The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G

The final step of molybdenum cofactor biosynthesis in plants is catalyzed by the two-domain protein Cnx1. The G domain of Cnx1 (Cnx1G) binds molybdopterin with high affinity and transfers molybdenum to molybdopterin. Here, we describe the functional and structural characterization of structure-based Cnx1G mutants. For molybdopterin binding residues Thr542 and Ser573 were found to be important because different mutations of those residues resulted in 7- to 26-fold higher k(D) values for molybdopterin binding. Furthermore, we showed that the terminal phosphate of molybdopterin is directly involved in protein-pterin interactions as dephosphorylated molybdopterin binds with one magnitude of order lower affinity to the wild-type protein. Molybdopterin binding was not affected in mutants defective in Ser476, Asp486, or Asp515. However, molybdenum insertion was completely abolished, indicating their important role for catalysis. Based on these results we propose the binding of molybdopterin to a large depression in the structure of Cnx1G formed by beta5, alpha5, beta6, and alpha6, whereas the negatively charged depression formed by the loop between beta3 and alpha4, the N-terminal end of alpha2, the 3(10) helix, and the region between beta6 and alpha6 is involved in catalysis.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase

The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.     

Chorein deficiency leads to upregulation of gephyrin and GABA(A) receptor

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. Recently, using a gene-targeting technique to delete exons 60-61, we produced a ChAc-model mouse that corresponds to a human disease mutation. In this study, a comparative microarray analysis of gene expression in the striatum revealed an increased level of gephyrin gene expression in the ChAc-model mice compared with wild type mice. Since gephyrin is known as a GABA(A) receptor-anchoring protein, we compared the protein-level expression and localization of gephyrin and the GABA(A) receptor alpha1 (GABRA1) and gamma2 (GABRG2) subunits. Gephyrin and GABRG2 immunoreactivities in the striatum and hippocampus of the ChAc-model mice were significantly higher than those in the wild types. Our results suggest that chorein functional loss may lead to a compensatory upregulation of gephyrin and GABRG2 in the pathologic condition in ChAc.     

The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli

In the second step of the molybdenum cofactor (Moco) biosynthesis in Escherichia coli, the l-cysteine desulfurase IscS was identified as the primary sulfur donor for the formation of the thiocarboxylate on the small subunit (MoaD) of MPT synthase, which catalyzes the conversion of cyclic pyranopterin monophosphate to molybdopterin (MPT). Although in Moco biosynthesis in humans, the thiocarboxylation of the corresponding MoaD homolog involves two sulfurtransferases, an l-cysteine desulfurase, and a rhodanese-like protein, the rhodanese-like protein in E. coli remained enigmatic so far. Using a reverse approach, we identified a so far unknown sulfurtransferase for the MoeB-MoaD complex by protein-protein interactions. We show that YnjE, a three-domain rhodanese-like protein from E. coli, interacts with MoeB possibly for sulfur transfer to MoaD. The E. coli IscS protein was shown to specifically interact with YnjE for the formation of the persulfide group on YnjE. In a defined in vitro system consisting of MPT synthase, MoeB, Mg-ATP, IscS, and l-cysteine, YnjE was shown to enhance the rate of the conversion of added cyclic pyranopterin monophosphate to MPT. However, YnjE was not an enhancer of the cysteine desulfurase activity of IscS. This is the first report identifying the rhodanese-like protein YnjE as being involved in Moco biosynthesis in E. coli. We believe that the role of YnjE is to make the sulfur transfer from IscS for Moco biosynthesis more specific because IscS is involved in a variety of different sulfur transfer reactions in the cell.     

Glycine receptor activation regulates short-term plasticity in CA1 area of hippocampal slices of rats

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their roles in synaptic transmission are unclear. In this study, we examined the effect of GlyR activation on paired-pulse stimulation of the whole-cell postsynaptic currents (PSCs) in the Schaffer-CA1 synapses in rat hippocampal slices. Bath application of glycine reduced the amplitude of PSCs, accompanied by an increase in holding current and resting conductance. Moreover, glycine application increased the paired-pulse ratio (PPR) of PSCs significantly, an effect largely abolished by the GlyR specific antagonist strychnine. Interestingly, glycine application had no significant effect on either the amplitude or the PPR of excitatory postsynaptic currents (EPSCs). Our findings suggest that GlyR activation regulates hippocampal short-term plasticity by altering GABAergic neurotransmission.     

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.     

Molecular mapping of the cnx2 locus involved in molybdenum cofactor biosynthesis in rice (Oryza sativa L.)

Molybdenum cofactor (Moco) is essential for nitrate reductase (NR), xanthine dehydrogenase (XDH), and aldehyde oxidase to perform their catalytic functions in plants. Moco biosynthesis is a complex process involving many genes. Little is known about the genetics and molecular aspects of Moco biosynthesis in plants and other eukaryotes. In rice, we previously isolated a Moco mutant C25 with a mutation in the CNX2 gene from a mutagenized indica cultivar IR30 and characterized its biochemical properties. This mutant was crossed with a japonica cultivar, Norin 8, to investigate the linkage of cnx2 to restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) markers. Chlorate resistance was used to trace the cnx2 mutation because of its cosegregation with the loss of NR and XDH activities observed earlier. RFLP and CAPS analyses show the location of the cnx2 locus on the long arm of chromosome 4. It is mapped between RFLP markers C513 and C377 with a distance of 9.5 and 13.1 cM, respectively. It is also linked with CAPS marker RA0738 at a distance of 30.3 cM. Received: 25 June 2000 / Accepted: 31 August 2000     

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

Summary The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 3₁₀-helicoid structure in which the E⁶...R¹⁰ ionic bridge stabilizes the C-terminus.     

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding tothe YLGYLEQLLR fragment of a casein has been examined in a sodium dodecylsulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamicssimulation. The decapeptide adopts an amphipathic 3₁₀-helicoidstructure in which theE⁶···R¹⁰ ionic bridgestabilizes the C-terminus.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Anxiolytic-like effects of sinapic acid in mice

Sinapic acid is a phenylpropanoid compound and is found in various herbal materials and high-bran cereals. With the exception of its antioxidant activities, the pharmacological properties of sinapic acid have been rarely reported. The purpose of this study was to characterize the putative anxiolytic-like properties of sinapic acid using an elevated plus-maze (EPM) and hole-board test. Control mice were orally treated with an equal volume of vehicle (10% Tween 80 solution), and positive control mice were treated with diazepam (1 mg/kg, i.p.). Sinapic acid (4 mg/kg, p.o.) significantly increased the percentages of time spent in the open arms of the EPM test (P<0.05). In the hole-board test, sinapic acid also significantly increased the number of head-dips at 4 mg/kg (P<0.05). In addition, the anxiolytic-like properties of sinapic acid examined in the EPM test were blocked by flumazenil or bicuculline, which are GABA(A) antagonists. Moreover, sinapic acid markedly potentiated GABA current in single cortical neurons in a dose-dependant manner, and reactive I(GABA) increased to 1.8 times at 1 muM of sinapic acid. These results suggested that sinapic acid is a prominent anxiolytic agent, and that its anxiolytic-like effects are mediated via GABA(A) receptors and potentiating Cl(-) currents.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.