Cholix toxin, an eukaryotic elongation factor 2 ADP‐ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

Abstract

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis.Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHBknockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholixinduced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.

Cell Death Signaling Pathway Induced by Cholix Toxin, a Cytotoxin and eEF2 ADP-Ribosyltransferase Produced by Vibrio cholerae

“…The suppression of Cholix-induced cell death by LRP1 knockdown was only partial. We also reported that Cholix-induced apoptosis was not suppressed at all by LRP1 knockdown in human hepatocytes [46]. Further, Cholix induced significant cell death in human colon cell line HCT116 cells, whose LRP1 gene was mutated by frameshifts [47,48].…”

“…Cholix-induced eEF2-ADP-ribosylation inhibits ribosomal protein synthesis, resulting in the death of host cells. In addition, by immunoblotting using an anti-Cholix antibody, we found that Cholix was detected in the mitochondrial fraction rather than in the cytosolic compartment, suggesting that Cholix can be translocated into mitochondria; however, there is no information on the translocation mechanism [46].…”

Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae

“…Though lipoprotein receptor-related protein has been shown to bind with Chx, the specific receptor for cholix bining is not known, as the sensitivity of human cell lines to cholix is variable. Recently, prohibitin (PHB) was identified acts as a Chx-binding protein (Yahiro et al, 2019 ), which is expressed by some cell membranes, and also by mitochondria and nucleus. V. cholerae ChxA has some homology with exotoxin A (ToxA) of Pseudomonas aeruginosa , but there is no evidence for lateral transfer (Purdy et al, 2010 ).…”

NAD metabolism in aging and cancer

“…79,81 mARTs serve as a common mechanism for several prokaryotic toxins. 82 ADP-ribosylation of an arginine residue on a membrane-bound GTP-binding protein by cholera toxin or E. coli heat-labile enterotoxin increases the activity of adenylyl cyclase resulting in intestinal fluid and electrolyte flux leading to severe fluid loss and dehydration. 83,84 Both diphtheria toxin and pseudomonas toxin ADP-ribosylate a modified histidine residue in eukaryotic elongation factor 2, which leads to cell death through the inhibition of protein synthesis.…”