scholarly journals V-ATPase is a universal regulator of LC3 associated phagocytosis and non-canonical autophagy

2021 ◽  
Author(s):  
Kirsty M Hooper ◽  
Elise Jacquin ◽  
Taoyingnan Li ◽  
Jonathan M Goodwin ◽  
John H Brumell ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Host Response ◽  
Common Mechanism ◽  
Drug Induced ◽  
Cellular Pathway ◽  
Wd40 Domain

Non-canonical autophagy is a key cellular pathway in immunity, cancer and neurodegeneration, characterised by Conjugation of ATG8 to endolysosomal Single-Membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1) and infection (influenza), dependent on the ATG16L1 WD40-domain, and specifically K490. However, the factor(s) associated with non-canonical ATG16L1 recruitment, and CASM induction, remain unknown. Here, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase directly recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis, and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM, and indicate that SopF evolved in part to evade non-canonical autophagy.

scholarly journals Subtractive CRISPR screen identifies factors involved in non-canonical LC3 lipidation

2020 ◽  
Author(s):  
Rachel Ulferts ◽  
Elena Marcassa ◽  
Lewis Timimi ◽  
Liam C Lee ◽  
Andrew Daley ◽  
...  
Keyword(s):  
Influenza A ◽  
Screening Strategy ◽  
Vacuolar Atpase ◽  
Proton Channel ◽  
Common Mechanism ◽  
Drug Induced ◽  
Atpase Subunit ◽  
Knock Out ◽  
Crispr Screen ◽  
Wd40 Domain

AbstractAlthough commonly associated with autophagosomes, LC3 can also be recruited to membranes in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. LC3 is attached to membranes by covalent lipidation that depends on recruitment of the ATG5-12-16L1 complex. Non-canonical LC3 lipidation requires the C-terminal WD40 domain of ATG16L1 that is dispensable for canonical autophagy. We devised a subtractive CRISPR knock-out screening strategy to investigate the requirements for non-canonical LC3-lipidation. This correctly identified the enzyme complexes directly responsible for LC3-lipidation. We additionally identified the RALGAP complex as important for M2-induced, but not ionophore drug induced LC3 lipidation. In contrast, we identified ATG4D as responsible for LC3 recycling in M2-induced and basal LC3-lipidation. Identification of a vacuolar ATPase subunit in the screen suggested a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug induced LC3-lipidation leads to association of the vacuolar ATPase and ATG16L1 and can be antagonised by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

Impact of Myeloperoxidase and NADPH-Oxidase Polymorphisms in Drug-Induced Agranulocytosis

2004 ◽  
Vol 24 (6) ◽  
pp. 613-617 ◽  
Author(s):  
Igor Mosyagin ◽  
Michael Dettling ◽  
Ivar Roots ◽  
Bruno Mueller-Oerlinghausen ◽  
Ingolf Cascorbi
Keyword(s):  
Nadph Oxidase ◽  
Drug Induced

scholarly journals Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase

1993 ◽  
Vol 290 (1) ◽  
pp. 41-49 ◽  
Author(s):  
V B O'Donnell ◽  
D G Tew ◽  
O T G Jones ◽  
P J England
Keyword(s):  
Nadph Oxidase ◽  
Human Neutrophil ◽  
Common Mechanism ◽  
Radical Mechanism ◽  
Dependent Inhibition ◽  
Enzyme Turnover ◽  
Covalent Adducts ◽  
Time Dependent Inhibition ◽  
Adjacent Amino Acid ◽  
Redox Centre

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

scholarly journals Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2766-2766 ◽  
Author(s):  
Antoni Domagala ◽  
Malgorzata Bobrowicz ◽  
Joanna Stachura ◽  
Marta Siernicka ◽  
Piotr Mrowka ◽  
...  
Keyword(s):  
Cell Death ◽  
Nadph Oxidase ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Raji Cell ◽  
Lymphocytic Leukemia ◽  
Common Mechanism ◽  
Concanamycin A ◽  
Diphenylene Iodonium ◽  
Direct Cell

Abstract Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death. In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination. Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). Disclosures No relevant conflicts of interest to declare.

