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Abstract:
ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.
Insights
Botulinum C2 toxin, a bacterial toxin, ADP-ribosylates actin, a key cellular protein. This modification significantly impacts actin
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Bacterial toxins often utilize ADP-ribosylation to disrupt eukaryotic cell functions.
- Diphtheria toxin, cholera toxin, and pertussis toxin are known examples affecting protein synthesis and adenylate cyclase regulation.
- Botulinum C2 toxin, distinct from neurotoxins, exhibits ADP-ribosyltransferase activity and causes various toxic effects.
Purpose of the Study:
- To identify the specific protein substrate targeted by Botulinum C2 toxin.
- To investigate the mechanism and consequences of Botulinum C2 toxin's ADP-ribosylation activity.
Main Methods:
- Incubation of intact cells and cell-free preparations with Botulinum C2 toxin.
- Analysis of protein modification using molecular mass determination.
- ADP-ribosylation assays using purified actin (G-actin and F-actin) from different sources (liver, skeletal muscle).
- Viscosity measurements of polymerized actin following toxin treatment.
Main Results:
- Botulinum C2 toxin was found to ADP-ribosylate a 43,000 molecular mass protein in cells and cell-free systems.
- Evidence suggests this 43K protein substrate is actin, undergoing mono-ADP-ribosylation.
- Purified liver G-actin was efficiently ADP-ribosylated, while liver F-actin and all forms of skeletal muscle actin showed minimal to no modification.
- ADP-ribosylation of liver G-actin led to a significant decrease in the viscosity of polymerized actin.
Conclusions:
- Botulinum C2 toxin specifically targets and ADP-ribosylates actin, primarily in its globular (G) form.
- This modification of actin by Botulinum C2 toxin alters its polymerization properties, potentially explaining some of the toxin's pathological effects.