Interaction of diphtheria toxin (fragment A) with actin

Bektaş, Muhammet; Varol, Başak; Nurten, Rüstem; Bermek, Engin

doi:10.1002/cbf.1590

Cited by 15 publications

(17 citation statements)

References 36 publications

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“…DT-FA can interact with both G-and F-actin. The binding to the latter appears to take place at the plus end of the filament, blocking further polymerisation [13]. Moreover, the role of F-actin and eEF2 in the endocytic trafficking of DT was determined in our findings [14].…”

Section: Introductionmentioning

confidence: 80%

“…The binding to the latter appears to take place at the plus end of the filament, blocking its further polymerisation [13]. In the endocytic process, the cargo role of actin has been identified.…”

Section: Discussionmentioning

confidence: 99%

“…The column was in turn equilibrated and run with F-actin buffer [13], and it was calibrated using ribonuclease (M r 13.7 kDa), G-actin (M r 42.5 kDa), bovine serum albumin (BSA) (M r 67 kDa), β-galactosidase (M r 116 kDa), and ferritin (M r 450 kDa). The reaction volume was 1 ml.…”

Section: Gel Filtration Analysismentioning

confidence: 99%

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The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Varol

Bektaş

Nurten

et al. 2012

Cellular and Molecular Biology Letters

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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 thepresence 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.

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Section: Introductionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Varol

Bektaş

Nurten

et al. 2012

Cellular and Molecular Biology Letters

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“…Here we achieved cochlear SC-specific ablation using Cre-mediated DTA expression, which produces cell-autonomous death via apoptosis due to disruption of protein synthesis and actin polymerization (Kochi and Collier, 1993; Collier, 2001; Bektas et al, 2009). This approach has been effectively used in retina, heart, dorsal root ganglia and utricle (Ivanova et al, 2005; Chen and Nathans, 2007; Abrahamsen et al, 2008; Burns et al, 2012) to dissect the roles of specific cell types.…”

Section: Discussionmentioning

confidence: 99%

Selective Ablation of Pillar and Deiters' Cells Severely Affects Cochlear Postnatal Development and Hearing in Mice

et al. 2013

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Mammalian auditory hair cells (HCs) are inserted into a well-structured environment of supporting cells (SCs) and acellular matrices. It has been proposed that when HCs are irreversibly damaged by noise or ototoxic drugs surrounding SCs seal the epithelial surface and likely extend the survival of auditory neurons. Because SCs are more resistant to damage than HCs the effects of primary SC loss on HC survival and hearing have received little attention. We used the Cre/loxP system in mice to specifically ablate pillar and Deiters’ cells (PCs and DCs). In Prox1CreERT2+/−;Rosa26DTA/+ (Prox1DTA) mice, CreER expression driven by the endogenous Prox1 promoter in presence of tamoxifen removes a stop-codon in the Rosa26DTA/+ allele and induces diphtheria toxin fragment A (DTA) expression. DTA produces cell-autonomous apoptosis. Prox1DTA mice injected with tamoxifen at postnatal days (P)0 and P1, show significant DC and outer PC loss at P2-P4, that reaches ~70% by one month. Outer HC loss follows at P14 and it is almost complete at one month, while inner HCs remain intact. Neural innervation to the outer HCs is disrupted in Prox1DTA mice and auditory brainstem response thresholds in adults are 40-50 dB higher than in controls. The hearing deficit correlates with loss of cochlear amplification. Remarkably, in Prox1DTA mice the auditory epithelium preserves the ability to seal the reticular lamina and spiral ganglion neuron counts are normal, a key requirement for cochlear implant success. Our results highlight that cochlear SC pools should be appropriately replenished during HC regeneration strategies.

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“…Previously, FA-actin interaction was displayed as in vitro and in vivo [20]. That FA broke the chain of F-actin was showed by using the techniques of chromatographic, electrophoretic and immunoflourescence.…”

Section: Discussionmentioning

confidence: 99%

The interaction between actin and FA fragment of diphtheria toxin

et al. 2012

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Actin protein has many other cellular functions such as movement, chemotaxis, secretion and cytodiaresis. Besides, it have structural function. Actin is a motor protein that it has an important role in the movement process of toxin in the cell. It is known that F-actin gives carriage support during the endosomal process. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerisation of monomeric G-actin molecules through sequential rowing, is called F-actin (FA). Actin interacts with a great number of cellular proteins along with cell skeleton and plasma membrane. It is also known that some bacterial toxins have ADP-ribosylation affect on actin. Diphteria toxin is the part which has the FA enzymatic activity corresponding the N-terminal section of the toxin, which inhibits the protein synthesis by ADP-ribosylating the elongation factor 2 in the presence of NAD. FA, taken into the cell by endocytosis inhibits protein synthesis by ADP-ribosyltransferase activity and breaks the cytoskeleton. In the studies both in vitro and in vivo, actin with interaction FA of diphteria toxin has been yet to be fully elucidated. The aim of this study was to determine the three dimensional structures of actin with interaction FA of diphteria toxin by the amprical methods and in paralel with the computing technology, theoretical methods have gained significant importance. In our study, actin with interaction FA of diphteria toxin has been determined as the most possible interaction area with the theoretical method; analogy modelling. This area has been closed in the presence of polypeptides and FA-actin interactions have been tested with the gel filtration chromatography techniques. As a result of the findings, we found that 15 amino acid artificial peptides (DAMYETMAQACAGNR) corresponding to 201–215 amino acid residues of FA interacts with G-actin and closes this area. Secondly, in the model formed with the analogy modelling, it appears that the most possible interaction area is between FA (tyr204) and G-actin (gly48). Results obtained from both theoretical and experimental data support the idea that the interaction occurs in this area.

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Interaction of diphtheria toxin (fragment A) with actin

Cited by 15 publications

References 36 publications

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Selective Ablation of Pillar and Deiters' Cells Severely Affects Cochlear Postnatal Development and Hearing in Mice

The interaction between actin and FA fragment of diphtheria toxin

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