Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase.

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with L-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

Effect of reserpine on Pseudomonas aeruginosa quorum sensing mediated virulence factors and biofilm formation.

This study aimed to evaluate the effect of reserpine, a plant-derived indole-alkaloid, against Pseudomonas aeruginosa PAO1 biofilms. The anti-biofilm activity of reserpine was evaluated by crystal violet staining, MTT assay, confocal laser scanning microscopy and scanning electron microscopy. Reserpine effects were also assessed by qRT-PCR of quorum sensing (QS)-regulated genes and biochemical quantification of the QS-mediated virulence factors pyocyanin, rhamnolipids, proteases and elastases. Reserpine reduced biofilm formation, cell motility, virulence factor production, and QS-controlled gene expression. Additionally, molecular docking analysis for AHL synthase LasI and QS transcriptional regulators LasR/MvfR revealed a plausible molecular mechanisms of reserpine QS inhibition. These findings provide insights into the underlying mode of action of reserpine, which may be useful in the development of new drugs against biofilm-related infections.

Inhibition of Pseudomonas aeruginosa quorum sensing by subinhibitory concentrations of curcumin with gentamicin and azithromycin.

OBJECTIVES: Pseudomonas aeruginosa quorum sensing (QS) circuits regulate virulence factors and co-ordinate bacterial pathogenicity. This study aimed to investigate the inhibitory activity of subinhibitory concentrations of curcumin with azithromycin and gentamicin against P. aeruginosa QS-related genes and virulence factors. METHODS: The minimum inhibitory concentrations (MICs) and synergistic activity of curcumin with azithromycin and gentamicin against P. aeruginosa PAO1 were determined using broth microdilution and checkerboard titration methods, respectively. The activity of sub-MICs (1/4× and 1/16× MIC) of curcumin on the QS signal molecules was assessed using a reporter strain assay. The influence of sub-MICs of curcumin, azithromycin and gentamicin alone and in combination on motility and biofilm formation was also determined and was confirmed by RT-PCR to test the expression of the QS regulatory genes lasI, lasR, rhlI and rhlR. RESULTS: Addition of curcumin drastically decreased the MIC of azithromycin and gentamicin. Curcumin showed synergistic effects with azithromycin and gentamicin. Treated PAO1 cultures in the presence of curcumin showed a significant reduction of signals C12-HSL and C4-HSL (P<0.05). Sub-MICs (1/4× and 1/16× MIC) of curcumin, azithromycin and gentamicin alone and in combination significantly reduced swarming and twitching motilities as well as biofilm formation. Expression of QS regulatory genes lasI, lasR, rhlI and rhlR using 1/4× MIC of curcumin, azithromycin and gentamicin alone and in combination was decreased significantly compared with untreated PAO1. CONCLUSIONS: These results indicate that a combination of sub-MIC of curcumin with azithromycin and gentamicin exhibited synergism against P. aeruginosa QS systems.

Subchronic exposure to arsenic disturbed the biogenic amine neurotransmitter level and the mRNA expression of synthetase in mice brains.

Little is known about the influence of arsenic (As) exposure on monoamine neurotransmitters and the underlying mechanisms, although arsenic toxicity on the central nervous system has been well documented. In the present study, the levels of norepinephrine (NE), dopamine (DA), and 5-HT were determined by high performance liquid chromatography in the cerebrum and cerebellum of mice exposed to 1, 2 and 4 ppm As2O3 through drinking water for 60 days. The ultra-structural change of vesicles in the synapses of mice brains was observed by transmission electron microscopy; the mRNA expressions of dopamine beta hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) as NE, DA and 5-HT synthetases were quantitatively assessed by real time reverse transcription-polymerase chain reaction. It was shown that the concentrations of NE, DA and 5-HT in the cerebrum or cerebellum of mice exposed to As were significantly lower than those in the control group. The number of synaptic vesicles significantly decreased in the brain of mice exposed to As. Moreover, the expressions of TH, TPH and DBH genes were significantly lower in the brains of mice exposed to As than those in the controls. These results suggested that subchronic exposure to As might decrease the concentrations of the three monoamine neurotransmitters in the mouse brain and downregulate TH, TPH and DBH gene expressions. It was also indicated that the decreased concentrations of the three monoamine neurotransmitters in the brain might be related to the down-regulated gene expressions of these synthetases by As.

