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Journal of Virology Oct 2018Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186)...
Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator. Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.
Topics: Amino Acid Substitution; Antigens, Viral; Antiviral Agents; Binding Sites; Cell Line, Tumor; DNA Methylation; DNA, Viral; Gene Expression Regulation; HEK293 Cells; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Lymphocytes; Membrane Glycoproteins; Mutation; Phosphonoacetic Acid; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Trans-Activators; Transcription Factor AP-1; Viral Proteins; Virus Replication
PubMed: 30021895
DOI: 10.1128/JVI.01062-18 -
Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor.Proceedings of the National Academy of... Feb 2010A novel concept in eukaryotic signal transduction is the use of nutrient transporters and closely related proteins as nutrient sensors. The action mechanism of these...
A novel concept in eukaryotic signal transduction is the use of nutrient transporters and closely related proteins as nutrient sensors. The action mechanism of these "transceptors" is unclear. The Pho84 phosphate transceptor in yeast transports phosphate and mediates rapid phosphate activation of the protein kinase A (PKA) pathway during growth induction. We have now identified several phosphate-containing compounds that act as nontransported signaling agonists of Pho84. This indicates that signaling does not require complete transport of the substrate. For the nontransported agonist glycerol-3-phosphate (Gly3P), we show that it is transported by two other carriers, Git1 and Pho91, without triggering signaling. Gly3P is a competitive inhibitor of transport through Pho84, indicating direct interaction with its phosphate-binding site. We also identified phosphonoacetic acid as a competitive inhibitor of transport without agonist function for signaling. This indicates that binding of a compound into the phosphate-binding site of Pho84 is not enough to trigger signaling. Apparently, signaling requires a specific conformational change that may be part of, but does not require, the complete transport cycle. Using Substituted Cysteine Accessibility Method (SCAM) we identified Phe(160) in TMD IV and Val(392) in TMD VIII as residues exposed with their side chain into the phosphate-binding site of Pho84. Inhibition of both transport and signaling by covalent modification of Pho84(F160C) or Pho84(V392C) showed that the same binding site is used for transport of phosphate and for signaling with both phosphate and Gly3P. Our results provide to the best of our knowledge the first insight into the molecular mechanism of a phosphate transceptor.
Topics: Binding Sites; Cyclic AMP-Dependent Protein Kinases; Glycerophosphates; Membrane Transport Proteins; Mutagenesis, Site-Directed; Phosphonoacetic Acid; Proton-Phosphate Symporters; Reproducibility of Results; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 20133652
DOI: 10.1073/pnas.0906546107 -
The Journal of Biological Chemistry Jan 2003Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active...
Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC(50) = 3.2 microm) but has no effect on DNA polymerization (IC(50) > 50 microm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the alpha-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn(2+) implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.
Topics: Butyrates; Enzyme Inhibitors; Foscarnet; HIV-1; Kinetics; RNA-Directed DNA Polymerase; Reverse Transcriptase Inhibitors; Ribonuclease H; Structure-Activity Relationship; Substrate Specificity; Thiophenes
PubMed: 12480948
DOI: 10.1074/jbc.C200621200 -
Scientific Reports Jun 2018Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior...
Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.
Topics: Animals; Aspartic Acid; Bacteria; Dermis; Drug Resistance, Multiple, Bacterial; HEK293 Cells; Humans; Immunity, Innate; Nod2 Signaling Adaptor Protein; Phosphonoacetic Acid; Pyrimidines; Signal Transduction; Skin Diseases, Bacterial; Swine
PubMed: 29880914
DOI: 10.1038/s41598-018-27012-0 -
Antimicrobial Agents and Chemotherapy Oct 1991The effects of 3'-azido-3'-deoxythymidine (AZT), phosphonoformate (PFA), and 2',3'-dideoxythymidine (ddT) and their combination on human immunodeficiency type 1 (HIV-1)...
Synergistic inhibition of human immunodeficiency virus type 1 replication in vitro by two-drug and three-drug combinations of 3'-azido-3'-deoxythymidine, phosphonoformate, and 2',3'-dideoxythymidine.
