Differences in Medium-Induced Conformational Plasticity Presumably Underlie Different Cytotoxic Activity of Ricin and Viscumin.

Pavel Volynsky,Alexander Tonevitsky,Eduard V Bocharov,Galina Zakharova,Valentin Tabakmakher,Roman Efremov,Maria Raygorodskaya,Diana Maltseva,Elena Britikova

doi:10.3390/biom12020295

Abstract

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

Highlights

AB-type protein toxins containing a catalytically active A subunit and a cell-bindingB subunit are often used to investigate various stages of intracellular transport [1,2]
The following reagents were used for cell culturing: McCoy’s cell culture medium (Gibco, Paisley, Scotland); fetal bovine serum (FBS; Gibco, Brazil); 100× PenStrep antibiotic mixture (Gibco, Grand Island, NE, USA); 1× Dulbecco’s phosphate buffer (DPBS; Gibco, Paisley, Scotland); 0.25% trypsin–EDTA solution with Hanks’ salts (PanEko, Moscow, Russia); 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS; Promega, Madison, WI, USA), and an electron coupling reagent phenazine methosulfate (PMS; Promega, Madison, WI, USA)
In order to assess the cytotoxic effect of ricin and viscumin, the colorectal adenocarcinoma HT29 cell line was treated by one of the ribosomeinactivating proteins (RIP) for only one hour and the cells were cultured under standard conditions in RIP-free medium for five days

Summary

Introduction

AB-type protein toxins containing a catalytically active A subunit and a cell-bindingB subunit are often used to investigate various stages of intracellular transport [1,2]. AB-type protein toxins containing a catalytically active A subunit and a cell-binding. Ricin is the most extensively studied AB-toxin, and its spatial structure is well described [3,4,5]. It represents a heterodimer consisting of two subunits linked by a disulfide bond. The catalytic A subunit of ricin (RTA) irreversibly inhibits ribosomes causing cell death [2]. The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes.

Methods

Results

Discussion

Conclusion