title: Auranofin Inhibits the Enzyme Activity of Pasteurella multocida Toxin PMT in Human Cells and Protects Cells from Intoxication.
abstract: The AB-type protein toxin fromPasteurella multocida(PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, Ulm 89081, Germany. stefan.carle@uni-ulm.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Carle
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany. thorsten.brink@pharmakol.uni-freiburg.de.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Brink
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany. joachim.orth@pharmakol.uni-freiburg.de.
authors.forename: Joachim H C
authors.initials: JH
authors.lastname: Orth
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany. klaus.aktories@pharmakol.uni-freiburg.de.|Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg 79104, Germany. klaus.aktories@pharmakol.uni-freiburg.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, Ulm 89081, Germany. holger.barth@uni-ulm.de.
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
mesh_terms: D001310:Auranofin / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics; D020134:Catalytic Domain; D018929:Cell Culture Techniques; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D016979:Pasteurella multocida / Q000201:enzymology* / Q000472:pathogenicity; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C062258:Pasteurella multocida toxin; D001310:Auranofin; D013880:Thioredoxin-Disulfide Reductase; D020962:Heterotrimeric GTP-Binding Proteins
keywords: G-protein; Pasteurella multocida toxin (PMT); auranofin; deamidation; thioredoxin reductase (TrxR)