanisms

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

The PRD I and PRD II domains of the eNOS promoter are heavily methylated in non- endothelial cells. As a rule, such methylation of DNA bases is a hallmark of transcriptionally inactive promoters. According to the eNOS promoter is hardly methylated in endothelial cells. In addition, nucleosomes active eNOS promoters are acetylated common to the histone proteins H3 and H4. This epigenetic mark is a characteristic of transcriptionally active promoters. Analogously, the force eNOS expression in non - endothelial cell types demethylating agents and histone deacetylase inhibitors.

MRNA stability

Since the half-life of eNOS mRNA encoding is usually between 10-35 hours (REF) will take some time formed for the immediate termination of transcription from the existing mRNA eNOS protein. Modulation of mRNA stability is therefore to a faster and more efficient way affect the protein expression. To date, three such mechanisms described (in 2008 ):

  • Polyadenylation at the 3 ' end of the mRNA is stabilized. Increased eNOS mRNA polyadenylation is observed upon treatment with drugs of the statin family or laminar shear forces exerted by a flowing blood on endothelial cells.
  • The binding of various proteins to the 3'- UTR region destabilisert mRNA. Among other things, cause the tumor necrosis factor - α (TNF- α ), bacterial lipopolysaccharides, oxidized low density lipoprotein ( oxLDL ) and thrombin such an effect.
  • An mRNA transcript called sone, which is from the non- codogenic strand of the eNOS gene that decreases cellular eNOS mRNA concentration. Since these antisense RNA ( aRNA ) is found mainly in non- endothelial cells, they probably helps here, largely to limit eNOS expression on the endothelium.

Post-translational modifications

Lipid anchor

The anchoring of eNOS in the cell membrane is mainly determined by fatty acids, which are appended in the context of post-translational modifications at the eNOS protein. One therefore speaks of so-called lipid anchors. Their association with eNOS occurs in two steps. First, eNOS is myristoylated irreversibly to Gly -2. Then take place in the Golgi apparatus, the palmitoylation of Cys -15 and Cys -26 which are the cause of the eNOS translocation in caveolae. eNOS activation, for example by phosphorylation may lead to the elimination of the palmitic acid, which eNOS is transported back to the Golgi apparatus.

Phosphorylations

ENOS phosphorylation play a major role in the regulation of eNOS activity. So far, six phosphorylation sites in eNOS protein known ( as of 2008). eNOS Ser -1177 is generally regarded as the most important phosphorylation site. Almost all of eNOS activators lead to phosphorylation at 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 Ca2 / calmodulin-dependent kinase. The phosphorylation at Ser -1177 accelerates the flow of electrons within the reductase domain and promotes the binding of calmodulin to eNOS. The protein phosphatase 2A can make the phosphorylation at Ser -1177 undo and revert eNOS after their activation back to the ground state.

Ser -633 is another important activating phosphorylation. It is located within the FMN binding site and subject to the control of PKA. Many PKA -activating eNOS agonists (for example, laminar shear forces, bradykinin, VEGF) also lead to the phosphorylation at Ser - 633rd Since the phosphorylation occurs there slightly delayed compared with Ser -1177 is suspected that the Ser -633 phosphorylation results in longer -lasting NO production. Phosphorylation at Ser -615, which is also in the FMN - binding site is mediated by AKT. Presumably by the binding of calmodulin is facilitated. However, this is not yet fully understood ( as of 2008). Thr -495 is the most important negative eNOS phosphorylation. It is usually constantly phosphorylated, what is primarily responsible protein kinase C ( PKC). Thr -495 phosphorylation reduces eNOS activity because they interfere with the binding of calmodulin to eNOS. Protein phosphatase 1, protein phosphatase 2A ( PP2A ) and protein phosphatase 2B can dephosphorylate eNOS Thr -495. In particular, a rapid increase in intracellular calcium concentration leading to the Thr -495 dephosphorylation. Thus, the negative impact is released to the calmodulin - binding and increases eNOS activity.

