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Genome-wide CRISPR loss-of-function screen for α-amanitin

ABSTRACT: The “death cap”, Amanita phalloides, is the world’s most poisonous mushroom, responsible for 90% of mushroom-related fatalities. The most fatal component of the death cap is α-amanitin(AMA). Despite its lethal effect, the exact mechanisms of how α-amanitin poisons humans remain unclear, leading to no specific antidote available for treatment. Here, we performed a genome-wide CRISPR loss-of-function screen to identify genes and pathways involved in AMA cytotoxicity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE226447 | GEO | 2023/03/07

REPOSITORIES: GEO

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Characterization of protein dynamics after alpha-amanitin exposure in hepatoma cells

Project description:Alpha-amanitin (α-AMA) is a cyclic peptide and is one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography-tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. We identified 1,828 proteins, quantified 1,563 proteins, and found a decrease of four subunits in T-complex protein 1-ring complex protein that was dependent on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global proteome abundance changes according to α-AMA concentration variations and suggests a possible novel molecular regulation mechanism for hepatotoxicity.

2021-09-09 | PXD018702 | Pride

Characterization of RAS/RAF/ERK signal cascade as a novel regulating factor in alpha-amanitin-induced liver injury

Project description:The well-known hepatotoxicity mechanism resulting from alpha-amanitin (-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from -AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses. Bioinformatics analysis showed that AMA exposure increased protein phosphorylation in a time-dependent AMA AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of -AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by -AMA exposure followed by aberrant splicing events leading to cell death.

2023-03-11 | PXD035758 | Pride

Characterization of RAS/RAF/ERK signal cascade as a novel regulating factor in alpha-amanitin-induced liver injury

2023-03-11 | PXD035817 | Pride

The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

Project description:The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study, however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in Drosophila melanogaster and perhaps in mycophagous Drosophila species has evolved as a cross-resistance to pesticides or other xenobiotic substances.

2015-12-01 | GSE52782 | GEO

Characterization of hepatocytes secretomes after alpha-amanitin exposure by quantitative proteomics

Project description:In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they serve as a representation of the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be applied to discover toxic mechanisms or exposure markers. The most widely reported amatoxin is alpha-amanitin (-AMA), which inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, the secretory proteins released by hepatic failure caused by -AMA are still not fully characterized. In this study, we analyzed secretory proteins in -AMA-induced hepatotoxicity after -AMA treatment to Huh-7 cells using a comparative proteomics technique. In the results, 1,505 and 1,440 were identified and quantified and significantly up- and down-regulated proteins were 4 and 127 proteins, respectively. Then, bioinformatics analysis was performed to get the GO, Interpro and KEGG pathways about significantly down-regulated proteins. Based on bioinformatics results, we picked complement component 3 (C3) as an exposure marker after -AMA-induced hepatotoxicity. Through western blot and C3 ELISA assay, we validated the C3 level was decreased resulting from -AMA. In conclusion, we found the C3 level in secretomes reduced after -AMA-induced hepatotoxicity using comparative proteomics and molecular biology techniques. We expect this research will be helpful to find new toxic mechanisms, therapeutic targets, and exposure markers in -AMA-induced hepatotoxicity.

2024-05-23 | PXD036476 | Pride

Characterization of complement C3 as a marker of alpha-amanitin toxicity by comparative secretome profiling

Project description:In the human body, proteins secreted into peripheral blood vessels are known as the secretome, and they represent the physiological or pathological status of cells. The unique response of cells to toxin exposure can be confirmed via secretome analysis, which can be used to discover toxic mechanisms or exposure markers. Alpha-amanitin (-AMA) is the most widely studied amatoxin and inhibits transcription and protein synthesis by directly interacting with RNA polymerase II. However, secretory proteins released during hepatic failure caused by -AMA have not been fully characterized. In this study, we analyzed secretome of -AMA-treated Huh-7 cells and mice using a comparative proteomics technique. Overall, 1,505 and 1,440 proteins were identified and quantified, respectively. Among them, 4 and 127 proteins were significantly upregulated and downregulated, respectively. Based on the bioinformatics results for the significantly downregulated proteins, we identified complement component 3 (C3) as a marker for -AMA-induced hepatotoxicity. Through western blot and C3 ELISA assays, we validated -AMA-induced downregulation of C3. In conclusion, using comparative proteomics and molecular biology techniques, we found that -AMA-induced hepatotoxicity reduced C3 levels in the secretome. We expect that this study will aid in identifying new toxic mechanisms, therapeutic targets, and exposure markers of -AMA-induced hepatotoxicity.

2024-05-23 | PXD038016 | Pride

Alpha-amanitin treatment effect on intronic transcription

Project description:We compared the gene expression profiles of prostate carcinoma cells, LNCaP, treated or not with an inhibitor of RNA Polymerase II (RNAP II), alpha-amanitin. These experiments pointed to a fraction of intronic messages that seems to be insensitive to alpha-amanitin or up-regulated in cells with blocked RNAP II transcription. Keywords: RNA-polymerase II inhibition experiments

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Transcription profiling of Xenopus laevis early gastrulation embryos injected with alpha-amanitin against RNA polymerase II activity

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2016-03-07 | E-MTAB-3614 | biostudies-arrayexpress

RNA expression analysis after RNA polymerase II transcriptional blocking with α-amanitin

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The NTCP receptor inhibitor Ezetimibe reduces the toxicity of α-amanitin: in vitro and in vivo study.

Project description:Background and Aims: To verify the protective effect of Ezetimibe, an sodium taurocholate co-transporting polypeptide (NTCP) inhibitor, on α-amanitin poisoning in vitro and in vivo by inhibiting NTCP to prevent α-amanitin into hepatocytes. Approach and Results: In animal experiments, the survival rate was significantly improved in the treatment group. The pathomorphological characteristics of liver and kidney in the treatment group were significantly improved. In cell experiments,The cell viability of the treatment group was significantly improved, and the expression of NTCP in the treatment group was significantly decreased by immunofluorescence. In molecular docking simulations, we demonstrated the potential of NTCP to bind Ezetimibe and α-amanitin, respectively. Transcriptomics in high-throughput sequencing was used to detect the differential metabolic genes between α-amanitin poisoning group and the treatment group, and signal pathway enrichment was used to analyze the significantly different signal pathways. Conclusions: Ezetimibe, as an inhibitor of NTCP, can reduce the entry of α-amanitin into hepatocytes to play a protective role and improve the cell viability and survival rate of mice.

2023-06-30 | GSE235740 | GEO

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