Site Of Nitric Oxide Synthase Research Articles

Biopterin metabolism and nitric oxide recoupling in cancer.

Tetrahydrobiopterin is a cofactor necessary for the activity of several enzymes, the most studied of which is nitric oxide synthase. The role of this cofactor-enzyme relationship in vascular biology is well established. Recently, tetrahydrobiopterin metabolism has received increasing attention in the field of cancer immunology and immunotherapy due to its involvement in the cytotoxic T cell response. Past research has demonstrated that when the availability of BH4 is low, as it is in chronic inflammatory conditions and tumors, electron transfer in the active site of nitric oxide synthase becomes uncoupled from the oxidation of arginine. This results in the production of radical species that are capable of a direct attack on tetrahydrobiopterin, further depleting its local availability. This feedforward loop may act like a molecular switch, reinforcing low tetrahydrobiopterin levels leading to altered NO signaling, restrained immune effector activity, and perpetual vascular inflammation within the tumor microenvironment. In this review, we discuss the evidence for this underappreciated mechanism in different aspects of tumor progression and therapeutic responses. Furthermore, we discuss the preclinical evidence supporting a clinical role for tetrahydrobiopterin supplementation to enhance immunotherapy and radiotherapy for solid tumors and the potential safety concerns.

Theophylline-based hybrids as acetylcholinesterase inhibitors endowed with anti-inflammatory activity: synthesis, bioevaluation, in silico and preliminary kinetic studies.

In this study, we investigated the conjugation of theophylline with different compounds of natural origin hoping to construct new hybrids with dual activity against cholinergic and inflammatory pathways as potential agents for the treatment of Alzheimer's disease (AD). Out of 28 tested hybrids, two hybrids, acefylline-eugenol 6d and acefylline-isatin 19, were able to inhibit acetylcholinesterase (AChE) at low micromolar concentration displaying IC50 values of 1.8 and 3.3 μM, respectively, when compared to the galantamine standard AChE inhibitor. Moreover, the prepared hybrids exhibited a significant anti-inflammatory effect against lipopolysaccharide induced inflammation in RAW 264.7 and reduced nitric oxide (NO), tumor necrosis alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels in a dose dependent manner. These hybrids demonstrated significant reductions in nitric oxide (NO), tumor necrosis alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels in RAW 264.7 cells induced by lipopolysaccharide (LPS). The findings of this study were further explained in light of network pharmacology analysis which suggested that AChE and nitric oxide synthase were the main targets of the most active compounds. Molecular docking studies revealed their ability to bind to the heme binding site of nitric oxide synthase 3 (NOS-3) and effectively occupy the active site of AChE, interacting with both the peripheral aromatic subsite and catalytic triad. Finally, the compounds demonstrated stability in simulated gastric and intestinal environments, suggesting potential absorption into the bloodstream without significant hydrolysis. These findings highlight the possible therapeutic potential of acefylline-eugenol 6d and acefylline-isatin 19 hybrids in targeting multiple pathological mechanisms involved in AD, offering promising avenues for further development as potential treatments for this devastating disease.

Calmodulin Induced Changes in the Active Site of Nitric Oxide Synthase

Nitric oxide is a transient signaling molecule in mammalian physiology, known as a vasodilating neurotransmitter and effector during innate immune responses. As such, studies about the family of nitric oxide synthases (NOS) responsible for the rapid conversion of amino acid L‐arginine into nitric oxide have been of great interest. The heme domain within the NOS active site critically binds molecular oxygen which combines with L‐arginine in a series of redox reactions. NOS activity is regulated by a number of cofactors, most notably the ubiquitous regulatory protein calmodulin (CaM) which, in the presence of calcium, binds NOS and promotes nitric oxide production. Though this relationship is well‐documented, the molecular mechanism is not understood. Previously, time resolved spectroscopy has been used to probe the active sites of heme proteins and observe differences in absorption on a nanosecond time frame during changes in the catalytic pockets. Our investigation studies the kinetic effects of CaM binding NOS during the photolysis of carbon monoxide from its heme domain. CaM has been shown to directly influence the active site, promoting availability to substrates and rebinding of ligands to the NOS heme, thus enhancing enzymatic activity. A major facet of our project involves further investigation of the various domains of NOS to determine their significance in CaM‐regulated binding. To this end, we prepared a number of knockout NOS variants which, through assaying via time‐resolved spectroscopy, further elucidate the molecular mechanism of active site regulation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Zinc Thiolate Reactivity toward Nitrogen Oxides: Insights into the Interaction of Zn2+ with S-Nitrosothiols and Implications for Nitric Oxide Synthase

