Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of pro...

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Bibliographic Details
Published in:Biochemical pharmacology 2004-05, Vol.67 (9), p.1721-1731
Main Authors: Bellisola, Giuseppe, Fracasso, Giulio, Ippoliti, Rodolfo, Menestrina, Gianfranco, Rosén, Anders, Soldà, Silvia, Udali, Silvia, Tomazzolli, Rossella, Tridente, Giuseppe, Colombatti, Marco
Format: Article
Language:English
Subjects:
Auranofin - pharmacology
Biological and medical sciences
Disulfide reduction
Humans
Immunotoxin
Immunotoxins - metabolism
Medical sciences
MEDICIN
MEDICINE
NADP
Oxidation-Reduction - drug effects
Pharmacology. Drug treatments
Protein disulfide isomerase
Protein Disulfide-Isomerases - metabolism
Ricin
Ricin - metabolism
Thioredoxin
Thioredoxin reductase
Thioredoxin-Disulfide Reductase - metabolism
U937 Cells
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Summary:Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS–PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls ( P
ISSN:0006-2952
1356-1839
1873-2968
DOI:10.1016/j.bcp.2004.01.013