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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