Up-regulation of NOX1/NADPH oxidase following drug-induced myocardial injury promotes cardiac dysfunction and fibrosis

2018 ◽  
Vol 120 ◽  
pp. 277-288 ◽  
Author(s):  
Kazumi Iwata ◽  
Kuniharu Matsuno ◽  
Ayumi Murata ◽  
Kai Zhu ◽  
Hitomi Fukui ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Cardiac Dysfunction ◽  
Myocardial Injury ◽  
Drug Induced

scholarly journals Dopamine Levels Induced by Substance Abuse Alter Efficacy of Maraviroc and Expression of CCR5 Conformations on Myeloid Cells: Implications for NeuroHIV

2021 ◽  
Vol 12 ◽  
Author(s):  
Stephanie M. Matt ◽  
Emily A. Nickoloff-Bybel ◽  
Yi Rong ◽  
Kaitlyn Runner ◽  
Hannah Johnson ◽  
...  
Keyword(s):  
Substance Abuse ◽  
Drugs Of Abuse ◽  
Receptor Gene ◽  
Myeloid Cells ◽  
Fluorescent Imaging ◽  
Public Health Issue ◽  
Common Mechanism ◽  
Drug Abusers ◽  
Drug Induced ◽  
Dopamine Signaling

Despite widespread use of antiretroviral therapy (ART), HIV remains a major public health issue. Even with effective ART many infected individuals still suffer from the constellation of neurological symptoms now known as neuroHIV. These symptoms can be exacerbated by substance abuse, a common comorbidity among HIV-infected individuals. The mechanism(s) by which different types of drugs impact neuroHIV remains unclear, but all drugs of abuse increase central nervous system (CNS) dopamine and elevated dopamine increases HIV infection and inflammation in human myeloid cells including macrophages and microglia, the primary targets for HIV in the brain. Thus, drug-induced increases in CNS dopamine may be a common mechanism by which distinct addictive substances alter neuroHIV. Myeloid cells are generally infected by HIV strains that use the chemokine receptor CCR5 as a co-receptor, and our data indicate that in a subset of individuals, drug-induced levels of dopamine could interfere with the effectiveness of the CCR5 inhibitor Maraviroc. CCR5 can adopt distinct conformations that differentially regulate the efficiency of HIV entry and subsequent replication and using qPCR, flow cytometry, Western blotting and high content fluorescent imaging, we show that dopamine alters the expression of specific CCR5 conformations of CCR5 on the surface of human macrophages. These changes are not affected by association with lipid rafts, but do correlate with dopamine receptor gene expression levels, specifically higher levels of D1-like dopamine receptors. These data also demonstrate that dopamine increases HIV replication and alters CCR5 conformations in human microglia similarly to macrophages. These data support the importance of dopamine in the development of neuroHIV and indicate that dopamine signaling pathways should be examined as a target in antiretroviral therapies specifically tailored to HIV-infected drug abusers. Further, these studies show the potential immunomodulatory role of dopamine, suggesting changes in this neurotransmitter may also affect the progression of other diseases.

scholarly journals ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1

2013 ◽  
Vol 304 (1) ◽  
pp. C102-C111 ◽  
Author(s):  
Daria V. Ilatovskaya ◽  
Tengis S. Pavlov ◽  
Vladislav Levchenko ◽  
Alexander Staruschenko
Keyword(s):  
Growth Factor ◽  
Nadph Oxidase ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fine Tuning ◽  
Common Mechanism ◽  
Ros Production ◽  
Cortical Collecting Duct ◽  
Nadph Oxidase Complex

The epithelial Na+ channel (ENaC) is a key transporter participating in the fine tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCDc14) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCDc14 cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H2DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCDc14 monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCDc14 monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.

Microscopic Analysis of Oxidative Stress in Cultured Cells. I. Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 634-635
Author(s):  
Beverly E. Maleeff ◽  
Tracy L. Gales ◽  
Padma K. Narayanan ◽  
Mark A. Tirmenstein ◽  
Timothy K. Hart
Keyword(s):  
Oxidative Stress ◽  
Hepg2 Cells ◽  
Cultured Cells ◽  
Cell Model ◽  
Hepatoma Cell ◽  
Common Mechanism ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Drug Induced

Cellular oxidative stress, a common mechanism of drug-induced toxicity, is a result of the formation of reactive oxygen species (ROS) in response to chemical stimuli. An endpoint of ROS production is lipid peroxidation, which can in turn lead to disruption of cellular membranes, loss of mitochondrial function, protein oxidation and DNA damage. This toxicity can be organ-specific due to the varying capacities of tissues to handle oxidative events. Liver is particularly sensitive to the effects of oxidative stress, and hepatic toxicity is seen clinically. HepG2 cells are an immortalized human hepatoma cell line used as an in vitro model for mammalian hepatotoxicity studies. The purpose of this study was to characterize the effects of chemically induced oxidative stress in this cultured cell model.HepG2 cells were grown to subconfluence in poly-L-lysine coated 4-well LabTek™ II chambered coverglasses (Nalge Nunc International).

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