Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro

Doeun Kim,Hye Suk Lee,Sunjoo Kim,Ann-Yae Na,Chang Hwan Sohn,Sangkyu Lee

doi:10.3390/toxins13030197

Abstract

Alpha-amanitin (α-AMA) is a cyclic peptide and 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. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending 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 changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Highlights

Intake of mushrooms continues to increase owing to their significant antioxidant properties that improve health; they are an abundant source of low-calorie food diet and proteins [1]
Increased Toxicity in Hepatoma Cells by α-Amanitin Treatment α-AMA was treated by concentration to evaluate cytotoxicity and determine the treatment concentration of α-AMA in hepatoma cells for quantitative proteomic studies
Tandem mass spectrometry analysis to demonstrate that the hepatic proteome changes after treatment with several concentrations of α-AMA

Summary

Introduction

Intake of mushrooms continues to increase owing to their significant antioxidant properties that improve health; they are an abundant source of low-calorie food diet and proteins [1]. As the habitat of poisonous mushrooms changes owing to climate change, there is a possibility of mixing poisonous and edible mushrooms owing to their similar appearance. Poisonous mushrooms may be misused when harvesting in nature. Certain poisonous mushrooms have deadly toxins, and their consumption leads to frequent poisoning accidents worldwide [1,2]. Amatoxins, as cyclopeptide hepatotoxin, are found in Amanita verna, Amanita phalloides, and Lepiota helveola. These toxins comprise alpha-amanitin (α-AMA), β-AMA, γ-AMA, amaninamide, amanin, etc. These toxins comprise alpha-amanitin (α-AMA), β-AMA, γ-AMA, amaninamide, amanin, etc. [3]

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