A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway.

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 µg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.

BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

Neopetromin, a Cyclic Tripeptide with a C-N Cross-Link, from the Marine Sponge Neopetrosia sp., That Causes Vacuole Fragmentation in Tobacco BY-2 Cells.

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.

Primary Mode of Action of the Novel Sulfonamide Fungicide against Botrytis cinerea and Field Control Effect on Tomato Gray Mold.

Botrytis cinerea is considered an important plant pathogen and is responsible for significant crop yield losses. With the frequent application of commercial fungicides, B. cinerea has developed resistance to many frequently used fungicides. Therefore, it is necessary to develop new kinds of fungicides with high activity and new modes of action to solve the increasingly serious problem of resistance. During our screening of fungicide candidates, one novel sulfonamide compound, N-(2-trifluoromethyl-4-chlorphenyl)-2-oxocyclohexyl sulfonamide (L13), has been found to exhibit good fungicidal activity against B. cinerea. In this work, the mode of action of L13 against B. cinerea and the field control effect on tomato gray mold was studied. L13 had good control against B. cinerea resistant to carbendazim, diethofencarb, and iprodione commercial fungicides in the pot culture experiments. SEM and TEM observations revealed that L13 could cause obvious morphological and cytological changes to B. cinerea, including excessive branching, irregular ramification or abnormal configuration, and the decomposition of cell wall and vacuole. L13 induced more significant electrolyte leakage from hyphae than procymidone as a positive control. L13 had only a minor effect on the oxygen consumption of intact mycelia, with 2.15% inhibition at 50 µg/mL. In two locations over 2 years, the field control effect of L13 against tomato gray mold reached 83% at a rate of 450 g ai ha-1, better than the commercial fungicide of iprodione. Moreover, toxicological tests demonstrated the low toxicological effect of L13. This research seeks to provide technical support and theoretical guidance for L13 to become a real commercial fungicide.

A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles.

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

Lactoferrin Protects against Methamphetamine Toxicity by Modulating Autophagy and Mitochondrial Status.

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker ßIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Inhibition of Autophagy Flux Promotes Secretion of Chondroitin Sulfate Proteoglycans in Primary Rat Astrocytes.

Following spinal cord injury (SCI), reactive astrocytes in the glial scar produce high levels of chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal regeneration. Transforming growth factor beta (TGFß) is a well-known factor that induces the production of CSPGs, and in this study, we report a novel mechanism underlying TGFß's effects on CSPG secretion in primary rat astrocytes. We observed increased TGFß-induced secretion of the CSPGs neurocan and brevican, and this occurred simultaneously with inhibition of autophagy flux. In addition, we show that neurocan and brevican levels are further increased when TGFß is administered in the presence of an autophagy inhibitor, Bafilomycin-A1, while they are reduced when cells are treated with a concentration of rapamycin that is not sufficient to induce autophagy. These findings suggest that TGFß mediates its effects on CSPG secretion through autophagy pathways. They also represent a potential new approach to reduce CSPG secretion in vivo by targeting autophagy pathways, which could improve axonal regeneration after SCI.

Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks.

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

Translocation of LSR from tricellular corners causes macropinocytosis at cell-cell interface as a trigger for breaking out of contact inhibition.

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.

Selenium triggers Nrf2-AMPK crosstalk to alleviate cadmium-induced autophagy in rabbit cerebrum.

Cadmium (Cd) is a toxic heavy metal that accumulates in the brain and causes a series of histopathological changes. Selenium (Se) exerts a crucial function in protecting damage caused by toxic heavy metals, but its potential mechanism is rarely studied. The main purpose of this study is to explore the protective effects of Se on Cd-induced oxidative stress and autophagy in rabbit cerebrum. Forty rabbits were randomly divided into four groups and treated as follows: Control group, Cd (1 mg/kg⋅BW) group, Se (0.5 mg/kg⋅BW) group and Cd (1 mg/kg⋅BW)+Se (0.5 mg/kg⋅BW) group, with 30 days feeding management. Our results suggested that Se treatment significantly suppressed the Cd-induced degenerative changes including cell necrosis, vacuolization, and atrophic neurons. In addition, Se decreased the contents of MDA and H2O2 and increased the activities of CAT, SOD, GST, GSH and GSH-Px, alleviating the imbalance of the redox system induced by Cd. Furthermore, Cd caused the up-regulation of the mRNA levels of autophagy-related genes (ATG3, ATG5, ATG7, ATG12 and p62), AMPK (Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag2, Prkag3) and Nrf2 (Nrf2, HO-1 and NQO1) signaling pathway, and the expression levels of LC3II/LC3I, p-AMPK/AMPK, Beclin-1, Nrf2 and HO-1 proteins, which were alleviated by Se, indicated that Se inhibited Cd-induced autophagy and Nrf2 signaling pathway activation. In conclusion, our study found that Se antagonized Cd-induced oxidative stress and autophagy in the brain by generating crosstalk between AMPK and Nrf2 signaling pathway.

