scholarly journals Molecular basis of V-ATPase inhibition by bafilomycin A1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rong Wang ◽  
Jin Wang ◽  
Abdirahman Hassan ◽  
Chia-Hsueh Lee ◽  
Xiao-Song Xie ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Tumor Metastasis ◽  
Unique Feature ◽  
Mammalian Species ◽  
Proton Translocation ◽  
Specific Inhibitor ◽  
Hydroxyl Group ◽  
Bafilomycin A1 ◽  
Binding Residues ◽  
Atpase Inhibition

AbstractPharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7’-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

Bafilomycin-sensitive acid secretion by mantle epithelium of the freshwater clam, Unio complanatus

1993 ◽  
Vol 264 (5) ◽  
pp. R946-R951 ◽  
Author(s):  
R. L. Hudson
Keyword(s):  
Acid Secretion ◽  
Short Circuit ◽  
Proton Translocation ◽  
Electrical Potential ◽  
Specific Inhibitor ◽  
Maximum Effect ◽  
Electrical Potential Difference ◽  
Bafilomycin A1 ◽  
Flux Chamber ◽  
Freshwater Clam

This study reports the effects of a series of putative inhibitors of proton translocation on electrogenic acid secretion by the isolated shell-facing mantle epithelium of the freshwater clam, Unio complanatus. In this epithelium, adenosine 3',5'-cyclic monophosphate (cAMP)-dependent acid secretion is electrogenic and equivalent to the cAMP-dependent short-circuit current (Isc) when mounted in a Ussing-type flux chamber. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPases (V-type ATPases), inhibited cAMP-dependent electrogenic acid secretion, with an IC50 of 7 nM and a maximum effect at 100 nM. Inhibition of acid secretion by bafilomycin A1 occurred only when added to the shell side and was not reversed by repeated washings. Concomitant with the inhibition of cAMP-dependent Isc by bafilomycin A1, the electrical potential difference across the shell-facing membrane depolarized, and the conductance ratio of the apical to basolateral membranes decreased. The cAMP-dependent Isc and cAMP-dependent acid secretion also were inhibited by dicyclohexyl-carbodiimide, diethylstilbestrol, N-ethylmaleimide, and tributyltin. Amiloride, omeprazole, oligomycin, venturicidin, and ouabain had no effect on either cAMP-dependent Isc or cAMP-dependent acid secretion. The cAMP-dependent Isc and cAMP-dependent acid secretion were not affected by Na(+)-free or K(+)-free solutions. Finally, the cAMP-dependent Isc was strongly dependent on the pH of the shell-facing compartment, with 50% inhibition of the maximum cAMP-dependent Isc occurring at pH 6.0 and complete inhibition at pH 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Label Free, Nontoxic Cu-GSH NCs as a Nanoplatform for Cancer Cell Imaging and Subcellular pH Monitoring Modulated by a Specific Inhibitor: Bafilomycin A1

2020 ◽  
Vol 3 (2) ◽  
pp. 1245-1257 ◽  
Author(s):  
Neeli Chandran ◽  
Prajit Janardhanan ◽  
Manikanta Bayal ◽  
Unnikrishnan Unniyampurath ◽  
Rajendra Pilankatta ◽  
...  
Keyword(s):  
Cancer Cell ◽  
Cell Imaging ◽  
Ph Monitoring ◽  
Specific Inhibitor ◽  
Label Free ◽  
Bafilomycin A1 ◽  
Cancer Cell Imaging

Mechanism of acidification along cortical distal tubule of the rat

1994 ◽  
Vol 266 (2) ◽  
pp. F218-F226 ◽  
Author(s):  
R. Fernandez ◽  
M. J. Lopes ◽  
R. F. de Lira ◽  
W. F. Dantas ◽  
E. J. Cragoe Junior ◽  
...  
Keyword(s):  
Channel Blocker ◽  
Exchange Resin ◽  
Rat Kidney ◽  
Specific Inhibitor ◽  
Distal Tubule ◽  
Ringer Solution ◽  
Na Channel ◽  
Bafilomycin A1 ◽  
Bicarbonate Reabsorption ◽  
Distal Tubules

