Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide synthases (NOSs) are composed of a flavin-containing reductase domain and a heme-containing oxygenase domain. Each NOS enzyme also contains a calmodulin (CaM) binding domain and requires bound calmodulin for enzymatic activity. The CaM binding properties of the different NOS isozymes differ in the need for free calcium ions (Ca2+). We investigated CaM binding using reductase domains from the human and mouse inducible as well as the rat neuronal isoforms of NOS. An Escherichia coli expression system was designed to generate truncated recombinant NOS proteins for each isoform in which an extended CaM binding domain was either included or deleted. The reductase domains with the extended N-terminal CaM binding domains of human iNOS (residues 480-1153) and mouse iNOS (residues 474-1144) show Ca2+ binding properties that are similar to their respective holoenzymes. In addition, the iNOS reductases were active in the presence or absence of CaM. Thus, CaM does not stimulate NADPH utilization of the reductase domain in iNOS enzymes. In contrast, the rat nNOS reductase enzymes showed Ca2+-dependent CaM binding and activation.
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PMID:The reductase domain of the human inducible nitric oxide synthase is fully active in the absence of bound calmodulin. 980 67

The primary sequences of the three mammalian nitric- oxide synthase (NOS) isoforms differ by the insertion of a 52-55-amino acid loop into the reductase domains of the endothelial (eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of.NO formation and calmodulin (CaM) binding (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777), the insert has been proposed to be an autoinhibitory element. We have examined the role of the insert in its native protein context by deleting the insert from both wild-type eNOS and from chimeras obtained by swapping the reductase domains of the three NOS isoforms. The Ca2+ concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of autoinhibition. Furthermore, removal of the insert greatly enhances the maximal activity of wild-type eNOS, the least active of the three isoforms. Despite the correlation between reductase and overall enzymatic activity for the wild-type and chimeric NOS proteins, the loop-free eNOS still requires CaM to synthesize.NO. However, the reductive activity of the CaM-free, loop-deleted eNOS is enhanced significantly over that of CaM-free wild-type eNOS and approaches the same level as that of CaM-bound wild-type eNOS. Thus, the inhibitory effect of the loop on both the eNOS reductase and. NO-synthesizing activities may have an origin distinct from the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and.NO-synthesizing activities. The eNOS insert not only inhibits activation of the enzyme by CaM but also contributes to the relatively low overall activity of this NOS isoform.
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PMID:Autoinhibition of endothelial nitric-oxide synthase. Identification of an electron transfer control element. 1032 64

Cimetidine (CIM) is an H2-receptor antagonist that has been used in racehorses in an attempt to reduce the occurrence of stress-related gastric ulceration. It has also been shown to produce several useful effects other than its gastric acid suppression properties. Further, it is a well documented antagonist of cytochrome P-450 (CYP) mediated oxygenation reactions. Nitric oxide (NO), a recently discovered mediator or modifier of numerous physiological functions, is generated by several forms of nitric oxide synthase (NOS), one of which is inducible (iNOS). Inducible NOS, expressed in neutrophils and macrophages as part of the inflammatory response to noxious stimuli, contains both a CYP and a CYP reductase domain. Because of the similarity of structure of iNOS and CYP, it was decided to determine whether CIM could reduce NO production, using a carrageenan inflammation model in the horse. Two experiments were conducted. In Trial 1, six female Thoroughbred horses each had three tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into three treatments: 0.9% NaCl (NaCI), CIM (3 mg/kg), and aminoguanidine (AG; 25 mg/kg), an inhibitor of iNOS. Each mare received three i.v. injections 12 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and tissue chamber fluid (TCF) were collected serially. Concentrations of NO3- (the major metabolite of NO), albumin, total protein, CIM and AG were measured and complete cell counts and differentials were conducted. Trial 2 also used six female Thoroughbred horses implanted with at least two tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into two treatments: NaCl (0.9%) and CIM (6 mg/kg). Each mare received seven i.v. injections of either NaCl or CIM 8 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and TCF were collected serially as before, and analysed for NO3- and CIM content. Areas under the curve (AUC) of the different parameters were calculated for the periods of -1-1, -1-3 and -1-7 days (Trial 1) and -2-1 for Trial 2. Absolute values were also compared at 4, 8 and 12 h postcarrageenan. Saline treatment did not reduce the elevated concentrations of NO3- in either plasma or TCF. Plasma, test chamber and control chamber NO3-concentrations rose from 0 to 12 h, and were very similar in all three sampled fluids. Cimetidine significantly (P< or =0.05) decreased NO3- production in plasma over the periods of -1-1, -1-3, and -1-7 days post inflammation when compared to NaCl treatment in Trial 1. Aminoguanidine and CIM decreased NO3-production in TCF for the periods -1-1, 1-3, and -1-7 days post inflammation in Trial 1 and -2-1 for Trial 2. Both CIM and AG also significantly reduced NO3-concentrations in plasma and TCF at 12 h postinitiation (Trials 1 and 2). Thus CIM, at the doses studied, was capable of reducing NO3- concentrations in this model as effectively as AG, a relatively specific inhibitor of iNOS activity.
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PMID:Cimetidine inhibits nitric oxide associated nitrate production in a soft-tissue inflammation model in the horse. 1037 98