Evolutionary root defines the structural basis of high and low level vancomycin resistance in enterococci.

D-alanyl-D-lactate (Dlac) and D-alanyl-D-serine (Dser) ligases respectively mediates high and low level vancomycin resistance among enterococci. To date, the evolutionary relationship of both ligases is largely unaddressed. Also poorly understood are the molecular differences in the magnitude of vancomycin resistance. To address the mention, we constructed the phylogenetic tree of all vancomycin resistance conferring ligases with the wild type ligases (Dala). Multiple sequence alignment and tertiary structures of the structurally unresolved proteins were constructed by homology modeling. Phylogenetic tree revealed that both Dlac and Dser are profoundly different from Dala as a result of continuous selection pressure. Separate clustering of Dlac and Dser also highlighted the structural basis of molecule in maintaining different level of resistance as exhibited by the bacteria. This notion was further augmented as the functionally key region, omega loop (ω-loop), was found relatively more structured in only Dlac. Moreover, the critically active residue, His-243/244, was also noticed to be restricted in Dlac and found replaced by non polar residues in Dser. The present study not only provides protein structural explanation of the different intensities of vancomycin resistance among enterococci, but also presents yet another example for the scope of evolutionary science in biomedicine.

Parkin is not regulated by the unfolded protein response in human neuroblastoma cells.

Mutations in the parkin gene cause the majority of cases of familial-linked Parkinson's disease, and mounting evidence suggests that parkin may play a role in idiopathic disease. Previous reports suggest that parkin may respond to and relieve, via E3-ligase activity, cellular stress at the endoplasmic reticulum caused by the accumulation of unfolded proteins. However, parkin's relationship to the mammalian unfolded protein response is unclear. Here, we comprehensively evaluate endogenous parkin in SH-SY5Y neuroblastomas at the promoter, RNA, and protein levels in response to unfolded protein stress induced by tunicamycin. While we find strong up-regulation of genes linked to the unfolded protein stress pathway, we detect no significant changes in parkin. These data suggest a lack of association between parkin and the unfolded protein response in SH-SY5Y cells.

The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer.

The von Hippel-Lindau tumor suppressor protein (pVHL) is the substrate-recognition module of an E3 ubiquitin ligase that targets the alpha subunits of hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. Recognition of HIF by pVHL is linked to enzymatic hydroxylation of conserved prolyl residues in the HIF alpha subunits by members of the EGLN family. Dysregulation of HIF-target genes such as vascular endothelial growth factor and transforming growth factor alpha has been implicated in the pathogenesis of renal cell carcinomas and of hemangioblastomas, both of which frequently lack pVHL function.

von Hippel Lindau tumor suppressor and HIF-1alpha: new targets of NSAIDs inhibition of hypoxia-induced angiogenesis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) block prostaglandin synthesis and impair healing of gastrointestinal ulcers and growth of colonic tumors, in part, by inhibiting angiogenesis. The mechanisms of this inhibition are incompletely explained. Here we demonstrate that both nonselective (indomethacin) and COX-2-selective (NS-398) NSAIDs inhibit hypoxia-induced in vitro angiogenesis in gastric microvascular endothelial cells via coordinated sequential events: 1) increased expression of the von Hippel-Lindau (VHL) tumor suppressor, which targets proteins for ubiquitination leading to 2) reduced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and, as a result, 3) reduced expression of vascular endothelial growth factor (VEGF) and its specific receptor Flt-1. Because HIF-1alpha is the major trigger for hypoxia-induced activation of the VEGF and Flt-1 genes, this could explain how NSAIDs inhibit hypoxia-induced angiogenesis. Exogenous VEGF and, to a lesser extent, exogenous prostaglandins partly reversed the NSAIDs inhibition of hypoxia-induced angiogenesis. Taken together, these results indicate that NSAIDs inhibit hypoxia-induced angiogenesis in endothelial cells by inhibiting VEGF and Flt-1 expression through increased VHL expression and the resulting ubiquitination and degradation of HIF-1alpha. This action of NSAIDs has both prostaglandin-dependent and prostaglandin-independent components.

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53.

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.

Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein.

BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.

Active-site mutants of the VanC2 D-alanyl-D-serine ligase, characteristic of one vancomycin-resistant bacterial phenotype, revert towards wild-type D-alanyl-D-alanine ligases.