Zinc thiolate complexes containing N(2)S tridentate ligands were prepared to investigate their reactivity toward reactive nitrogen species, chemistry proposed to occur at the zinc tetracysteine thiolate site of nitric oxide synthase (NOS). The complexes are unreactive toward nitric oxide (NO) in the absence of dioxygen, strongly indicating that NO cannot be the species directly responsible for S-nitrosothiol formation and loss of Zn(2+) at the NOS dimer interface in vivo. S-Nitrosothiol formation does occur upon exposure of zinc thiolate solutions to NO in the presence of air, however, or to NO(2) or NOBF(4), indicating that these reactive nitrogen/oxygen species are capable of liberating zinc from the enzyme, possibly through generation of the S-nitrosothiol. Interaction between simple Zn(2+) salts and preformed S-nitrosothiols leads to decomposition of the -SNO moiety, resulting in release of gaseous NO and N(2)O. The potential biological relevance of this chemistry is discussed.

Endogenous Nitric Oxide Synthase Inhibitors in the Biology of Disease

The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring, and modulating circulating asymmetric dimethylarginine alone is inadequate. This review outlines the basic biochemistry and physiology of endogenous methylarginines, examines both the experimental and observational evidence for a role in disease pathogenesis, and examines the potential for therapeutic regulation of these molecules.

Targeting the NADPH Binding Site of Nitric Oxide Synthase by a Ligand with Two-Photon Absorption Properties

Synthesis of nitric oxide is performed by NO-synthase. The catalysis is initiated by the combined transfer of electrons/proton from NADPH to the flavin FAD. We have previously characterized one photoactive compound (named Nanotrigger)1,2 allowing to trigger and synchronize NOS activity upon light illumination. Here, we described a new compound (C1) with two-photon absorption properties suitable to assess the binding to the NOS protein. This family of compounds combines a docking moiety (NADPH analog) and a chromophore moiety responsive for light illumination. C1 was characterized in DMSO by an absorption maximum at 460nm and was found to be fluorescent with an emission peak centered at 740nm upon one-photon excitation. However, the fluorescence emission was strongly sensitive to solvent polarity as evidenced by the significant decrease in the emission intensity in polar solvents. The same behaviour was observed under two-photon excitation (Exc, 940nm). One- and two-photon fluorescence approaches were used to assess the binding of C1 to the neuronal NOS. Under one-photon excitation, the emission properties of the complex were rather difficult to interpret due to the overlapping between the intrinsic fluorescence of nNOS and C1 fluorescence. As the two-photon fluorescence of nNOS protein is very weak, the complex formation was measured by monitoring the two-photon fluorescence recovery of C1 upon binding to nNOS. The calculated Kd value was found to be consistent with the value characterizing NADPH binding. However, competition experiments suggest that the competition between C1 and NADPH is more complex than expected from a one-binding site model. Our results suggest that C1 represents a promising compound for cellular applications in two-photon fluorescence imaging.1 Beaumont et al., J. Am. Chem. Soc. 2007, 129, 2178-2186.2 Beaumont et al., ChemBioChem 2009, 10, 690-701.

Effect of Keto Acids on Asymmetric Dimethylarginine, Muscle, and Fat Tissue in Chronic Kidney Disease and After Kidney Transplantation

ASYMMETRIC DIMETHYLARGININE (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. By competitively displacing L-arginine from the substrate-binding site of NO synthase, ADMA interferes with many physiological functions of NO, such as endothelium-dependent vasodilatation and leukocyte adhesion. Asymmetric dimethylarginine and its biologically inactive regioisomer, symmetric dimethylarginine (SDMA), can be found in human plasma and urine in low concentrations. Concentrations of both dimethylarginines are increased in patients with end-stage renal disease, which may explain, at least in part, the endothelial dysfunction and cardiovascular complications in this patient population. In addition, the metabolism of ADMA, but not of SDMA, occurs via hydrolytic degradation to citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH).

Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase

Nitric oxide (NO) is synthesised by a two-step oxidation of l-arginine ( l-Arg) in the active site of nitric oxide synthase (NOS) with formation of an intermediate, N ω -hydroxy- l-Arg (NOHA). Crystal structures of NOSs have shown the importance of an active-site Val567 residue (numbered for rat neuronal NOS, nNOS) interacting with non-amino acid substrates. To investigate the role of this Val residue in substrate recognition and NO-formation activity by nNOS, we generated and purified four Val567 mutants of nNOS, Val567Leu, Val567Phe, Val567Arg and Val567Glu. We characterized these proteins and tested their ability to generate NO from the oxidation of natural substrates l-Arg and NOHA, and from N-hydroxyguanidines previously identified as alternative substrates for nNOS. The Val567Leu mutant displayed lower NO formation activities than the wild type (WT) in the presence of all tested compounds. Surprisingly, the Val567Phe mutant formed low amounts of NO only from NOHA. These two mutants displayed lower affinity for l-Arg and NOHA than the WT protein. Val576Glu and Val567Arg mutants were much less stable and did not lead to any formation of NO. These results suggest that Val567 is an important residue for preserving the integrity of the active site, for substrate binding, and subsequently for NO-formation in nNOS.

ADMA: a novel risk factor that explains excess cardiovascular event rate in patients with end-stage renal disease.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. By competitively displacing L-arginine from the substrate binding site of NO synthase, ADMA interferes with many of the physiological functions of NO, like endothelium-dependent vasodilation and leukocyte adhesion. ADMA, like its biologically inactive regioisomer, symmetric dimethylarginine (SDMA), can be found in human plasma and urine in low concentrations. The concentrations of both dimethylarginines are increased in patients with end-stage renal disease, which may explain at least in part endothelial dysfunction and cardiovascular complications in this patient population. In addition, the metabolism of ADMA, but not SDMA, occurs via hydrolytic degradation to citrulline and dimethylamine by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Data from experimental studies suggest that ADMA inhibits vascular NO elaboration at concentrations that can be measured in plasma of patients with renal disease. Interestingly, ADMA and SDMA are poorly eliminated during hemodialysis. This is probably due to a high level of binding of both molecules to plasma proteins. High ADMA concentrations in patients with end-stage renal disease may contribute to their excess cardiovascular event rate, as in clinical studies a relationship between ADMA and carotid artery intimal thickening was found. Moreover, in a prospective study we demonstrated recently that determination of ADMA plasma concentration is useful to predict future cardiovascular event rate and total mortality in this patient population. As other researchers reported observations that are in line with our findings, there is evidence that ADMA may be a novel cardiovascular risk factor.

Target recognition of apocalmodulin by nitric oxide synthase I peptides.

An increasing number of proteins are found that are regulated by the Ca(2+)-free state of calmodulin, apocalmodulin. Many of these targets harbor a so-called IQ motif within their primary sequence, but several target proteins of apocalmodulin lack this motif. We investigated whether the Ca(2+)-dependent calmodulin-binding site of nitric oxide synthase I could be transformed into a target site of apocalmodulin. Synthetic peptides representing the wild-type amino acid sequence and several peptides carrying mutations were studied by isothermal titration calorimetry and fluorescence spectroscopy. A single amino acid substitution of a negative charge to a positive charge can convert a classical Ca(2+)-dependent binding site of calmodulin into a target site for apocalmodulin. In addition, the introduction of hydrophobic amino acids increases the apparent binding affinity from the micromolar to the nanomolar range. Binding of wild-type and mutant peptides to Ca(2+)-calmodulin was enthalpically driven, and binding to apocalmodulin was entropically driven. Our data indicate that only a few selected amino acid positions in a calmodulin-binding site determine its Ca(2+) dependency.

Application of Different Surface Plasmon Resonance Biosensor Chips to Monitor the Interaction of the CaM-Binding Site of Nitric Oxide Synthase I and Calmodulin

Surface plasmon resonance biosensors depend on modified gold surfaces to allow immobilization of proteins or peptides for interaction analysis. We investigated sensor chip surfaces that differ in the geometry of the immobilization matrix: two contain a three-dimensional coupling matrix and two have a surface with immobilization sites on a two-dimensional plane. Properties of sensor chips were compared by studying the interaction of calmodulin with a peptide representing the calmodulin-binding site of nitric oxide synthase I. Apparent KD values were determined by three different procedures in order to apply tests for self-consistency. At low surface densities (5–8 fmol/mm2) on three of the four tested surfaces, estimated KD values were within one order of magnitude and similar to the value found in solution (KD = 1–3 nM). When immobilization densities were increased by one to two orders of magnitude, apparent association rate constants were less distorted on a flat carboxymethylated surface than on dextran-coated sensor chips.