The cellular mechanism of luminal acidification (bicarbonate reabsorption) was studied in cortical distal tubules of rat kidney. The stopped-flow microperfusion technique was applied to early distal (ED) and late distal (LD) segments, perfused with bicarbonate Ringer solution to which specific inhibitors were added, to measure bicarbonate reabsorption [HCO3 flux (JHCO3)]. pH and transepithelial potential difference (Vt) were recorded by double-barreled H+ exchange resin/reference (1 M KCl) electrodes. Amiloride increased stationary pH and reduced Vt in both early and late segments. Hexamethylene-amiloride (HMA), a specific Na(+)-H+ exchange blocker, reduced JHCO3 in both segments (ED by 43.6 and LD by 40.3%) without affecting Vt. Benzamil, an Na(+)-channel blocker, reduced Vt by 75.9 in ED and 74.9% in LD but had no significant effect on acidification in both segments. The specific inhibitor of H(+)-ATPase, bafilomycin A1, inhibited LD JHCO3 at a concentration of 2 x 10(-7) M by 49%, but ED was inhibited by 24% only at 2 x 10(-6) M. Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase, reduced JHCO3 by 35% in LD of K(+)-depleted rats but not in control rats and had no effect on ED. These data indicate that, in ED, bicarbonate reabsorption is mediated mostly by Na(+)-H+ exchange. In LD, there is evidence for contribution of Na(+)-H+ exchange, vacuolar H(+)-ATPase, and H(+)-K(+)-ATPase (in K(+)-depleted rats) to bicarbonate reabsorption.

scholarly journals Acidocalcisomes in Toxoplasma gondii tachyzoites

1996 ◽  
Vol 313 (2) ◽  
pp. 655-659 ◽  
Author(s):  
Silvia N. J. MORENO ◽  
Li ZHONG
Keyword(s):  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Mitochondrial Atpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Extracellular Medium ◽  
Acidic Compartment ◽  
Acidic Organelles

Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca2+-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H+-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca2+-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin+NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.

scholarly journals Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Shiou-Ling Lu ◽  
Chih-Feng Kuo ◽  
Hao-Wen Chen ◽  
Yi-Shuan Yang ◽  
Ching-Chuan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Bacterial Growth ◽  
Group A Streptococcus ◽  
Multiorgan Failure ◽  
Specific Inhibitor ◽  
Low Ph ◽  
Bafilomycin A1 ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells.IMPORTANCEPrevious reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.

Necroptosis modulated by autophagy is a predominant form of melanoma cell death induced by sanguilutine

2012 ◽  
Vol 393 (7) ◽  
pp. 647-658 ◽  
Author(s):  
Jindřiška Hammerová ◽  
Stjepan Uldrijan ◽  
Eva Táborská ◽  
Alena Hyršlová Vaculová ◽  
Iva Slaninová
Keyword(s):  
Cell Death ◽  
Therapeutic Potential ◽  
Apoptotic Cell ◽  
Specific Inhibitor ◽  
Melanoma Cells ◽  
Bafilomycin A1 ◽  
Autophagy Induction ◽  
Predominant Type ◽  
Predominant Form ◽  
Lc3 Puncta

Abstract We show that the plant quaternary benzo[c]phenanthridine alkaloid sanguilutine (SL) is a strong inducer of caspase-independent non-apoptotic death in human melanoma cells. Necrostatin-1, a specific inhibitor of necroptosis, completely reversed the cytotoxic effect of SL, suggesting that necroptosis was a predominant type of cell death induced by SL in these cells. In addition, we showed that SL can trigger an autophagic response, as confirmed by GFP-LC3 puncta formation and LC3-II accumulation. Interestingly, we observed a significant decrease in the viability of melanoma cells treated with combination of autophagy inhibitors (3-methyladenine, bafilomycin-A1 and LY294002) and SL. Our results further indicated that autophagy may serve as a pro-survival mechanism, delaying the induction of necroptosis in melanoma cells. The ability of SL to induce caspase-independent non-apoptotic cell death (necroptosis) suggests its possible therapeutic potential in the treatment of apoptosis-resistant melanoma tumours. Furthermore, SL might serve as a useful tool for studying the mechanisms of necroptosis and autophagy induction and the interplay between these two processes.

scholarly journals Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection

1998 ◽  
Vol 72 (12) ◽  
pp. 9645-9655 ◽  
Author(s):  
Nora Bayer ◽  
Daniela Schober ◽  
Elisabeth Prchla ◽  
Robert F. Murphy ◽  
Dieter Blaas ◽  
...  
Keyword(s):  
Viral Infection ◽  
Conformational Change ◽  
Hela Cells ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Inhibitory Effect ◽  
Bafilomycin A1 ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Ph

ABSTRACT Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

scholarly journals Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher B. Mulholland ◽  
Atsuya Nishiyama ◽  
Joel Ryan ◽  
Ryohei Nakamura ◽  
Merve Yiğit ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Unique Feature ◽  
Mammalian Species ◽  
Dna Demethylation ◽  
Evolutionary Divergence ◽  
Mammalian Development ◽  
Active Demethylation ◽  
Global Hypomethylation ◽  
Genome Wide

AbstractGenome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

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