Identification of α-amanitin effector proteins in hepatocytes by limited proteolysis-coupled mass spectrometry

초록

The misuse of poisonous mushrooms containing amatoxins causes acute liver failure (ALF) in patients and is a cause of significant mortality. Although the toxic mechanisms of alpha-amanitin (alpha-AMA) and its interactions with RNA polymerase II (RNAP II) have been studied, alpha-AMA effector proteins that can interact with alpha-AMA in hepatocytes have not been systematically studied. Limited proteolysis-coupled mass spectrometry (LiP-MS) is an advanced technology that can quickly identify protein-ligand interactions based on global comparative proteomics. This study identified the alpha-AMA effector proteins found in human hepatocytes, following the detection of conformotypic peptides using LiP-MS coupled with tandem mass tag (TMT) technology. Proteins that are classified into protein processing in the endoplasmic reticulum and the ribosome during the KEGG pathway can be identified through affinity evaluation, according to alpha-AMA concentration-dependent LiP-MS and LiP-MS in hepatocytes derived from humans and mice, respectively. The possibility of interaction between alpha-AMA and proteins containing conformotypic peptides was evaluated through molecular docking studies. The results of this study suggest a novel path for alpha-AMA to induce hepatotoxicity through interactions with various proteins involved in protein synthesis, as well as with RNAP II.

키워드