Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.
J Mol Biol 2000 Oct 27
PMID:Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain. 1103 Nov 13

We recently described that melatonin and some kynurenines modulate the N-methyl-D-aspartate-dependent excitatory response in rat striatal neurons, an effect that could be related to their inhibition of nNOS. In this report, we studied the effect of melatonin and these kynurenines on nNOS activity in both rat striatal homogenate and purified rat brain nNOS. In homogenates of rat striatum, melatonin inhibits nNOS activity, whereas synthetic kynurenines act in a structure-related manner. Kynurenines carrying an NH(2) group in their benzenic ring (NH(2)-kynurenines) inhibit nNOS activity more strongly than melatonin itself. However, kynurenines lacking the NH(2) group or with this group blocked do not affect enzyme activity. Kinetic analysis shows that melatonin and NH(2)-kynurenines behave as noncompetitive inhibitors of nNOS. Using purified rat brain nNOS, we show that the inhibitory effect of melatonin and NH(2)-kynurenines on the enzyme activity diminishes with increasing amounts of calmodulin in the incubation medium. However, changes in other nNOS cofactors such as FAD or H(4)-biopterin, do not modify the drugs' response. These data suggest that calmodulin may be involved in the nNOS inhibition by these compounds. Studies with urea-polyacrylamide gel electrophoresis further support an interaction between melatonin and NH(2)-kynurenines, but not kynurenines lacking the NH(2) group, with Ca(2+)-calmodulin yielding Ca(2+)-calmodulin-drug complexes that prevent nNOS activation. The results show that calmodulin is a target involved in the intracellular effects of melatonin and some melatonin-related kynurenines that may account, at least in part, for the neuroprotective properties of these compounds.
Mol Pharmacol 2000 Nov
PMID:Structure-related inhibition of calmodulin-dependent neuronal nitric-oxide synthase activity by melatonin and synthetic kynurenines. 1104 43

We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [(3)H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [(3)H]2-amino-4-picoline bound saturably and with high affinity to human NOSs. Affinity constants (K(D) values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS isoforms (iNOS, nNOS, eNOS). Binding of [(3)H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity. L-NIL [L-N(6)-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective while L-NA (N(G)-nitro-L-arginine) and L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate assay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzyme inhibition and the inhibition of [(3)H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.
Mol Pharmacol 2000 Nov
PMID:The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes. 1104 50

Nitric oxide (NO) has been proposed as a mediator of cervical ripening. We investigated the expression, using Western blotting, and localization, using immunohistochemistry, of the nitric oxide synthase (NOS) enzymes, inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (bNOS) in the human cervix during pregnancy and parturition. Cervical biopsies were obtained from non-pregnant women, women in the first trimester of pregnancy, and pregnant women at term before and after the onset of labour. Each of the NOS isoforms was localized in the cervices of both non-pregnant and pregnant subjects using immunohistochemistry. iNOS expression was significantly greater in early pregnancy compared with the non-pregnant state (P: < 0.005). iNOS expression was up-regulated further in samples obtained in the third trimester compared with the first trimester. bNOS expression was greater in samples from the first trimester of pregnancy than in non-pregnant samples (P: < 0. 005), but showed no additional increase in late pregnancy or with the onset of labour. eNOS expression was increased in samples obtained in the third trimester both before (P: = 0.002) and after the onset of labour (P: < 0.002) when compared with non-pregnant samples. The increased expression of NOS isoforms in late pregnancy supports the hypothesis that NO is involved in the process of cervical ripening.
Mol Hum Reprod 2000 Nov
PMID:Changes in the expression of nitric oxide synthase in the human uterine cervix during pregnancy and parturition. 1104 68

Cyclooxygenase 2 (COX-2) inhibits nerve growth factor (NGF) withdrawal apoptosis in differentiated PC12 cells. The inhibition of apoptosis by COX-2 was concomitant with prevention of caspase 3 activation. To understand how COX-2 prevents apoptosis, we used cDNA expression arrays to determine whether COX-2 regulates differential expression of apoptosis-related genes. The expression of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase [PIN]) was significantly stimulated in PC12 cells overexpressing COX-2. The COX-2-dependent stimulation of DLC expression was, at least in part, mediated by prostaglandin E(2). Overexpression of DLC also inhibited NGF withdrawal apoptosis in differentiated PC12 cells. Stimulation of DLC expression resulted in an increased association of DLC/PIN with neuronal nitric oxide synthase (nNOS), thereby reducing nNOS activity. Furthermore, nNOS expression and activity were significantly increased in differentiated PC12 cells after NGF withdrawal. This increased nNOS activity as well as increased nNOS dimer after NGF withdrawal were inhibited by COX-2 or DLC/PIN overexpression. An nNOS inhibitor or a membrane-permeable superoxide dismutase (SOD) mimetic protected differentiated PC12 cells from NGF withdrawal apoptosis. In contrast, NO donors induced apoptosis in differentiated PC12 cells and potentiated apoptosis induced by NGF withdrawal. The protective effects of COX-2 on apoptosis induced by NGF withdrawal were also overcome by NO donors. These findings suggest that COX-2 promotes cell survival by a mechanism linking increased expression of prosurvival genes coupled to inhibition of NO- and superoxide-mediated apoptosis.
Mol Cell Biol 2000 Nov
PMID:Cyclooxygenase 2 promotes cell survival by stimulation of dynein light chain expression and inhibition of neuronal nitric oxide synthase activity. 1104 52

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.
Mol Cells 2000 Oct 31
PMID:5'-Flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene. 1110 Nov 49