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

S -nitrosylation

From S -nitrosylation is defined as the reversible binding of nitric oxide, or derived compounds, such as free-radical peroxynitrite, of thiol groups. For proteins, the thiol groups probably added from the amino acid cysteine. At rest, the catalytic activity of eNOS is inhibited by Nitrosylierungen to Cys -93 and Cyst -98. It is unclear whether this stability of the homodimer is impaired or is this more of the flow of electrons between two monomers inhibits (as of 2008 ). After eNOS activation leads to a rapid, transient denitrosylation. This acts as a signal to shift the eNOS protein into the cytosol. While there, the eNOS activity subsides, the protein again increasingly nitrosylated and finally transported back in caveolae.

Acetylation

There is preliminary evidence that acetylation also can affect the eNOS activity. ENOS is apparently to Lys -496 and Lys -506 (based on the amino acid sequence of bovine eNOS) permanently acetylated. Two lysine residues are in the calmodulin - binding region, and may therefore have a similar adverse effect on calmodulin binding are as phosphorylation at Thr- 495th SIRT1, a histone deacetylase class III may cancel this acetylation.

Glycosylation

Cell proteins may be modified 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 the GlcNAc on eNOS Ser -1177, and has the consequence that less Ser -1177 can be phosphorylated. Thus, the eNOS activity is permanently impaired. Perhaps these eNOS glycosylation contributes to the for diabetes mellitus typical vascular comorbidities.

Protein -protein interactions

There are numerous proteins known to interact with eNOS and thus affect endothelial NO production. These include protein kinases, phosphatases, membrane proteins, and adapter and cytoskeletal proteins. Calmodulin was the first protein, the interaction is detected by eNOS. Calmodulin binding is a prerequisite for maximum catalytic activity of eNOS. At low intracellular calcium concentrations, the calmodulin binding by an auto - inhibitory loop of the FMN binding domain is affected. Increasing the intracellular calcium concentration, this loop moves calmodulin binds to and displaces beyond eNOS or caveolin -1, an important negative regulator of the eNOS. All this accelerates the flow of electrons between reductase and oxygenase domain ( see also: catalytic mechanism of eNOS). Since many signaling pathways is lead to a rapid increase of intracellular calcium, the calmodulin - binding, a very frequently occurring mechanism for the rapid increase in eNOS activity. The heat shock protein 90 ( Hsp90 ) facilitates the binding of calmodulin to eNOS. It also functions as a protein framework that brings together eNOS inter alia with the protein kinase B. Protein kinases and phosphatases generally have great influence on the eNOS activity (see also: eNOS phosphorylation). In caveolae still other proteins in the immediate vicinity eNOS accumulate. G -protein coupled receptors, such as bradykinin (B2- receptor), acetylcholine (M2 receptor) or histamine transmitted eNOS activating effects of their extracellular ligand into the cell. The cationic amino acid transporter CAT -1 ( cationic amino acid transporter ) supplied eNOS due to its proximity directly with the substrate L- arginine. This may be 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

For maximum cellular NO production, the substrate L- arginine must be available in sufficient quantity. For the Michaelis constant of the eNOS values ​​of 3-30 uM L- arginine were measured. Although arginine in the cell in much higher concentrations to occur ( up to 2 mM ), an additional administration of arginine, for example, by infusion, increasing the endothelial NO production. Currently, three hypotheses are discussed, which are intended to provide an explanation for this so-called " arginine paradox " (as of 2008 ):

  • ENOS is permanently by asymmetric dimethylarginine (ADMA ) is inhibited, because it competes with L-arginine for the binding site in the catalytic center. ADMA may promote eNOS uncoupling 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 due to the spatial proximity of eNOS and arginine transporter CAT -1 differs from that of the cytosol.
  • High local concentrations of arginine, such as occur with infusions activate eNOS by binding to α2 - Adrenozezeptoren. This also applies to much lower concentrations of arginine degradation product agmatine.

Cellular arginine concentration also depends on other arginine - processing enzymes, such as arginase. Arginase converts arginine to urea and ornithine in. The messengers TNF- α and thrombin enhance the expression of arginase, thereby increasing arginine consumption in endothelial cells and affect so maybe eNOS function.

Human Genome Organisation Biocatalysis Smooth muscle tissue Thrombus GTP-Cyclohydrolase I Angiotensin II receptor antagonist Proteinkinase B TATA-Box Enhancer (genetics) DNA methylation Antisense-RNA Proteinkinase A Proteinkinase C Bradykinin receptor Histamine receptor Biological Chemistry (journal) Digital Object Identifier
308252
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