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

Abstract

Alpha-amanitin (alpha-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. alpha-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of alpha-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 alpha-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 alpha-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of alpha-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in alpha-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.