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PubMed1999

Interference with brachyury function inhibits convergent extension, causes apoptosis, and reveals separate requirements in the FGF and activin signalling pathways.

Brachyury plays a key role in mesoderm formation during vertebrate development. Absence of the gene results in loss of posterior mesoderm and failure of the notochord to differentiate, while misexpression of Brachyury in the prospective ectoderm of Xenopus results in ectopic mesoderm formation. Brachyury is therefore both necessary and sufficient for posterior mesoderm formation. Here we present a detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development. Our results show that Brachyury is required for the convergent extension movements of gastrulation, for mesoderm differentiation in response to FGF, and for the survival of posterior mesodermal cells in both Xenopus and mouse.

Activins↗
PubMed2003

Identification and expression of alpha cDNA encoding human 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD): a key enzyme for the tryptophan-niacine pathway and quinolinate hypothesis.

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from 2-amino-3-carboxymuconate-6-semialdehyde (ACMS). 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) is the only known enzyme which can process ACMS to a benign catabolite and thus prevent the accumulation of quinolinate from ACMS. ACMSD seems to be regulated by nutritional and hormonal signals, but its molecular mechanism has, to date, been largely unknown. Utilizing partial amino acid sequences obtained from highly purified porcine kidney ACMSD, a cDNA encoding human ACMSD was cloned and characterized. The cDNA encodes a unique open reading frame of 336 amino acids and displays little homology to any known enzymes or motifs in mammalian databases, suggesting that ACMSD may contain a new kind of protein fold. Real-time PCR-based quantification of ACMSD revealed very low but significant levels of the expression in the brain. Brain ACMSD messages was down- and up-regulated in response to low protein diet and streptozocin-induced diabetes, respectively. Expression of QPRT, another enzyme which catabolizes quinolinate, was also found in the brain. This suggests that a pathway does exist by which the levels of quinolinate in the brain are regulated. In this report, we address the molecular basis underlying quinolinate metabolism and the regulation of ACMSD expression.

Amino Acid Sequence↗
PubMed2000

Relaxation of human ureteral smooth muscle in vitro by modulation of cyclic nucleotide-dependent pathways.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

Colforsin↗
PubMed2006

Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.

Animals↗
PubMed2003

Computational analysis of the evolution of the structure and function of 1-deoxy-D-xylulose-5-phosphate synthase, a key regulator of the mevalonate-independent pathway in plants.

We investigated molecular evolution of 1-deoxy-D-xylulose-5-phosphate synthase (DXS), an important regulatory enzyme of the mevalonate-independent pathway involved in terpenoid biosynthesis. Sequence alignment showed that some regions, likely to be functionally important, were highly conserved among all of the plant DXS sequences analysed. Phylogenetic trees were inferred using DXS sequences from 11 species of Angiosperms and showed the division of the sequences into two classes. Clustering of DXS sequences did not correspond to phylogenetic relationships among the plant species studied. There was no consistency in the similarity of the variable regions in the secondary structure of the DXS functional protein except for Capsicum and Lycopersicon, both members of the Solanaceae. Hydrophobicity plots for the functional region of DXS revealed great similarity in their hydrophobic structure, consistent with the phylogenetic trees inferred, and with eight prominent hydrophilic and hydrophobic peaks. We also observed a consistent set of features common to the DXS transit peptides studied. These features were the same hydrophobic slope, a hydrophobic region in residues 35-45, and, in eight of 12 sequences, a Pro-Pro-Thr sequence at the C-terminal end. The transit sequences are likely bipartite and contain features that suggest the DXS protein is not only targeted to the chloroplast, but also to the thylakoid. To our knowledge this is the first suggestion that DXS is located specifically in the chloroplast thylakoid.

Amino Acid Sequence↗
PubMed2003

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.

Cell Death↗
PubMed1999

Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Acetyl Coenzyme A↗
PubMedDate not supplied

WIN55212-2 docking to the CB1 cannabinoid receptor and multiple pathways for conformational induction.

Key pharmacophoric elements for the (aminoalkyl)indole (AAI) CB1 cannabinoid receptor agonists are the aminoalkyl moiety, the lipophilic aroyl group, and the heterocyclic indole ring. In the present study, the docking space allowed for (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2; 1) within the CB1 receptor was extensively explored by a docking approach that combines Monte Carlo (MC) and molecular dynamics (MD) simulations. The goals were to understand the key binding interactions of AAIs within the CB1 receptor and to examine the role of the ligand in inducing a receptor conformational change. From the findings of extensive SAR studies on the cannabinoid compounds and correlation between AAI binding affinity data and calculated binding energies, we proposed two alternative binding conformations, aroyl-up1 and aroyl-up2. These denote the directionality of the ligand naphthyl ring within the receptor upward with respect to the extracellular side. A comprehensive structural analysis of 1 demonstrated that the aroyl ring moiety could be important as the steric trigger for inducing CB1 receptor conformational change. Thus, it appears that aromatic-aromatic interactions are important not only for the binding of 1 but also for inducing receptor conformational change. It is possible that differences in the nature of the ligand binding could contribute to ligand-specific conformational changes in the receptor.