The effects of 3'-azido-3'-deoxythymidine (AZT), phosphonoformate (PFA), and 2',3'-dideoxythymidine (ddT) and their combination on human immunodeficiency type 1 (HIV-1) replication were studied by measuring the HIV-1 p24 antigen expression and reverse transcriptase (RT) release in HIV-1-infected MT4 cells in vitro. RT activity was also measured in a cell-free system by using poly(rA)-oligo(dT) as the primer-template, and cell growth inhibition was measured in noninfected MT4 cells. The interactions of these two- and three-drug combinations were evaluated by the combination index (CI) method and isobologram techniques. The 50% effective concentrations (EC50s) of AZT, PFA, and ddT were 0.014 to 0.005, 9.4 to 8.8, and 8.4 to 2.5 microM, respectively, for p24 enzyme-linked immunosorbent assays (ELISAs) and 0.005 to 0.0034, 1.43 to 1.37, and 2.87 to 2.83 microM, respectively, for RT activity in vitro; for RT activity in the cell-free system, the EC50s were 0.00019 to 0.00024, 0.012 to 0.02, and 0.00074 to 0.0005 microM, for AZT-5'-triphosphate, PFA, and ddT-5'-triphosphate, respectively. AZT in combination with PFA (1:200) or ddT (1:5) as well as the combination of these three drugs (1:200:5) synergistically inhibited HIV-1 replication and RT activity in the cell-free system over a wide range of drug concentrations, with the CIs ranging from 0.5 to 0.09, in which CIs of less than 1, 1, and greater than 1 indicate synergism, additive effect, and antagonism, respectively. Three- and two-drug combinations of AZT, PFA, and ddT showed similar degrees of synergism against HIV-1 replication in p24 assays and RT release assays, whereas the combination of AZT and ddT was found to be the most selective in terms of its anti-HIV-1 effect versus cytotoxicity. Dose reduction indices calculated from both HIV-1 replication inhibition, as measured by p24 ELISA and by RT activity in the cell-free system, indicated that two- and three-drug combinations at high effect levels and the selected combination ratios allow 2- to 240-fold dose reduction over the single drug alone in terms of their anti-HIV-1 effects. The three-drug combination showed the highest dose reduction index. These finding suggest that increased efficacy and reduced toxicity may be achieved in AIDS therapy by using AZT, PFA, and ddT in two- or three-drug combinations.
Topics: Antiviral Agents; Cell Line; Cell Survival; Cell-Free System; Dideoxynucleosides; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Foscarnet; HIV-1; Humans; Phosphonoacetic Acid; Reverse Transcriptase Inhibitors; Virus Replication; Zidovudine
PubMed: 1722077
DOI: 10.1128/AAC.35.10.2003 -
Nature Aug 2012Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We...
Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally deleted gene is a member of a functionally redundant family of genes carrying out an essential function. The glycolytic gene enolase 1 (ENO1) in the 1p36 locus is deleted in glioblastoma (GBM), which is tolerated by the expression of ENO2. Here we show that short-hairpin-RNA-mediated silencing of ENO2 selectively inhibits growth, survival and the tumorigenic potential of ENO1-deleted GBM cells, and that the enolase inhibitor phosphonoacetohydroxamate is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger-deleted genes encoding functionally redundant essential activities and provide an effective treatment strategy for cancers containing such genomic events.
Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 1; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Essential; Genes, Tumor Suppressor; Glioblastoma; Homozygote; Humans; Hydroxamic Acids; Mice; Molecular Targeted Therapy; Neoplasm Transplantation; Phosphonoacetic Acid; Phosphopyruvate Hydratase; RNA, Small Interfering; Sequence Deletion; Tumor Suppressor Proteins
PubMed: 22895339
DOI: 10.1038/nature11331 -
ACS Chemical Biology Aug 2016HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase...
HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.
Topics: Crystallography, X-Ray; Diphosphates; Drug Design; Foscarnet; HIV Reverse Transcriptase; Hydrolysis; Nucleotides; Protein Conformation; Reverse Transcriptase Inhibitors
PubMed: 27192549
DOI: 10.1021/acschembio.6b00187 -
European Journal of Biochemistry Jun 1984Pyrimidine nucleosides in blood plasma of rats were identified by different procedures, including chemical peak shift methods, before their quantification by...
