Molecular basis of V-ATPase inhibition by bafilomycin A1.
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ABSTRACT:
SUBMITTER: Wang R
PROVIDER: S-EPMC7979754 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zika virus (ZIKV) is a highly transmissive virus that belongs to the Flaviviridae family, which comprises several other pathogens that threaten human health. This re-emerging virus gained attention during the outbreak in Brazil in 2016, where a considerable number of microcephaly cases in newborns was associated with ZIKV infection during pregnancy. Lacking a preventive vaccine or antiviral drugs, efforts have been made to better understand the viral life cycle. In light of this, the relevance of the endosomal-lysosomal compartment for the ZIKV life cycle was investigated. A549 and SH-SY5Y cells were infected with either the African strain (associated with mild symptoms) or the French Polynesia strain (associated with neurological complications). For both strains, the V-ATPase inhibitor, bafilomycin A1, efficiently inhibited ZIKV entry and prevented the spread of the infection by interfering with viral maturation. Additionally, affecting cholesterol metabolism and transport with the drug U18666A, which inactivates late endosomes and lysosomes, impairs the viral life cycle. The data presented show a clear antiviral effect of two compounds that target the same compartments in different ways. This highlights the relevance of the endosomal-lysosomal compartment for the viral life cycle that should be considered as a target for antivirals.
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Project description:Quiescent cells (in G0) can be stimulated to enter the cell cycle and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H(+)-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A1, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A1 at successively later times during G1 progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A1 was added at the onset of S-phase. Bafilomycin A1 also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A1. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G1 phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.
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Project description:Reduction of ATP hydrolysis activity of vacuolar-type ATPase/synthase (V0V1) as a result of ADP inhibition occurs as part of the normal mechanism of V0V1 of Thermus thermophilus but not V0V1 of Enterococcus hirae or eukaryotes. To investigate the molecular basis for this difference, domain-swapped chimeric V1 consisting of both T. thermophilus and E. hirae enzymes were generated, and their function was analyzed. The data showed that the interaction between the nucleotide binding and C-terminal domains of the catalytic A subunit from E. hirae V1 is central to increasing binding affinity of the chimeric V1 for phosphate, resulting in reduction of the ADP inhibition. These findings together with a comparison of the crystal structures of T. thermophilus V1 with E. hirae V1 strongly suggest that the A subunit adopts a conformation in T. thermophilus V1 different from that in E. hirae V1. This key difference results in ADP inhibition of T. thermophilus V1 by abolishing the binding affinity for phosphate during ATP hydrolysis.
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Project description:Analysis of mRNA expression changes in human colon carcinoma cell lines (HCT116 H2B-GFP) after treatment bafilomycin A1.
2014-02-19 | GSE47836 | GEO
Project description:Analysis of mRNA expression changes in human colon carcinoma cell lines (HCT116 H2B-GFP) after treatment bafilomycin A1. mRNA expression analysis of HCT116 H2B-GFP treated with bafilomycin A1 compared to untreated HCT116 H2B-GFP. Four biological replicates were performed for treated samples.
2014-02-19 | E-GEOD-47836 | biostudies-arrayexpress
Project description:Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P ≤ 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments.IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.
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Project description:Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0-10 ?M, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1? and HIF-2?, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-? stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-? stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.
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