The Ca(2+)-calmodulin system controls the neuronal and endothelial isoforms of NOS, whereas the inducible isoform is calcium independent apparently because CaM is a tightly bound subunit of iNOS. The canonical CaM-binding site is located between the oxygenase and reductase NOS domains. CaM controls eNOS dimerization rather then iNOS one. The proteins with the so-called "IQ" motif bind calmodulin in a Ca(2+)-independent manner. This group of proteins does not include iNOS, which has the canonical CaM-binding motif. In the experiments with synthetic peptides was demonstrated that the interaction between the calmodulin and CaM-binding site of iNOS does not depend on the Ca2+ concentration. On the other hand, in the experiments with fusion, mutant and truncated NOSs was shown that these features of CaM-binding region of iNOS is not enough for the enzyme to bind calmodulin Ca(2+)-independently; this interaction requires the additional binding sites both in reductase and oxygenase domains of iNOS. In the experiments with fusion calmodulins the mechanism of calmodulin regulation of electron transfer in NOS was elaborated. The concept of autoinhibitory control element in the FMN-binding site of constitutive NOS is discussed.
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PMID:[Mechanisms of regulation by calmodulin of nitric oxide synthase]. 1043 53

Nitric oxide synthase oxygenase domains (NOS(ox)) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS(ox) reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal beta-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra- to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS(ox) molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.
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PMID:N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization. 1056 39

Statins are lipid-lowering agents which act by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme is responsible for the conversion of HMG-CoA to mevalonate. Products of mevalonate metabolism are critical for several cellular processes of eukaryotic cells, and inhibition of the mevalonate pathway by statins has pleiotropic effects. It has been reported that statins inhibit the migration and proliferation of vascular smooth cells (VSMCs) and macrophages, decrease interleukin-6 and inducible nitric oxide synthase expression in VSMCs, improve endothelial function and up-regulate endothelial nitric oxide synthase expression. The above effects of statins are independent of plasma cholesterol levels, and are completely blocked by exogenous mevalonate and some isoprenoids. These findings suggest that, in addition to their effects on plasma lipids, statins exert direct antiatherosclerotic effects on the cells primarily involved in atherosclerosis.
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PMID:Direct effects of statins on cells primarily involved in atherosclerosis. 1077 Feb 67

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H(2)O(2)-supported oxidation of N(omega)-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280-Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280-Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.
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PMID:Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions. 1151 17