BACKGROUND: The rising number of vancomycin-resistant enterococci (VREs) is a major concern to modern medicine because vancomycin is currently the 'last resort' drug for life-threatening infections. The D-alanyl-D-X ligases (where X is an hydroxy or amino acid) of bacteria catalyze a critical step in bacterial cell-wall peptidoglycan assembly. In bacteria that produce glycopeptide antibiotics and in opportunistic pathogens, including VREs, D-, D-ligases serve as switches that confer antibiotic resistance on the bacteria themselves. Peptidoglycans in vancomycin-sensitive bacteria end in D-alanyl-D-alanine, whereas in vancomycin-resistant bacteria they end in D-alanyl-D-lactate or D-alanyl-D-serine. RESULTS: We demonstrate that the selective utilization of D-serine by the Enterococcus casseliflavus VanC2 ligase can be altered by mutagenesis of one of two residues identified by homology to the X-ray structure of the Escherichia coli D-alanyl-Dalanine ligase (DdlB). The Arg322-->Met (R322M) and Phe250-->Tyr (F250Y) ligase mutants show a 36-44-fold decrease in the use of D-serine, as well as broadened specificity for utilization of other D-amino acids in place of D-serine. The F250Y R322M double mutant is effectively disabled as a D-alanyl-D-serine ligase and retains 10% of the catalytic activity of wild-type D-alanyl-D-alanine ligases, reflecting a 6,000-fold switch to the D-alanyl-D-alanine peptide. Correspondingly, the Leu282-->Arg mutant of the wild-type E. coli DdlB produced a 560-fold switch towards D-alanyl-D-serine formation. CONCLUSIONS: Single-residue changes in the active-site regions of D-, D-ligases can cause substantial changes in recognition and activation of hydroxy or amino acids that have consequences for glycopeptide antibiotic efficacy. The observations reported here should provide an approach for combatting antibiotic-resistant bacteria.

Identification of a vitamin K-dependent carboxylase in the venom duct of a Conus snail.

Peptides from the venom ducts of cone snails (genus Conus) contain gamma-carboxyglutamate residues. The gamma-glutamyl carboxylase responsible for this post-translational modification is localized in the microsomal fraction, strictly dependent on vitamin K, activated by ammonium sulfate, and is associated with endogenous substrate. The K(m) of the enzyme for vitamin K is comparable to that for the bovine carboxylase. However, a propeptide containing substrate related to the blood coagulation protein factor IX, a highly efficient substrate for the bovine enzyme, was poorly carboxylated by the Conus enzyme, suggesting differences in gamma-carboxylase recognition signal sequences and/or structural requirements at the carboxylation site.

Correcting temperature-sensitive protein folding defects.

Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein. Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy. Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected. These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities. We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases.

Gemcitabine: a modulator of intracellular nucleotide and deoxynucleotide metabolism.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) is a deoxycytidine (dCyd) analog that extensively modulates intracellular CTP and dCTP metabolism. In Chinese hamster ovary (CHO) cells, a 4-hour exposure to gemcitabine (100 mumol/L) reduced cellular CTP and dCTP concentrations to 5.9% and 50%, respectively. Intracellular UTP concentrations increased, indicating a metabolic block at CTP synthetase. Pool-sizes of ATP and GTP remained unaffected. In contrast, a CHO mutant deficient in deoxycytidine kinase, and thus unable to accumulate dFdCTP, maintained its CTP pools under identical conditions, suggesting that the CTP pool depletion was dependent on dFdC phosphorylation. Neither 100 mumol/L arabinosylcytosine nor 5 mmol/L hydroxyurea affected CTP levels, indicating that inhibition of DNA synthesis by analog incorporation or by depletion of dNTP pools were not the causes of the CTP pool perturbation. Metabolic studies demonstrated that incorporation of [3H]uridine into the UTP pool was not impaired by dFdC treatment, whereas the specific activity of the CTP pools decreased as a function of increasing gemcitabine concentration and time of exposure. Comparable results were obtained using 3-deazauridine, a known inhibitor of CTP synthetase. We conclude that high cellular concentrations of dFdCTP deplete cellular CTP concentrations by inhibition of the dCTP pool and also may be a limiting factor for RNA synthesis.

Stimulation of glycosphingolipid biosynthesis by L-threo-1-phenyl-2-decanoylamino-1-propanol and its homologs in B16 melanoma cells.