Recent data suggest that the neuronal isoform of nitric oxide synthase (nNOS) and glutamate receptors of the N-methyl-D-aspartate (NMDA) type are physically coupled and, hence, functionally interrelated. Several alternatively spliced isoforms of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit and the neuronal nitric oxide synthase (nNOS) are known, and recent studies have shown that a spliced C-terminal may be responsible for the coupling of NMDAR's to nNOS via its PDZ domain and the postsynaptic density protein PSD95. However, little is known about whether and to what extent changes in nNOS expression influence NMDA receptor density or function. We have therefore compared the localization of nNOS alpha, beta and gamma with that of two relevant NMDAR1 splice variants in wild-type mice versus knockout mice deficient in nNOS alpha, generated by homologous recombination with a targeted deletion of exon 2, containing one PDZ domain (nNOS alpha(Delta/Delta) mice). Whereas nNOS alpha was completely absent in nNOS alpha(Delta/Delta) mice, nNOS beta and gamma were expressed in both wild-type and knockout animals. nNOS gamma mRNA, though, was hardly detectable, if at all, mainly within the olfactory bulb, the cerebellum and mesencephalic nuclei of knockout animals. The expression of the NMDAR1-1 splice variant (without any short carboxy-terminal amino acid motif, recognized by PDZ domains) was remarkably decreased in striatal, cortical, hippocampal and cerebellar tissue in nNOS alpha(Delta/Delta) animals, but no changes in NMDAR1-4 (with an alternatively spliced C-terminal and thus with a PDZ binding motif) mRNA and protein levels were observed. While NMDAR1-4 may be related to receptor targeting and clustering to PSD95 and to nNOS, our data suggest that differences in nNOS expression obviously do not directly influence gene expression of this particular NMDAR splice variant. Otherwise, the observed diminution of NMDAR1-1 splice variant mRNA and protein levels may, at least partially, explain the decreased vulnerability of nNOS alpha(Delta/Delta) mice to glutamate-mediated neurotoxicity.
Brain Res Mol Brain Res 2000 Dec 28
PMID:Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS alpha (nNOS alpha(Delta/Delta) mice). 1114 2

Caveolin-3 is the muscle-specific isoform of the caveolin protein family, which is a major component of caveolae, small membrane invaginations found in most cell types. Caveolins play important roles in the formation of caveola membranes, acting as scaffolding proteins to organize and concentrate lipid-modified signaling molecules, and modulate a signaling pathway. For instance, caveolin-3 interacts with neuronal nitric oxide synthase (nNOS) and inhibits its catalytic activity. Recently, specific mutations in the caveolin-3 gene, including the Pro104Leu missense mutation, have been shown to cause an autosomal dominant limb-girdle muscular dystrophy (LGMD1C), which is characterized by the deficiency of caveolin-3 in the sarcolemma. However, the molecular mechanism by which these mutations cause the deficiency of caveolin-3 and muscle cell degeneration remains elusive. Here we generated transgenic mice expressing the Pro104Leu mutant caveolin-3. They showed severe myopathy accompanied by the deficiency of caveolin-3 in the sarcolemma, indicating a dominant negative effect of mutant caveolin-3. Interestingly, we also found a great increase of nNOS activity in their skeletal muscle, which, we propose, may play a role in muscle fiber degeneration in caveolin-3 deficiency.
Hum Mol Genet 2001 Feb 01
PMID:Transgenic mice expressing mutant caveolin-3 show severe myopathy associated with increased nNOS activity. 1115 34

Dyneins are multi-subunit molecular motors that translocate molecular cargoes along microtubules. Other than acting as an essential component of the dynein motor complex, the 89-residue subunit of dynein light chain (DLC8) also regulates a number of other biological events by binding to various proteins and enzymes. Currently known DLC8 targets include neuronal nitric oxide synthase; the proapoptotic Bcl-2 family member protein designated Bim; a Drosophila RNA localization protein Swallow, myosin V, neuronal scaffolding protein GKAP, and IkappaBalpha, an inhibitor of the NFkappaB transcription factor. The DLC8-binding domains of the various targets are confined within a short, continuous stretch of amino acid residues. However, these domains do not share any obvious sequence homology with each other. Here, the three-dimensional structures of DLC8 complexed with two peptides corresponding to the DLC8-binding domains of neuronal nitric oxide synthase and Bim, respectively, were determined by NMR spectroscopy. Although the two DLC8-binding peptides have entirely different amino acid sequences, both peptides bind to the protein with a remarkable similar conformation by engaging the symmetric DLC8 dimer through antiparallel beta-sheet augmentation via the beta2 strand of the protein. Structural comparison indicates that the two target peptides use different regions within the conformational flexible peptide-binding channels to achieve binding specificity. We have also re-determined the apo-form solution structure of DLC8 in this work. The structures of the DLC8/target peptide complexes, together with the dynamic properties of the protein, provide a molecular basis of DLC8's diverse amino acid sequence-dependent target recognition.
J Mol Biol 2001 Feb 09
PMID:Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain. 1117 96

We used Western blotting to analyse the expression of different isoforms of nitric oxide synthase (NOS) in the rat vestibular nucleus complex (VNC) at various times following unilateral vestibular deafferentation (UVD), together with a radioenzymatic assay to compare NOS activity at the same time points. nNOS expression did not change significantly in the ipsilateral or contralateral VNC at any time following UVD. However, eNOS expression decreased significantly (P<0.05) in the contralateral VNC at 6 h post-UVD, recovering to normal levels by 50 h. iNOS was not expressed at any time following UVD. NOS activity demonstrated a significant increase in the contralateral VNC at 6 h post-UVD (P<0.05), recovering toward normal levels by 50 h.
Brain Res Mol Brain Res 2001 Mar 31
PMID:Differences in NOS protein expression and activity in the rat vestibular nucleus following unilateral labyrinthectomy. 1129 43


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