Postischemic acute renal failure (ARF) is common and often fatal. Cellular mechanisms include cell adhesion, cell infiltration and generation of oxygen free radicals, and inflammatory cytokine production. Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ("statins") directly influence inflammatory mechanisms. The hypothesis that ischemia-induced ARF could be ameliorated with statin treatment was investigated and possible molecular mechanisms were analyzed in a uninephrectomized rat model. Male Sprague-Dawley rats were pretreated with cerivastatin (0.5 mg/kg) or vehicle for 3 d. Ischemic ARF was induced by left renal artery clipping for 45 min, while the right kidney was being removed. After 24 h of ARF, serum creatinine levels were increased 7.5-fold in vehicle-treated control animals with ARF, compared with sham-operated animals (P < 0.005). Statin treatment reduced the creatinine level elevation by 40% (P < 0.005). Simultaneously, ischemia-induced severe decreases in GFR were significantly ameliorated by statin treatment (sham operation, 0.95 +/- 0.09 ml/min, n = 13; ischemia without treatment, 0.06 +/- 0.02 ml/min, n = 9; ischemia with statin pretreatment, 0.21 +/- 0.03 ml/min, n = 11; P < 0.001). Furthermore, statin pretreatment prevented the occurrence of tubular necrosis, with marked loss of the brush border, tubular epithelial cell detachment, and tubular obstruction in the S3 segment of the outer medullary stripe. In addition, monocyte and macrophage infiltration was almost completely prevented, intercellular adhesion molecule-1 upregulation was greatly decreased, and inducible nitric oxide synthase expression was reduced. Fibronectin and collagen IV expression was reduced, approaching levels observed in sham-operated animals. In vehicle-treated rats with ARF, mitogen-activated protein kinase extracellular activated kinase-1/2 activity was increased and the transcription factors nuclear factor-kappaB and activator protein-1 were activated. Statin treatment reduced this activation toward levels observed in sham-operated rats. The data suggest that hydroxy-3-methylglutaryl coenzyme A reductase inhibition protects renal tissue from the effects of ischemia-reperfusion injury and thus reduces the severity of ARF. The chain of events may involve anti-inflammatory effects, with inhibition of mitogen-activated protein kinase activation and the redox-sensitive transcription factors nuclear factor-kappaB and activator protein-1.
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PMID:Postischemic acute renal failure is reduced by short-term statin treatment in a rat model. 1219 73

Relatively new targets in drug design projects in cancer pharmacology include cytostatic agents, immune system modulators, and angiogenesis inhibitors. Preventive oncology applies pharmacological agents to reverse, retard, or halt progression of neoplastic cells to invasive malignancy. Prevention of cancer, however, can be accomplished through many strategies, including changes in diet and lifestyle. For example, the vast majority of lung cancers (80-90%) can be attributed to cigarette smoking and therefore, the most effective primary preventive strategy for lung cancer is to quit smoking. Chemoprevention through interruption of multistage careinogenesis include different molecular targets. Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists. Ligands for the peroxisome proliferator-activated receptor gamma (PPAR-gamma) suppress breast carcinogenesis in experimental models and induce differentiation of human liposarcoma cells. Selective PPAR modulators (SPARMs), by analogy to the SERM concept, are designed to have desired effects on specific genes relevant to carcinogenesis. Enzymatic approach in endocrine-related tumors involve inhibition of aromatase to prevent breast cancer and inhibition of 5-alpha-reductase to prevent prostate cancer. Down-regulation of inflammatory prostaglandin synthesis by inhibition of cyclooxygenase-2 (COX-2). inhibition of the inducible nitric oxide synthase (iNOS), and stimulation of phase II detoxication system, are currently examined in experimental models and clinical trials. Overall, potential targets in preventive strategies to reduce the risk of cancer involve agonists of endocrine receptors, factors down-regulating inflammation, factors inducing programmed cell death (PCD)/apoptosis, enzymatic inhibitors and gene therapy.
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PMID:Current strategies for anticancer chemoprevention and chemoprotection. 1266 76

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.
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PMID:Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity. 1280 87


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