Myosin-5a facilitates stress granule formation by interacting with G3BP1.

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process.

Sodium arsenite induces aggresome formation by promoting PICK1 BAR domain homodimer formation.

The aggresome is a perinuclear structure that sequesters misfolded proteins. It is implicated in various neurodegenerative diseases. The perinuclear structure enriched with protein interacting with C kinase 1 (PICK1) was found to be inducible by cellular stressors, colocalizing with microtubule-organizing center markers and ubiquitin, hence classifying it as an aggresome. Sodium arsenite but not arsenate was found to potently induce aggresome formation through an integrated stress response-independent pathway. In HEK293T cells, under arsenite stress, PICK1 localization to the aggresome was prioritized, and formation of PICK1 homodimers was favored. Additionally, PICK1 could enhance protein entry into aggresomes. This study shows that arsenite can induce the formation of both RNA stress granules and aggresomes at the same time, and that PICK1 shows conditional localization to aggresomes, suggesting a possible involvement of PICK1 in neurodegenerative diseases.

A Sb(III)-specific efflux transporter from Ensifer adhaerens E-60.

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysRars, which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysRars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysRars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysRars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysRars, respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Sodium arsenite and arsenic trioxide differently affect the oxidative stress of lymphoblastoid cells: An intricate crosstalk between mitochondria, autophagy and cell death.

Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.

Identification of a novel RNA transcript TISPL upregulated by stressors that stimulate ATF4.

Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5' rapid amplification of cDNA ends (5' RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.

Investigation of the protective effect of chitosan against arsenic-induced nephrotoxicity and oxidative damage in rat kidney tissue.

Arsenic is an important metalloid that can cause poisoning in humans and domestic animals. Exposure to arsenic causes cell damage, increasing the production of reactive oxygen species. Chitosan is a biopolymer obtained by deacetylation of chitin with antioxidant and metal ion chelating properties. In this study, the protective effect of chitosan on arsenic-induced nephrotoxicity and oxidative damage was investigated. 32 male Wistar-albino rats were divided into 4 groups of 8 rats each as control group (C), chitosan group (CS group), arsenic group (AS group), and arsenic+chitosan group (AS+CS group). The C group was given distilled water by oral gavage, the AS group was given 100 ppm/day Na-arsenite ad libitum with drinking water, the CS group was given 200 mg/kg/day chitosan dissolved in saline by oral gavage, the AS+CS group was given 100 ppm/day Na-arsenite ad libitum with drinking water and 200 mg/kg/day chitosan dissolved in saline by oral gavage for 30 days. At the end of the 30-day experimental period, 90 mg/kg ketamine was administered intraperitoneally to all rats, and blood samples and kidney tissues were collected. Urea, uric acid, creatinine, P, Mg, K, Ca, Na, Cystatin C (CYS-C), Neutrophil Gelatinase Associated Lipocalin (NGAL) and Kidney Injury Molecule 1 (KIM-1) levels were measured in serum samples. Malondialdehyde (MDA), Glutathione (GSH), Catalase (CAT) and Superoxide dismutase (SOD) levels in the supernatant obtained from kidney tissue were analyzed by ELISA method. Compared with AS group, uric acid and creatinine levels of the AS+CS group were significantly decreased (p<0.001), urea, KIM-1, CYS-C, NGAL, and MDA levels were numerically decreased and CAT, GSH, and SOD levels were numerically increased (p>0.05). In conclusion, based on both biochemical and histopathological-immunohistochemical- immunofluorescence findings, it can be concluded that chitosan attenuates kidney injury and protects the kidney.

Novel insights into the co-selection of metal-driven antibiotic resistance in bacteria: a study of arsenic and antibiotic co-exposure.

The simultaneous development of antibiotic resistance in bacteria due to metal exposure poses a significant threat to the environment and human health. This study explored how exposure to both arsenic and antibiotics affects the ability of an arsenite oxidizer, Achromobacter xylosoxidans CAW4, to transform arsenite and its antibiotic resistance patterns. The bacterium was isolated from arsenic-contaminated groundwater in the Chandpur district of Bangladesh. We determined the minimum inhibitory concentration (MIC) of arsenite, cefotaxime, and tetracycline for A. xylosoxidans CAW4, demonstrating a multidrug resistance (MDR) trait. Following this determination, we aimed to mimic an environment where A. xylosoxidans CAW4 was exposed to both arsenite and antibiotics. We enabled the strain to grow in sub-MIC concentrations of 1 mM arsenite, 40 µg/mL cefotaxime, and 20 µg/mL tetracycline. The expression dynamics of the arsenite oxidase (aioA) gene in the presence or absence of antibiotics were analyzed. The findings indicated that simultaneous exposure to arsenite and antibiotics adversely affected the bacteria's capacity to metabolize arsenic. However, when arsenite was present in antibiotics-containing media, it promoted bacterial growth. The study observed a global downregulation of the aioA gene in arsenic-antibiotic conditions, indicating the possibility of increased susceptibility through co-resistance across the entire bacterial population of the environment. This study interprets that bacterial arsenic-metabolizing ability can rescue the bacteria from antibiotic stress, further disseminating environmental cross-resistance. Therefore, the co-selection of metal-driven antibiotic resistance in bacteria highlights the need for effective measures to address this emerging threat to human health and the environment.