Unusual Role of Tyr588 of Neuronal Nitric Oxide Synthase in Controlling Substrate Specificity and Electron Transfer

Nitric oxide (NO) is synthesized from l-Arg via NG-hydroxyl-l-Arg (NHA) in the heme active site of nitric oxide synthase (NOS). According to the crystal structure of other NOS isoforms, the carboxylate group of l-Arg hydrogen bonds to the hydroxyl group of the conserved Tyr588 residue in the heme distal site of neuronal NOS (nNOS). Indeed, the nNOS mutations Tyr588His, Tyr588Ser, and Tyr588Phe markedly increased the dissociation constants for l-Arg and NHA by 2.2–8.2-fold and 1.5–3.9-fold, respectively. Similarly, Tyr588His and Tyr588Ser mutations markedly decreased the l-Arg-driven NO formation rates by 50 and 30% than that of the wild type, respectively. However, the catalytic activities of the same mutants using NHA were higher than that of the wild type by up to 136%. As a result, the turnover ratio of NHA to l-Arg was 4.12 for the Tyr588Ser mutant, compared with 1.07 for the wild-type enzyme. Intriguingly, heme reduction rates for the Tyr588 mutants were much lower than for wild type by two orders of magnitude.

Chiral Recognition at the Heme Active Site of Nitric Oxide Synthase Is Markedly Enhanced byl-Arginine and 5,6,7,8-Tetrahydrobiopterin

The effects of substrate,l-Arg and cofactors, (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for theS-andR-enantiomers of ligand I (S/R) was 30, while theS/Rratio for ligand II and theR/Sratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 μM H4B. However, in the presence of 1 mMl-Arg, theS/Rratio of theKdvalues for ligand I was decreased down to 5.9. In the presence of both 1 mMl-Arg and 0.1 mM H4B, theS/Rratios for ligands I and II and theR/Sratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.

Astrocytes and Bergmann glia as an important site of nitric oxide synthase I

In the central nervous system nitric oxide appears to be critically involved in a number of physiological and pathological processes. Although there is convincing evidence for expression of nitric oxide synthase in cultured glial cells, demonstration of this enzyme in glial cells in situ remained largely unsatisfactory. In the present study we applied immunostaining to freeze-dried sections of snap-frozen hippocampi and cerebella of rats and to sections of freeze-dried brain tissue in order to minimize diffusion artefacts and thus to obtain more precise information about the true in situ localization of nitric oxide synthase. Here we show that astrocytes and Bergmann glia react strongly with antibodies raised against cerebellar nitric oxide synthase and against a type I nitric oxide synthase-specific C-terminal peptide, respectively. This finding was further substantiated by histochemical localization of NADPH-diaphorase activity in astrocytes and Bergmann glia as well as by immunoreactivity of both types of glia cells with antibodies to the NADPH-delivering enzyme glucose-6-phosphate dehydrogenase. We conclude, that astrocytes are important sites of nitric oxide synthase I in brain, suggesting that these cells might use nitric oxide as gaseous messenger molecule for various aspects of glia-neuron signalling. © 1996 Wiley-Liss, Inc.

Astrocytes and Bergmann glia as an important site of nitric oxide synthase I.

In the central nervous system nitric oxide appears to be critically involved in a number of physiological and pathological processes. Although there is convincing evidence for expression of nitric oxide synthase in cultured glial cells, demonstration of this enzyme in glial cells in situ remained largely unsatisfactory. In the present study we applied immunostaining to freeze-dried sections of snap-frozen hippocampi and cerebella of rats and to sections of freeze-dried brain tissue in order to minimize diffusion artefacts and thus to obtain more precise information about the true in situ localization of nitric oxide synthase. Here we show that astrocytes and Bergmann glia react strongly with antibodies raised against cerebellar nitric oxide synthase and against a type I nitric oxide synthase-specific C-terminal peptide, respectively. This finding was further substantiated by histochemical localization of NADPH-diaphorase activity in astrocytes and Bergmann glia as well as by immunoreactivity of both types of glia cells with antibodies to the NADPH-delivering enzyme glucose-6-phosphate dehydrogenase. We conclude, that astrocytes are important sites of nitric oxide synthase I in brain, suggesting that these cells might use nitric oxide as gaseous messenger molecule for various aspects of glia-neuron signalling.