ABSTRACT
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
Subject(s)
Active Transport, Cell Nucleus/physiology , Amyotrophic Lateral Sclerosis/pathology , Ataxin-2/metabolism , C9orf72 Protein/genetics , Frontotemporal Dementia/pathology , Active Transport, Cell Nucleus/drug effects , Aged , Amyotrophic Lateral Sclerosis/metabolism , Arsenites/toxicity , Ataxin-2/antagonists & inhibitors , Ataxin-2/genetics , C9orf72 Protein/metabolism , DNA Repeat Expansion/genetics , Female , Frontotemporal Dementia/metabolism , HEK293 Cells , Humans , Male , Membrane Glycoproteins/metabolism , Middle Aged , Nuclear Pore Complex Proteins/metabolism , Oxidative Stress/drug effects , RNA Interference , RNA, Small Interfering/metabolism , Sodium Compounds/toxicity , alpha Karyopherins/antagonists & inhibitors , alpha Karyopherins/genetics , alpha Karyopherins/metabolism , beta Karyopherins/antagonists & inhibitors , beta Karyopherins/genetics , beta Karyopherins/metabolism , ran GTP-Binding Protein/antagonists & inhibitors , ran GTP-Binding Protein/genetics , ran GTP-Binding Protein/metabolismABSTRACT
Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Intranuclear Inclusion Bodies/metabolism , Neurons/metabolism , RNA, Long Noncoding/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Animals, Genetically Modified , Arsenites/pharmacology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/ultrastructure , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/ultrastructure , DNA-Binding Proteins/metabolism , Disease Models, Animal , Drosophila melanogaster , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Intranuclear Inclusion Bodies/drug effects , Intranuclear Inclusion Bodies/ultrastructure , Mice , Mutation , Neurons/drug effects , Neurons/ultrastructure , Primary Cell Culture , Protein Transport/drug effects , RNA, Long Noncoding/metabolism , Signal Transduction , Stress, PhysiologicalABSTRACT
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.
Subject(s)
Cytoplasm , Homeostasis , RNA, Messenger , Stress Granules , Humans , RNA, Messenger/metabolism , RNA, Messenger/genetics , Stress Granules/metabolism , Cytoplasm/metabolism , RNA Caps/metabolism , Arsenites/pharmacology , Oxidative Stress , Active Transport, Cell Nucleus , RNA Nucleotidyltransferases/metabolism , RNA Nucleotidyltransferases/genetics , Sodium Compounds/pharmacology , Exportin 1 Protein , Karyopherins/metabolism , Karyopherins/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Cytoplasmic Granules/metabolism , RNA Stability , Cell Nucleus/metabolism , Cell Line, Tumor , NucleotidyltransferasesABSTRACT
RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.
Subject(s)
Protein Binding , RNA-Binding Proteins , RNA , Humans , RNA/metabolism , RNA/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Immunoprecipitation/methods , ELAV-Like Protein 1/metabolism , ELAV-Like Protein 1/genetics , Cell Nucleus/metabolism , Cell Nucleus/genetics , Cytoplasmic Granules/metabolism , Arsenites , HeLa Cells , Cytosol/metabolism , HEK293 CellsABSTRACT
Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.
Subject(s)
Arsenites/toxicity , Cytoplasmic Granules/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Sodium Compounds/toxicity , Stress, Physiological , TNF Receptor-Associated Factor 2/metabolism , Autophagy/drug effects , Cytoplasmic Granules/enzymology , Cytoplasmic Granules/pathology , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Proteasome Endopeptidase Complex/genetics , Protein Transport , Proteolysis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2/geneticsABSTRACT
Upon stress, cytoplasmic mRNA is sequestered to insoluble ribonucleoprotein (RNP) granules, such as the stress granule (SG). Partially due to the belief that translationally suppressed mRNAs are recruited to SGs in bulk, stress-induced dynamic redistribution of mRNA has not been thoroughly characterized. Here, we report that endoplasmic reticulum (ER) stress targets only a small subset of translationally suppressed mRNAs into the insoluble RNP granule fraction (RG). This subset, characterized by extended length and adenylate-uridylate (AU)-rich motifs, is highly enriched with genes critical for cell survival and proliferation. This pattern of RG targeting was conserved for two other stress types, heat shock and arsenite toxicity, which induce distinct responses in the total cytoplasmic transcriptome. Nevertheless, stress-specific RG-targeting motifs, such as guanylate-cytidylate (GC)-rich motifs in heat shock, were also identified. Previously underappreciated, transcriptome profiling in the RG may contribute to understanding human diseases associated with RNP dysfunction, such as cancer and neurodegeneration.