Pyrimidine nucleosides in blood plasma of rats were identified by different procedures, including chemical peak shift methods, before their quantification by reversed-phase high-performance liquid chromatography. The concentrations of uridine, cytidine, and deoxycytidine were 1.0 +/- 0.2, 10.6 +/- 1.9, and 33.4 +/- 5.4 mumol/l, respectively. Six hours after the administration of D-galactosamine, the level of circulating cytidine was severely depressed to 25% of control values; uridine decreased to 54% while deoxycytidine remained unchanged. 24 h after the dose of the amino sugar, the levels of cytidine and uridine returned to control values in blood plasma. Total acid-soluble uridine, cytidine, guanosine, and adenosine was determined by reversed-phase HPLC after treatment of the neutralized acid-soluble supernatant of freeze-clamped rat livers with phosphodiesterase and alkaline phosphatase. Six hours after its administration, D-galactosamine induced a 2.2-fold and a 1.6-fold rise in total acid-soluble uridine and cytidine, respectively. Co-administration of N-(phosphonoacetyl)-L-aspartate, an inhibitor of de novo pyrimidine synthesis, suppressed the increase in total acid-soluble uridine observed after D-galactosamine alone, but was without effect on the enhancement of total cytidine. Three hours after D-galactosamine and 15 min after [2-14C] cytidine, there was a rapid fall of the labeled nucleoside in blood plasma to 49% of control animals accompanied by a 2.8-fold rise in the total radioactivity of rat liver homogenates. From these results it can be concluded that the hepatocellular rise in total acid-soluble cytidine after D-galactosamine, in contrast to the increase in total acid-soluble uridine, originates from the phosphorylation of blood plasma cytidine via the salvage pathway. The depletion of circulating cytidine in the presence of hepatocellular UTP deficiency points to the importance of the liver and the hepatic UTP level for the clearance of blood plasma cytidine.
Topics: Animals; Antimetabolites; Aspartic Acid; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cytidine; Female; Galactosamine; Liver; Nucleosides; Nucleotides; Phosphonoacetic Acid; Rats; Rats, Inbred Strains; Solubility; Uracil Nucleotides; Uridine Triphosphate
PubMed: 6734601
DOI: 10.1111/j.1432-1033.1984.tb08197.x -
Physiological Research Aug 2020Lithium is mainly excreted into urine, and a large fraction of lithium filtered through glomeruli is reabsorbed in the proximal tubule. However, the mechanisms...
Lithium is mainly excreted into urine, and a large fraction of lithium filtered through glomeruli is reabsorbed in the proximal tubule. However, the mechanisms responsible for lithium reabsorption remain unclear. We previously reported that the reabsorption of lithium was biphasic in rats, and that foscarnet inhibited lithium reabsorption with a high affinity type. We herein evaluated the effects of acetazolamide and foscarnet on the renal excretion of lithium in rats treated with lithium chloride at 2 doses. In rats intravenously injected with a bolus of 25 mg/kg lithium chloride, acetazolamide facilitated the urinary excretion of lithium, and increased the fractional excretion of lithium from 0.446 to 0.953, near the theoretically maximum value. At a dose of 2.5 mg/kg lithium chloride, the fractional excretion of lithium was 0.241 in control rats, 0.420 in rats administered acetazolamide, and 0.976 in rats administered acetazolamide and foscarnet. These results showed the potent inhibition of lithium reabsorption by acetazolamide and foscarnet in rats. And, it was exhibited that the effects of acetazolamide on lithium reabsorption differed with the dosages of lithium administered.
Topics: Acetazolamide; Animals; Antiviral Agents; Disease Models, Animal; Diuretics; Drug Interactions; Foscarnet; Kidney Tubules, Proximal; Lithium Chloride; Male; Rats; Rats, Wistar; Renal Reabsorption
PubMed: 32584131
DOI: 10.33549/physiolres.934285 -
Biochimica Et Biophysica Acta.... Sep 2019Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter...
Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K = 1.387 ± 0.1674 mM Pi and V = 198.6 ± 10.23 Pi × h × mg protein hence reflecting a low affinity Pi transport. H-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H-dependent Pi transport was five-fold higher than the Na-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.
Topics: Breast Neoplasms; Cadherins; Cell Adhesion; Cell Line; Cell Proliferation; Down-Regulation; Female; Humans; Hydrogen-Ion Concentration; Ion Transport; Kinetics; Phosphate Transport Proteins; Phosphates; Phosphonoacetic Acid; Sodium-Phosphate Cotransporter Proteins, Type IIb; Tumor Microenvironment; Up-Regulation
PubMed: 31034992
DOI: 10.1016/j.bbadis.2019.04.015