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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 -
Journal of Virology Jun 2019In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we...
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B. Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
Topics: Antiviral Agents; DNA Topoisomerases; DNA, Circular; DNA, Viral; Foscarnet; Genome, Viral; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; RNA, Small Interfering; Virion; Virus Replication
PubMed: 30867306
DOI: 10.1128/JVI.02230-18 -
Gut Jan 2020Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk...
OBJECTIVE
Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent.
DESIGN
Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection.
RESULTS
Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets.
CONCLUSION
The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.
Topics: Antiviral Agents; Aspartate Carbamoyltransferase; Aspartic Acid; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Line; Dihydroorotase; Estrogen Receptor Antagonists; Estrogen Receptor alpha; Fulvestrant; Gene Silencing; Hepatitis D, Chronic; Hepatitis Delta Virus; Hepatocytes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin Resistance; Life Cycle Stages; Loss of Function Mutation; Phosphonoacetic Acid; Pyrimidines; RNA Interference; RNA, Small Interfering; RNA, Viral; Signal Transduction; Virus Replication
PubMed: 30833451
DOI: 10.1136/gutjnl-2018-317065 -
Archivos Argentinos de Pediatria Feb 2019Herpes simplex encephalitis (HSE) is the most common cause of sporadic focal encephalitis worldwide. Acyclovir is the treatment of choice of HSE since the 1980s. After...
Herpes simplex encephalitis (HSE) is the most common cause of sporadic focal encephalitis worldwide. Acyclovir is the treatment of choice of HSE since the 1980s. After the widespread use of acyclovir, HSE related mortality rate had reduced but resistant strains emerged. Acyclovir resistant HSV incidence was reported as about 0.5 % and 3.5 %-10 % in immunocompetent and immunocompromised patients, respectively. Herein, a 12-year-old immunocompetent patient with HSV-1 encephalitis who was successfully treated with combined acyclovir and foscarnet therapy is described. In the case of deteriorating clinical condition under acyclovir treatment even if the absence of demonstration of increased CSF HSV viral load, the possibility of acyclovir resistant HSE and the addition of foscarnet to the acyclovir treatment might be considered.
Topics: Acyclovir; Antiviral Agents; Child; Drug Combinations; Drug Resistance, Viral; Encephalitis, Herpes Simplex; Foscarnet; Humans; Male; Remission Induction; Simplexvirus; Treatment Failure
PubMed: 30652455
DOI: 10.5546/aap.2019.eng.e47 -
American Journal of Transplantation :... Jan 2019
Topics: Antiviral Agents; Control Groups; Cord Blood Stem Cell Transplantation; Encephalitis; Foscarnet; Ganciclovir; Herpesvirus 6, Human; Humans; Prospective Studies; Syndrome
PubMed: 30102463
DOI: 10.1111/ajt.15069 -
Scientific Reports Jul 2018Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of...
Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T ↔ R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.
Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Aspartate Carbamoyltransferase; Aspartic Acid; Catalytic Domain; Cytidine Triphosphate; Enzyme Stability; Models, Molecular; Phosphonoacetic Acid; Protein Multimerization; Protein Subunits; Substrate Specificity
PubMed: 30038211
DOI: 10.1038/s41598-018-27992-z -
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 -
Blood Advances Jun 2018HDAC inhibitors might induce ciHHV-6 reactivation. In ciHHV-6 HSCT recipients posttransplant viral load can estimate persistent host chimerism when the donor is ciHHV-6...
HDAC inhibitors might induce ciHHV-6 reactivation. In ciHHV-6 HSCT recipients posttransplant viral load can estimate persistent host chimerism when the donor is ciHHV-6 negative.
Topics: Chimerism; Female; Foscarnet; Hematopoietic Stem Cell Transplantation; Herpesvirus 6, Human; Histone Deacetylase Inhibitors; Humans; Middle Aged; Tissue Donors; Viral Load; Virus Activation
PubMed: 29898877
DOI: 10.1182/bloodadvances.2018015982 -
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 -
PLoS Pathogens May 2018Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results...
Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.
Topics: Azacitidine; DNA Demethylation; DNA Replication; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Gammaherpesvirinae; Gene Expression Regulation, Viral; Genes, Immediate-Early; Kinetics; Nucleic Acid Synthesis Inhibitors; Phosphonoacetic Acid; Promoter Regions, Genetic; Transcription, Genetic; Virus Replication
PubMed: 29813138
DOI: 10.1371/journal.ppat.1007070