Subject(s)
Cytoplasmic Granules/metabolism , Endoplasmic Reticulum Stress , Heat-Shock Response , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Transcriptome , AU Rich Elements , Animals , Arsenites/toxicity , Binding Sites , Cytoplasmic Granules/genetics , Endoplasmic Reticulum Stress/drug effects , HCT116 Cells , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Protein Binding , Proto-Oncogenes , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/genetics , Solubility , Thapsigargin/toxicity , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/drug effectsABSTRACT
Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state NMR spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-EM. 15N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved ß-grasp fold homologous to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain of p97, leading to a partial loss of the cryo-EM density of the N-terminal domain of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance.
Subject(s)
Intracellular Signaling Peptides and Proteins , Models, Molecular , Stress Granules , Valosin Containing Protein , Humans , Arsenites/metabolism , Arsenites/chemistry , Crystallography, X-Ray , Protein Binding , Protein Domains , Stress Granules/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/metabolism , Valosin Containing Protein/chemistry , Valosin Containing Protein/genetics , Zinc Fingers , Protein Folding , Magnetic Resonance Imaging , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni2+. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin.
Subject(s)
Arsenate Reductases , Bacteroidetes , Catalytic Domain , Oxidation-Reduction , Arsenate Reductases/metabolism , Arsenate Reductases/genetics , Arsenate Reductases/chemistry , Bacteroidetes/enzymology , Bacteroidetes/genetics , Arsenates/metabolism , Kinetics , Phosphoric Monoester Hydrolases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/chemistry , Catalysis , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Arsenites/metabolismABSTRACT
Cisplatin is an effective platinum-based chemotherapeutic with several side effects, including ototoxicity. Cochlear cells have low rates of proliferation yet are highly susceptible to cisplatin. We hypothesised that cisplatin ototoxicity might be caused by cisplatin-protein interactions rather than cisplatin-DNA interactions. Two known cisplatin-binding proteins are involved in the stress granule (SG) response. SGs are a pro-survival mechanism involving formation of transient ribonucleoprotein complexes during stress. We examined the effects of cisplatin on SG dynamics and composition in cell lines derived from the cochlea and retinal pigment epithelium. Cisplatin-induced SGs are significantly diminished in size and quantity compared to arsenite-induced SGs and are persistent after 24â h recovery. Additionally, cisplatin pre-treated cells were unable to form a typical SG response to subsequent arsenite stress. Cisplatin-induced SGs had significant reductions in the sequestration of eIF4G and the proteins RACK1 and DDX3X. Live-cell imaging of Texas Red-conjugated cisplatin revealed its localisation to SGs and retention for at least 24â h. We show cisplatin-induced SGs have impaired assembly, altered composition and are persistent, providing evidence of an alternate mechanism for cisplatin-induced ototoxicity via an impaired SG response.
Subject(s)
Arsenites , Ototoxicity , Humans , Cisplatin/pharmacology , Arsenites/toxicity , Arsenites/metabolism , Ototoxicity/metabolism , Stress Granules , Cytoplasmic Granules/metabolismABSTRACT
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.
Subject(s)
Arsenites , Tumor Suppressor Protein p53 , Apoptosis , Arsenites/metabolism , Arsenites/pharmacology , I-kappa B Kinase/metabolism , I-kappa B Kinase/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Exoribonucleases/metabolismABSTRACT
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.
Subject(s)
Arsenites , Calcineurin , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Calcineurin/metabolism , Calcineurin/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Arsenites/toxicity , Arsenites/pharmacology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Signal Transduction , DNA-Binding Proteins , Muscle ProteinsABSTRACT
Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.