Benzoxazines↗
PubMed2005

Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway.

17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90, results in cell type-specific inhibition of proliferation of leukemic cells. GTP14564 is a tyrosine kinase inhibitor actively against FLT3. The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the role of FLT3 in their inhibitory effects. The importance of FLT3 mutations was demonstrated using small interfering RNA (siRNA) targeted to FLT3. Similar to FLT3 siRNA, GTP14564 inhibited FLT3 internal tandem duplication (ITD) cells (MV4;11) and FLT3 amplified wild-type cells (SEMK2-M1), but not wild-type FLT3 cells (RS4;11). However, when RS4;11 cells were stimulated with FLT3-ligand, phosphorylation of STAT5 and GTP14564 inhibition were observed. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells. We observed synergistic effects of combined 17-AAG and GTP14564 in cell lines with FLT3-ITD and amplified wild-type FLT3. Combined treatment with 17-AAG and GTP14564 reduced the levels of p-FLT3 and p-STAT5, enhanced G0/G1 arrest and apoptosis in FLT3-ITD and amplified wild-type FLT3. The combination of 17-AAG with FLT3 kinase inhibitors can enhance targeted therapy in leukemias with FLT3 mutations, such as MLL fusion gene leukemias.

Apoptosis↗
PubMed2001

Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway.

The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l-galactosyl residues, catalyzed by a largely unknown GDP-d-mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-d-mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-d-mannose 5"-epimerase. The plant GDP-d-mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

Amino Acid Sequence↗
PubMed1997

Heterologous expression, isolation, and characterization of versicolorin B synthase from Aspergillus parasiticus. A key enzyme in the aflatoxin B1 biosynthetic pathway.

Aflatoxin B1 is a potent environmental carcinogen produced by certain strains of Aspergillus. Central to the biosynthesis of this mycotoxin is the reaction catalyzed by versicolorin B synthase (VBS) in which a racemic substrate, versiconal hemiacetal, is cyclized to an optically active product whose absolute configuration is crucial to the interaction of aflatoxin B1 with DNA. Attempted over-production of VBS in Escherichia coli led principally to protein aggregated into inclusion bodies but also small amounts of soluble but catalytically inactive enzyme. Comparisons to wild-type VBS by SDS-polyacrylamide gel electrophoresis and after N-glycosidase F treatment revealed that extensive glycosylation accounted for the mass discrepancy (7,000+/-1,500 Da) between the native and bacterially expressed proteins. Several over-expression systems in Saccharomyces cerevisiae were surveyed in which one that incorporated a secretion signal was found most successful. VBS of indistinguishable mass on SDS-polyacrylamide gel electrophoresis and kinetic properties from the wild-type enzyme could be obtained in 50-100-fold greater amounts and whose catalytic behavior has been examined. The translated protein sequence of VBS showed three potential N-glycosylation sites (Asn-Xaa-Ser/Thr) consistent with the modifications observed above and unexpectedly revealed extensive homology to the ADP-binding region prominently conserved in the glucose-methanol-choline (GMC) family of flavoenzymes. Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex biosynthetic pathway.

Aflatoxin B1↗
PubMed2002

Identification and expression of a cDNA encoding human alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD). A key enzyme for the tryptophan-niacine pathway and "quinolinate hypothesis".

Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS). Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is the only known enzyme that can process ACMS to a benign catabolite and thus prevent the accumulation of quinolinate from ACMS. ACMSD seems to be regulated by nutritional and hormonal signals, but its molecular mechanism has, to date, been largely unknown. Utilizing partial amino acid sequences obtained from highly purified porcine kidney ACMSD, a cDNA encoding human ACMSD was cloned and characterized. The cDNA encodes a unique open reading frame of 336 amino acids and displays little homology to any known enzymes or motifs in mammalian databases, suggesting that ACMSD may contain a new kind of protein fold. Real-time PCR-based quantification of ACMSD revealed very low but significant levels of the expression in the brain. Brain ACMSD messages were down- and up-regulated in response to low protein diet and streptozocin-induced diabetes, respectively. The enzyme activities measured from partially purified brains were closely correlated with the changes in the message levels. Expression of quinolinate phosphoribosyltransferase (QPRT), another enzyme that catabolizes quinolinate, was also found in the brain. This suggests that a pathway does exist by which the levels of quinolinate in the brain are regulated. In this report, we address the molecular basis underlying quinolinate metabolism and the regulation of ACMSD expression.

Amino Acid Sequence↗
PubMed2003

The transcription factors HIF-1 and HNF-4 and the coactivator p300 are involved in insulin-regulated glucokinase gene expression via the phosphatidylinositol 3-kinase/protein kinase B pathway.

