title: The cytotoxic effect of diphtheria toxin on the actin cytoskeleton.
abstract: Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as K(m) = 2.2 nM; V(max) = 0.25 pmol.min(-1). The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In the presence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60-65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.
authors.affiliation: İstanbul University, İstanbul Faculty of Medicine, Department of Biophysics, Çapa-İstanbul, Türkiye.
authors.forename: Başak
authors.initials: B
authors.lastname: Varol
authors.forename: Muhammet
authors.initials: M
authors.lastname: Bektaş
authors.forename: Rüstem
authors.initials: R
authors.lastname: Nurten
authors.forename: Engin
authors.initials: E
authors.lastname: Bermek
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000187:drug effects*; D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D004167:Diphtheria Toxin / Q000633:toxicity*; D054736:Elongation Factor 2 Kinase / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000633:toxicity*
chemical_list: D000199:Actins; D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; D036002:ADP Ribose Transferases; C517689:EEF2K protein, human; D054736:Elongation Factor 2 Kinase