Previous studies have demonstrated that the ceramide analog D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) inhibits glucosylceramide (GlcCer) synthase and thus leads to extensive depletion of glycosphingolipids (GSLs) biosynthesized from GlcCer [reviewed by Radin, N.S., Shayman, J.A., and Inokuchi, J. (1993) Adv. Lipid Res. 26, 183-213). In the present study, stereospecificity of PDMP activity was demonstrated with an enantiomeric pair, D-threo-PDMP and L-threo-PDMP. Treatment of B16 melanoma cells with the D-threo or L-threo isomer produced contrasting changes of GSL biosynthesis, as monitored by metabolic labeling with [3H]Gal. D-PDMP markedly inhibited incorporation of radioactivity into GlcCer, LacCer, and GM3 as expected, whereas the L-threo isomer significantly increased it. Homologs of L-PDMP having different N-acyl chains were synthesized and also tested for their effects. Among them, the compounds having C8-C14 acyl chains increased incorporation of the radioactivity into GSLs to different degrees, demonstrating that the stimulatory effect of the L-threo homologs depends on acyl chain length. In order to elucidate the biochemical mechanisms of these PDMP effects, the activities of GlcCer synthase, LacCer synthase, and GM3 synthase in B16 cell lysates were measured in the presence of PDMP. D-Threo-PDMP but not the L-threo isomer inhibited both LacCer and GM3 synthases as well as GlcCer synthase, suggesting that the ceramide-like structure of the D-PDMP molecule interacted stereospecifically with these GSL-synthesizing enzymes. On the other hand, L-PDMP had no effect in the in vitro assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of 3-methylcrotonyl-coenzyme-A carboxylase from leaves of Zea mays.

3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol.min-1.mg-1 protein, representing an approximately 5000-fold purification of the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a molecular weight of 853,000 +/- 34,000 and is composed of two types of subunits, a biotin-containing subunit of 80 +/- 2 kDa and a non-biotin-containing subunit of 58.5 +/- 1.5 kDa. These data suggest that the enzyme has an alpha 6 beta 6 configuration. The optimum pH for activity is 8.0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO3- are 11 microM, 20 microM, and 0.8 mM, respectively. Kinetic studies of the 3-methylcrotonyl-CoA carboxylase reaction with variable concentrations of two substrates confirmed that ATP and HCO3- bind sequentially to the enzyme and that ATP and 3-methylcrotonyl-CoA bind in ping-pong fashion. However, similar analyses indicate that the binding of HCO3- at the first site is affected by 3-methylcrotonyl-CoA. Kinetic studies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase reaction establish that Mg.ATP is the substrate for the enzyme, that free ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2+ and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so. In addition to carboxylating 3-methylcrotonyl-CoA, the maize carboxylase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, acetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is independent of the active sites. The monovalent cations K+, Cs+, Rb+, and NH4+ activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ being the most potent activator. The inhibition of 3-methylcrotonyl-CoA carboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which suggests that sulfhydryl and arginyl residues may be involved in catalysis.

The effect of some platinum compounds on the activity of the CTP synthetase of Ehrlich ascites tumor cells: in vitro and in vivo studies.

The present work investigates the effect of cis-DDP (DDP, diamminedichloroplatinum(II)), trans-DDP, SPC (spermine platinum(II) complex), and K2PtCl4 on the activity of the CTP synthetase in the cytosol of Ehrlich ascites tumor cells. To study their in vitro effect, the platinum compounds were supplemented to the incubation mixture for the enzyme assay. A concentration dependent inhibition of the CTP synthetase was found which was strongest in the case of trans-DDP. When ascites cells collected from mice, pretreated in vivo with platinum compounds, were used, the enzyme assay showed that the inhibition is strongest in the case of cis-DDP and K2PtCl4 (about 90% inhibition). This distinct inhibitory effect of the platinum compounds in the present experiments may be explained with the metabolic conversions of the compounds in the organism to their more active forms and/or with the inhibition of the protein biosynthesis under their influence because the lifetime of the CTP synthetase is short. This last assertion is proved in this work by control experiments with the antibiotic cycloheximide, which is an inhibitor of the protein biosynthesis.

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation.

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m2 over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50-60% of pretreatment values after 2-5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however due to C4b-binding protein levels higher than total PS levels (p less than 0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 less than p less than 0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.