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Molecular Cell May 2024The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal...
The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.
Topics: Humans; Ubiquitination; HEK293 Cells; Ubiquitin-Protein Ligases; Prodrugs; Nicotinamide-Nucleotide Adenylyltransferase; Substrate Specificity; Cryoelectron Microscopy; Protein Binding
PubMed: 38759627
DOI: 10.1016/j.molcel.2024.04.014 -
Journal of Virological Methods Aug 2017Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic...
Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC) assays with dyes in uninfected proliferating cells produced similar CC values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F (P<0.001). Curiously, the CC values for 3-deazaguanine were 83-93μM with ALB-F versus 2.4-7.0μM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC values, this impacted on the resulting in vitro selectivity indexes (calculated as CC/EC ratio).
Topics: Animals; Antiviral Agents; Cell Survival; Chikungunya virus; Chlorocebus aethiops; Coloring Agents; Cytopathogenic Effect, Viral; Dengue Virus; Junin virus; Oxazines; RNA Viruses; Vero Cells; Virus Replication; Viruses; Xanthenes
PubMed: 28359770
DOI: 10.1016/j.jviromet.2017.03.012 -
Experimental Parasitology Feb 2015Leishmania amazonensis undergoes apoptosis-like programmed cell death (PCD) under heat shock conditions. We identified a potential role for inosine 5' monophosphate...
Leishmania amazonensis undergoes apoptosis-like programmed cell death (PCD) under heat shock conditions. We identified a potential role for inosine 5' monophosphate dehydrogenase (IMPDH) in L. amazonensis PCD. Trypanosomatids do not have a "de novo" purine synthesis pathway, relying on the salvage pathway for survival. IMPDH, a key enzyme in the purine nucleotide pathway, is related to cell growth and apoptosis. Since guanine nucleotide depletion triggers cell cycle arrest and apoptosis in several organisms we analyzed the correlation between IMPDH and apoptosis-like death in L. amazonensis. The L. amazonensis IMPDH inhibition effect on PCD was evaluated through gene expression analysis, mitochondrial depolarization and detection of Annexin-V labeled parasites. We demonstrated a down-regulation of impdh expression under heat shock treatment, which mimics the natural mammalian host infection. Also, IMPDH inhibitors ribavirin and mycophenolic acid (MPA) prevented cell growth and generated an apoptosis-like phenotype in sub-populations of L. amazonensis promastigotes. Our results are in accordance with previous results showing that a subpopulation of parasites undergoes apoptosis-like cell death in the nutrient poor environment of the vector gut. Here, we suggest the involvement of purine metabolism in previously observed apoptosis-like cell death during Leishmania infection.
Topics: Animals; Apoptosis; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Heat-Shock Response; IMP Dehydrogenase; Leishmania mexicana; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mycophenolic Acid; Nucleosides; Purine Nucleotides; RNA, Protozoan; Ribavirin
PubMed: 25499513
DOI: 10.1016/j.exppara.2014.12.006 -
Nucleosides, Nucleotides & Nucleic Acids 2012The novel aza-analogues of tiazofurin (TZF) with 2-[5,5-bis(hydroxymethyl)pyrrolidin-2-yl] moiety, as sugar mimic, were synthesized from O,O-cyclohexylidene derivative...
The novel aza-analogues of tiazofurin (TZF) with 2-[5,5-bis(hydroxymethyl)pyrrolidin-2-yl] moiety, as sugar mimic, were synthesized from O,O-cyclohexylidene derivative of 4,4-bis(hydroxymethyl)-4-nitrobutanal in multi-gram scale. The synthetic route consisted of three stages: (i) the synthesis of corresponding derivative of 5,5-bis(hydroxymethyl)pyrrolidine-2-carbonitrile, (ii) the construction of ethyl thiazole-2-carboxylate part by the conversion of the pyrrolidine-2-carbonitrile into the N-trifluoroacetyl derivative followed by cyclocondensation with L-cysteine ethyl ester and then by dehydrogenation, and (iii) the final transformation of the ethyl thiazole-4-carboxylate into the aza-analogues of TZF. The TZF aza-analogues were evaluated for their antiviral activities in cell-culture-based assays.
Topics: Animals; Antiviral Agents; Aza Compounds; Carbohydrates; Chlorocebus aethiops; Cysteine; DNA Viruses; Fibroblasts; HeLa Cells; Humans; Molecular Mimicry; Pyrrolidines; RNA Viruses; Ribavirin; Vero Cells
PubMed: 22257212
DOI: 10.1080/15257770.2011.643848 -
Toxins Jun 2011We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure.... (Clinical Trial)
Clinical Trial
4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?
We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.
Topics: Adolescent; Biomarkers; Child; Chromatography, High Pressure Liquid; Erythrocytes; Female; Glomerular Filtration Rate; Humans; Kidney Failure, Chronic; Male; Mass Spectrometry; NAD; Nucleotides; Renal Dialysis; Severity of Illness Index; Uremia
PubMed: 22069723
DOI: 10.3390/toxins3060520 -
Biochemical and Biophysical Research... Oct 2008The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates...
The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m(7)G) cap moiety on the 5'-end of mRNAs. We demonstrate that a physical mimic of the m(7)G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m(7)G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m(7)G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.
Topics: Animals; Antimetabolites, Antineoplastic; Antiviral Agents; Apoptosis; Biological Transport; Cell Cycle Proteins; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Humans; Mice; Nuclear Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Regulon; Ribavirin
PubMed: 18706892
DOI: 10.1016/j.bbrc.2008.07.163 -
Journal of Virology Nov 2007The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent...
The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5'-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.
Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Dose-Response Relationship, Drug; Gene Frequency; Genome, Viral; Guanosine Triphosphate; Hantaan virus; Mutation; Mycophenolic Acid; Organoselenium Compounds; RNA, Viral; Ribavirin; Ribonucleosides; Vero Cells
PubMed: 17699579
DOI: 10.1128/JVI.00874-07 -
Structure (London, England : 1993) Aug 2007Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer...
Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.
Topics: Adenosine Diphosphate; Amino Acid Sequence; Antineoplastic Agents; Binding Sites; Catalysis; Crystallography, X-Ray; Escherichia coli; Humans; Hydrogen Bonding; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Mutation; Nicotinamide Mononucleotide; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Structure, Secondary; Ribavirin; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 17698003
DOI: 10.1016/j.str.2007.06.017 -
Organic Letters Sep 2005[reaction: see text] A tandem dimerization-macrocyclization approach using 1,3-dipolar azide-alkyne cycloaddition reactions has been employed in the facile and...
[reaction: see text] A tandem dimerization-macrocyclization approach using 1,3-dipolar azide-alkyne cycloaddition reactions has been employed in the facile and convergent solution phase syntheses of C2 symmetric cyclic peptide scaffolds bearing triazole epsilon2-amino acids as dipeptide surrogates.
Topics: Heterocyclic Compounds; Molecular Structure; Peptides, Cyclic; Ribavirin
PubMed: 16178569
DOI: 10.1021/ol0518028 -
Antimicrobial Agents and Chemotherapy Jun 2005The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard...
The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; IMP Dehydrogenase; Kinetics; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Toxoplasma; Transcription, Genetic
PubMed: 15917510
DOI: 10.1128/AAC.49.6.2172-2179.2005