Subject(s)
Protein Biosynthesis , Transcriptome , Humans , Arsenites/toxicity , Proteomics , RNA/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes' peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5'-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.
Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin , Adenosine Triphosphate/metabolism , Arsenites/metabolism , Arsenites/pharmacology , Enzyme Activation/drug effects , HeLa Cells , Heat-Shock Response , Humans , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Signal Transduction , Ubiquitin/metabolism , Ubiquitinated Proteins/metabolism , UbiquitinationABSTRACT
Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The â¼50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of transfer RNA (tRNA) wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human hepatocellular carcinoma cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to demonstrating an epitranscriptomic facet of arsenite toxicity and response, our results highlight the links between environmental exposures, stress tolerance, RNA modifications, and micronutrients.
Subject(s)
Arsenites , Epigenesis, Genetic , Guanine , RNA, Transfer , Transcriptome , Arsenites/toxicity , Cell Line, Tumor , Codon/genetics , Guanine/analogs & derivatives , Guanine/metabolism , Humans , Mitochondria/drug effects , Oxidation-Reduction , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , RNA Processing, Post-Transcriptional/drug effects , RNA, Transfer/geneticsABSTRACT
Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.
Subject(s)
Arsenic , Arsenites , Humans , Antimony , Oxidation-ReductionABSTRACT
Vascular endothelial cells play a critical role in maintaining the health of blood vessels, but dysfunction can lead to cardiovascular diseases. The impact of arsenite exposure on cardiovascular health is a significant concern due to its potential adverse effects. This study aims to explore how NBR1-mediated autophagy in vascular endothelial cells can protect against oxidative stress and apoptosis induced by arsenite. Initially, our observations revealed that arsenite exposure increased oxidative stress and triggered apoptotic cell death in human umbilical vein endothelial cells (HUVECs). However, treatment with the apoptosis inhibitor Z-VAD-FMK notably reduced arsenite-induced apoptosis. Additionally, arsenite activated the autophagy pathway and enhanced autophagic flux in HUVECs. Interestingly, inhibition of autophagy exacerbated arsenite-induced apoptotic cell death. Our findings also demonstrated the importance of autophagy receptor NBR1 in arsenite-induced cytotoxicity, as it facilitated the recruitment of caspase 8 to autophagosomes for degradation. The protective effect of NBR1 against arsenite-induced apoptosis was compromised when autophagy was inhibited using pharmacological inhibitors or through genetic knockdown of essential autophagy genes. Conversely, overexpression of NBR1 facilitated caspase 8 degradation and reduced apoptotic cell death in arsenite-treated HUVECs. In conclusion, our study highlights the vital role of NBR1-mediated autophagic degradation of caspase 8 in safeguarding vascular endothelial cells from arsenite-induced oxidative stress and apoptotic cell death. Targeting this pathway could offer a promising therapeutic approach to mitigate cardiovascular diseases associated with arsenite exposure.