LPS-induced autophagy in human dental pulp cells is associated with p38.

Lipopolysaccharides (LPS), which are components of the cell wall of Gram-negative bacteria, are among the important factors that induce inflammation, including pulpitis. Autophagy in human dental pulp cells (hDPCs) acts as a protective mechanism that promotes cell survival under adverse conditions through different signaling pathways. In this study, we examined whether LPS increases autophagy in hDPCs and investigated the role of mitogen-activated protein kinases signaling and nuclear factor κB (NF-κB) in this process. We found that stimulation of hDPCs with 0.1 µg/mL LPS increased the protein and mRNA levels of autophagy markers, beclin1 and microtubule associated protein light chain 3II (LC3II). In addition, acridine orange staining and transmission electron microscopy demonstrated the induction of autophagy upon the treatment of LPS. Furthermore, LPS affected phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and the nuclear translocation of NF-κB. While p38 inhibitor suppressed the LPS-induced increase in protein levels of beclin1 and LC3-II. Our results suggest that LPS induced autophagy in hDPCs and affected the phosphorylation of p38, ERK, and JNK, as well as the nuclear translocation of NF-κB. Phosphorylation of p38 may be involved in LPS-induced autophagy in hDPCs.

Norepinephrine Protects against Methamphetamine Toxicity through ß2-Adrenergic Receptors Promoting LC3 Compartmentalization.

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane ß2-adrenergic receptors (ARs). Evidence indicates that ß2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

Early Life Antibiotics Influence In Vivo and In Vitro Mouse Intestinal Epithelium Maturation and Functioning.

BACKGROUND & AIMS: The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. METHODS: Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. RESULTS: We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. CONCLUSIONS: Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.

The acetone crude extract of Quercus infectoria (Olivier) galls alters pH of the digestive vacuole of the malaria parasite, Plasmodium falciparum.

The reduced efficacy of the mainstay antimalarial drugs due to the widespread of drugresistant Plasmodium falciparum has necessitated efforts to discover new antimalarial drugs with new targets. Quercus infectoria (Olivier) has long been used to treat various ailments including fever. The acetone extract of the plant galls has recently been reported to have a promising antimalarial activity in vitro. This study was aimed to determine the effect of the Q. infectoria gall acetone crude extract on pH of the digestive vacuole of Plasmodium falciparum. A ratiometric fluorescent probe, fluorescein isothiocyanate-dextran (FITC-dextran) was used to facilitate a quantitative measurement of the digestive vacuole pH by flow cytometry. Mid trophozoite stage malaria parasites grown in resealed erythrocytes containing FITC-dextran were treated with different concentrations of the acetone extract based on the 50% inhibitory concentration (IC50). Saponin-permeabilized parasites were analyzed to obtain the ratio of green/yellow fluorescence intensity (Rgy) plotted as a function of pH in a pH calibration curve of FITC-dextran. Based on the pH calibration curve, the pH of the digestive vacuole of the acetone extract-treated parasites was significantly altered (pH values ranged from 6.35- 6.71) in a concentration-dependent manner compared to the untreated parasites (pH = 5.32) (p < 0.001). This study provides a valuable insight into the potential of the Q. infectoria galls as a promising antimalarial candidate with a novel mechanism of action.

Plasmodium falciparum Atg18 localizes to the food vacuole via interaction with the multi-drug resistance protein 1 and phosphatidylinositol 3-phosphate.

Autophagy, a lysosome-dependent degradative process, does not appear to be a major degradative process in malaria parasites and has a limited repertoire of genes. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 bind PI3P and play an essential role in autophagosome formation. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is likely a shared feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, wild type ScAtg18 interacted with PI3P, but its expression in P. falciparum showed complete cytoplasmic localization, indicating additional requirement for food vacuole localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the immunoprecipitates of PfAtg18 and P. berghei Atg18, and also interacted with PfAtg18. In contrast with PfAtg18, ScAtg18 did not interact with MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine caused cytoplasmic localization of PfAtg18, suggesting that these target PfAtg18 transport pathway. Thus, PI3P and MDR1 are critical mediators of PfAtg18 localization.

Autophagy of the m6A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis.

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.