Rapid arsenite oxidation by Paenarthrobacter nicotinovorans strain SSBW5: unravelling the role of GlpF, aioAB and aioE genes.

A novel arsenite resistant bacterial strain SSBW5 was isolated from the battery waste site of Corlim, Goa, India. This strain interestingly exhibited rapid arsenite oxidation with an accumulation of 5 mM arsenate within 24 h and a minimum inhibitory concentration (MIC) of 18 mM. The strain SSBW5 was identified as Paenarthrobacter nicotinovorans using 16S rDNA sequence analysis. Fourier-transformed infrared (FTIR) spectroscopy of arsenite-exposed cells revealed the interaction of arsenite with several important functional groups present on the cell surface, possibly involved in the resistance mechanism. Interestingly, the whole genome sequence analysis also clearly elucidated the presence of genes, such as GlpF, aioAB and aioE encoding transporter, arsenite oxidase and oxidoreductase enzyme, respectively, conferring their role in arsenite resistance. Furthermore, this strain also revealed the presence of several other genes conferring resistance to various metals, drugs, antibiotics and disinfectants. Further suggesting the probable direct or indirect involvement of these genes in the detoxification of arsenite thereby increasing its tolerance limit. In addition, clumping of bacterial cells was observed through microscopic analysis which could also be a strategy to reduce arsenite toxicity thus indicating the existence of multiple resistance mechanisms in strain SSBW5. In the present communication, we are reporting for the first time the potential of P. nicotinovorans strain SSBW5 to be used in the bioremediation of arsenite via arsenite oxidation along with other toxic metals and metalloids.

The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.

Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

Pevonedistat Inhibits SOX2 Expression and Sphere Formation but Also Drives the Induction of Terminal Differentiation Markers and Apoptosis within Arsenite-Transformed Urothelial Cells.

Urothelial cancer (UC) is a common malignancy and its development is associated with arsenic exposure. Around 25% of diagnosed UC cases are muscle invasive (MIUC) and are frequently associated with squamous differentiation. These patients commonly develop cisplatin (CIS) resistance and have poor prognosis. SOX2 expression is correlated to reduced overall and disease-free survival in UC. SOX2 drives malignant stemness and proliferation in UC cells and is associated with development of CIS resistance. Using quantitative proteomics, we identified that SOX2 was overexpressed in three arsenite (As3+)-transformed UROtsa cell lines. We hypothesized that inhibition of SOX2 would reduce stemness and increase sensitivity to CIS in the As3+-transformed cells. Pevonedistat (PVD) is a neddylation inhibitor and is a potent inhibitor of SOX2. We treated non-transformed parent and As3+-transformed cells with PVD, CIS, or in combination and monitored cell growth, sphere forming abilities, apoptosis, and gene/protein expression. PVD treatment alone caused morphological changes, reduced cell growth, attenuated sphere formation, induced apoptosis, and elevated the expression of terminal differentiation markers. However, the combined treatment of PVD with CIS significantly elevated the expression of terminal differentiation markers and eventually led to more cell death than either solo treatment. Aside from a reduced proliferation rate, these effects were not seen in the parent. Further research is needed to explore the potential use of PVD with CIS as a differentiation therapy or alternative treatment for MIUC tumors that may have become resistant to CIS.

Gene regulation and ionome homeostasis in rice plants in response to arsenite stress: potential connection between transcriptomics and ionomics.

Ionomics and transcriptomics were applied to demonstrate response of rice to arsenite [As(III)] stress in the current study. Rice plants were cultured in nutrient solutions treated with 0, 100 and 500 µg/L As(III) coded as CK, As1 and As5, respectively. The rice ionomes exhibited discriminatory response to environmental disturbances. Solid evidence of the effects of As(III) stress on binding, transport or metabolism of P, K, Ca, Zn and Cu was obtained in this work. Differentially expressed genes (DEGs) in the shoots were identified in three datasets: As1 vs CK, As5 vs CK and As5 vs As1. DEGs identified simultaneously in two or three datasets were selected for subsequent interaction and enrichment analyses. Upregulation of genes involved in protein kinase activity, phosphorus metabolic process and phosphorylation were detected in the rice treated with As(III), resulting in the maintenance of P homeostasis in the shoots. Zn and Ca binding genes were up-regulated since excess As inhibited the translocation of Zn and Ca from roots to shoots. Increased expression of responsive genes including HMA, WRKY, NAC and PUB genes conferred As tolerance in the rice plants to cope with external As(III) stress. The results suggested that As(III) stress could disturb the uptake and translocation of macro and essential elements by rice. Plants could regulate the expression of corresponding genes to maintain mineral nutrient homeostasis for essential metabolic processes.

Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Arsenite-induced Radiosensitization of Glioma Cells Is Dependent on p53 Deficiency.

BACKGROUND/AIM: Arsenite is a radiosensitizer of glioma cells both in vitro and in vivo; however, the underlying mechanism of action is unclear. Radiosensitizers specific for p53-deficient tumors are a promising adjunct to radiotherapy because, unlike normal cells, many tumor cells lack p53. Previously, we demonstrated that arsenite sensitizes the p53-deficient glioma cell line U87MG-E6 to X-rays. MATERIALS AND METHODS: Using flowcytometry, we expand these findings to p53-proficient U87MG cells exposed to heavy ion beams, including carbon and iron ions. RESULTS: Arsenite sensitized U87MG-E6, but not U87MG, cells to heavy ion beams and X-rays. Cell cycle analysis indicated that sensitization of U87MG-E6 was related to an increase in the percentage of cells in the late S/G2/M phases after combined treatment with arsenite, especially when carbon ion beams were used. Induction of γH2AX was significant in U87MG-E6, but not in U87MG, cells after irradiation with carbon ion beams plus arsenite. CONCLUSION: Arsenite sensitizes cells by increasing the percentage of cells in the late S/G2/M phases after irradiation, possibly via inhibition of DNA repair in the context of p53 deficiency. The findings provide information that may be useful for the development of advanced radiotherapy protocols.

ISG20L1 acts as a co-activator of DAPK1 in the activation of the p53-dependent cell death pathway.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKß-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKß/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA-binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1-mediated necroptosis in a manner dependent on stress granule formation.

Arsenic exposure alters the expression of genes related to metabolic diseases in differentiated adipocytes and in newborns and children.

The mechanisms underlying the association between prenatal arsenic exposure and the development of metabolic diseases remain unclear. Aberrant adipogenesis and adipokine production are associated with increased risk for the development of metabolic diseases in susceptible populations. Generation of mature adipocytes is tightly regulated by the expression of genes encoding: peroxisome proliferator-activated receptor γ (PPARG), fatty acid-binding protein (FABP4), and glucose transporter-4 (SLC2A4), and adipokines such as leptin (LEP) and adiponectin (ADIPOQ). This study aimed to investigate the expression of these genes, which are associated with the pathogenesis of metabolic diseases in newborns and children exposed to arsenic in utero. A high arsenic exposed group showed significantly decreased PPARG and FABP4 expression in cord blood samples from newborns and in saliva samples from children. By contrast, the expression of the SLC2A4 and ADIPOQ mRNA was significantly decreased in high-arsenic exposed children. Furthermore, the levels of toenail arsenic were negatively correlated with the salivary mRNA expression levels of PPARG (r = -0.412, p < 0.01), aP2 (r = -0.329, p < 0.05), and SLC2A4 (r = -0.528, p < 0.01). In vitro studies utilizing umbilical cord derived mesenchymal stem cells (UC-MSCs) as a surrogate for fetal MSCs showed that arsenite treatment (0.5 µM and 1 µM) significantly impaired adipogenic differentiation in a concentration dependent manner. Such impairment may be related to a significant decrease in the expression of: PPARγ, FABP4, and SLC2A4 observed at 1 µM arsenite. Arsenite treatment also promoted inflammation through a significant increase in the mRNA expression levels of the pro-inflammatory adipokine, LEP, and the inflammatory cytokines: CXCL6, IL-1ß, and CXCL8. Collectively, our results suggests that such alterations may be a consequence of the effects of arsenic exposure on fetal MSCs eventually leading to impaired adipogenic differentiation and the promotion of inflammation, both of which contribute to the development of metabolic diseases later in life.

Arsenite enhances ERO1α expression via ryanodine receptor dependent and independent mechanisms.

Arsenite is a potent carcinogen and toxic compound inducing an array of deleterious effects via different mechanisms, which include the Ca2+-dependent formation of reactive oxygen species. The mechanism whereby the metalloid affects Ca2+ homeostasis involves an initial stimulation of the inositol 1, 4, 5-triphosphate receptor, an event associated with an endoplasmic reticulum (ER) stress leading to increased ERO1α expression, and ERO1α dependent activation of the ryanodine receptor (RyR). Ca2+ release from the RyR is then critically connected with the mitochondrial accumulation of Ca2+. We now report that the resulting formation of mitochondrial superoxide triggers a second mechanism of ER stress dependent ERO1α expression, which however fails to impact on Ca2+ release from the RyR or, more generally, on Ca2+ homeostasis. Our results therefore demonstrate that arsenite stimulates two different and sequential mechanisms leading to increased ERO1α expression with different functions, possibly due to their different subcellular compartmentalization.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.