Subject(s)
Apoptosis , Arsenites , Autophagy , Caspase 8 , Human Umbilical Vein Endothelial Cells , Oxidative Stress , Humans , Arsenites/toxicity , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Apoptosis/drug effects , Autophagy/drug effects , Caspase 8/metabolism , Caspase 8/genetics , Oxidative Stress/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Proteolysis/drug effects , Cells, CulturedABSTRACT
OBJECTIVE: Long-term exposure to arsenic has been linked to several illnesses, including hypertension, diabetes, hepatic and renal diseases and cardiovascular malfunction. The aim of the current investigation was to determine whether zingerone (ZN) could shield rats against the hepatotoxicity that sodium arsenite (SA) causes. METHODS: The following five groups of thirty-five male Sprague Dawley rats were created: I) Control; received normal saline, II) ZN; received ZN, III) SA; received SA, IV) SA + ZN 25; received 10 mg/kg body weight SA + 25 mg/kg body weight ZN, and V) SA + ZN 50; received 10 mg/kg body weight SA + 50 mg/kg body weight ZN. The experiment lasted 14 days, and the rats were sacrificed on the 15th day. While oxidative stress parameters were studied by spectrophotometric method, apoptosis, inflammation and endoplasmic reticulum stress parameters were measured by RT-PCR method. RESULTS: The SA disrupted the histological architecture and integrity of the liver and enhanced oxidative damage by lowering antioxidant enzyme activity, such as those of glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) level and increasing malondialdehyde (MDA) level in the liver tissue. Additionally, SA increased the mRNA transcript levels of Bcl2 associated x (Bax), caspases (-3, -6, -9), apoptotic protease-activating factor 1 (Apaf-1), p53, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), c-Jun NH2-terminal kinase (JNK), mitogen-activated protein kinase 14 (MAPK14), MAPK15, receptor for advanced glycation endproducts (RAGE) and nod-like receptor family pyrin domain-containing 3 (NLRP3) in the liver tissue. Also produced endoplasmic reticulum stress by raising the mRNA transcript levels of activating transcription factor 6 (ATF-6), protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and glucose-regulated protein 78 (GRP-78). These factors together led to inflammation, apoptosis, and endoplasmic reticulum stress. On the other hand, liver tissue treated with ZN at doses of 25 and 50 mg/kg showed significant improvement in oxidative stress, inflammation, apoptosis and endoplasmic reticulum stress. CONCLUSIONS: Overall, the study's data suggest that administering ZN may be able to lessen the liver damage caused by SA toxicity.
Subject(s)
Arsenites , Chemical and Drug Induced Liver Injury , NF-kappa B , NLR Family, Pyrin Domain-Containing 3 Protein , Rats, Sprague-Dawley , Signal Transduction , Sodium Compounds , Tumor Necrosis Factor-alpha , Animals , Male , Signal Transduction/drug effects , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury/prevention & control , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Arsenites/toxicity , Sodium Compounds/toxicity , Rats , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/genetics , Liver/drug effects , Liver/metabolism , Liver/pathology , Caspase 3/metabolism , Caspase 3/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/genetics , Oxidative Stress/drug effects , Apoptosis/drug effects , Protective Agents/pharmacology , Protective Agents/therapeutic use , Endoplasmic Reticulum Chaperone BiP , Endoribonucleases , Multienzyme Complexes , Protein Serine-Threonine KinasesABSTRACT
Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.
Subject(s)
Arsenic , Dihydrolipoamide Dehydrogenase , Fatty Acids , Homeostasis , Oryza , Plant Proteins , Stress, Physiological , Adaptation, Physiological/drug effects , Adaptation, Physiological/genetics , Arsenic/toxicity , Arsenites/toxicity , Dihydrolipoamide Dehydrogenase/metabolism , Dihydrolipoamide Dehydrogenase/genetics , Fatty Acids/biosynthesis , Gene Expression Regulation, Plant/drug effects , Mutation/genetics , Oryza/genetics , Oryza/drug effects , Oryza/metabolism , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Roots/drug effects , Plant Roots/metabolism , Plastids/metabolism , Plastids/drug effects , Reactive Oxygen Species/metabolism , Stress, Physiological/drug effects , Stress, Physiological/geneticsABSTRACT
Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.
Subject(s)
Arsenites , Cytochrome P-450 Enzyme System , Drug Interactions , Rats, Sprague-Dawley , Animals , Arsenites/toxicity , Arsenites/pharmacokinetics , Male , Rats , Cytochrome P-450 Enzyme System/metabolism , Intestinal Absorption/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Omeprazole/pharmacology , Omeprazole/pharmacokinetics , Midazolam/pharmacokinetics , Caffeine/pharmacokinetics , Chlorzoxazone/pharmacokinetics , Metoprolol/pharmacokinetics , Metoprolol/pharmacology , Tolbutamide/pharmacokinetics , Sodium Compounds/toxicity , Sodium Compounds/pharmacokineticsABSTRACT
This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or EPHX1) and soluble epoxide hydrolase (sEH or EPHX2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. SIGNIFICANCE STATEMENT: Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.