Glucokinase plays a key role in the regulation of glucose utilization in liver and its expression is strongly enhanced by insulin and modulated by venous pO(2). In primary rat hepatocytes, pO(2) modulated insulin-dependent glucokinase (GK) gene expression was abolished by wortmannin an inhibitor of phosphatidylinositol 3-kinase (PI3K). Transfection of vectors encoding the p110 catalytic subunit of PI3K or constitutively active protein kinase B (PKB) stimulated GK mRNA and protein expression. The transfection of GK promoter constructs together with expression vectors for p110 or constitutively active PKB revealed that the GK promoter region -87/-80 mediates the response to PI3K/PKB. Transfection experiments and gel shift assays show that this element is able to bind hypoxia-inducible factor-1 (HIF-1) in a hypoxia- and PKB-dependent manner. The ability of HIF-1alpha to activate the GK promoter was enhanced by hepatocyte nuclear factor-4alpha (HNF-4alpha), acting via the sequence -52/-39, and by the coactivator p300. Stimulation of the GK promoter by insulin was dependent on the intact -87/-80 region and maximal stimulation was achieved when HIF-1alpha, HNF-4, and p300 were cotransfected with the -1430 GK promoter Luc construct in primary hepatocytes. Maximal stimulation of GK promoter activity by insulin was inhibited when a p300 vector was used containing a mutation within a PKB phosphorylation site. Thus, a regulatory transcriptional complex consisting of HIF-1, HNF-4, and p300 appears to be involved in insulin-dependent GK gene activation.

Animals↗
PubMed2003

Facile protection of phenols and hydroxypyridines: key components of natural products--a new pathway to orellanine.

The (1,3,6-trioxa-heptyl) aromatic compounds are easily prepared from various phenols and hydroxypyridines. Protection by 2-methoxyethoxymethyl chloride (MEM-Cl) was achieved by phase transfer catalysis with Aliquat 336, subsequent removal of the protecting group was completed with Amberlyst 15. The 2-bromo-3-hydroxy-pyridine 3o constitutes the appropriate starting material for the synthesis of orellanine, the most potent nephrotoxin found in some Cortinarius mushrooms species.

2,2'-Dipyridyl↗
PubMed2003

From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803.

Activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphofructokinase (PFK), enolase, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) carboxylase were determined in extracts of photoautotrophic, mixotrophic, and heterotrophic cultures of Synechocystis sp. PCC 6803. Annotated genomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were analyzed for the respective predicted physical properties of each enzyme investigated here. Enzymatic activity was largely unaffected by nutritional mode, with the exception of glucokinase and PK whose activities were significantly elevated in heterotrophic cultures of Synechocystis sp. PCC 6803. PFK activity was insensitive to bacterial PFK-A (allosteric) effectors such as PEP, implying that Synechocystis PFK should be classified as a PFK-B (non-allosteric). Immunoblot and kinetic studies indicated that irrespective of nutritional mode, the Synechocystis PK corresponds to a PK-A (AMP activated) rather than PK-F (fructose-1,6-bisphosphate activated).

Anabaena↗
PubMed2006

The maize root plasma membrane H(+)-ATPase is regulated by a sugar-induced transduction pathway.

H(+)-ATPase, the key enzyme for the energization of ion and nutrient transport across the plasma membrane, is activated by phosphorylation-dependent 14-3-3 binding. Since the involvement of 14-3-3 proteins in sugar sensing-regulated processes has recently emerged, here we address the question as to whether sugar sensing plays a role in the regulation of H(+)-ATPase. The data reported here show that sugar depletion inhibits the association of 14-3-3 proteins with H(+)-ATPase by hampering phosphorylation of the 14-3-3 binding site of the enzyme. By using non-metabolizable disaccharides, we show that H(+)-ATPase regulation by 14-3-3 proteins can involve a specific sugar perception and transduction mechanism.

14-3-3 Proteins↗
PubMed2006

Glutathiolation regulates tumor necrosis factor-alpha-induced caspase-3 cleavage and apoptosis: key role for glutaredoxin in the death pathway.

Caspase-3 cleavage and activation are known to play central roles in apoptosis. However, the mechanisms that regulate caspase-3 cleavage remain elusive. Glutaredoxin (Grx) is a ubiquitous redox molecule that is unique in its ability to regulate S-glutathiolation (glutathiolation) of proteins. Here we show the essential role of Grx in caspase-3 cleavage via regulation of caspase-3 glutathiolation. Grx activity was significantly upregulated by tumor necrosis factor-alpha in endothelial cells. Small interference RNA knock down of Grx significantly inhibited tumor necrosis factor-alpha-induced endothelial cell death because of attenuated caspase-3 cleavage concomitant with increased caspase-3 glutathiolation. Enhanced caspase-3 cleavage by wild-type Grx overexpression was reversed by catalytically inactive Grx (C22S), demonstrating a requirement for thioltransferase activity. Cysteine-to-serine mutations (C163S, C184S, and C220S) of caspase-3 that were predicted to prevent glutathiolation showed increased cleavage compared with wild-type caspase-3. This inverse correlation between caspase-3 glutathiolation and cleavage was further confirmed by the observation that in vitro glutathiolation of caspase-3 inhibited its cleavage with recombinant caspase-8. Furthermore, Grx association with caspase-3 was decreased by tumor necrosis factor-alpha. These findings demonstrate a novel mechanism of caspase-3 regulation by Grx in tumor necrosis factor-alpha-induced apoptosis.

Animals↗