No abstract

CRM197, cross-reacting material 197, is a mutant of diphtheria toxin (DTx). CRM197 is used in pharmacology as a carrier protein. It has been recently shown that CRM197 causes breakdown in actin filaments. In order to show intracellular localization of CRM197 and visualize cell structure via actin cytoskeleton, endothelial cells were cultured and subjected to CRM197 in vitro. To address the interaction between CRM197 and actin both experimental and theoretical studies were carried out. Colocalization of CRM197 with actin filaments was determined by immunofluorescence microscopy. Following 24-hour incubation, the loss of cell-cell contact between cells was prominent. CRM197 was shown to bind to G-actin by gel filtration chromatography, and this binding was confirmed by Western blot analysis of eluted samples obtained following chromatography. Based on crystal structure, docked model of CRM197-actin complex was generated. Molecular dynamics simulation revealed that Lys42, Cys218, Cys233 of CRM197 interacts with Gly197, Arg62 and Ser60 of G-actin, respectively. CRM197 binding to G-actin, colocalization of CRM197 with actin filament, and actin cytoskeleton rearrangement resulting in the loss of cell-cell contact show that actin comes into sight as target molecule for CRM197.

No abstract

Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70-75 % was observed in IL-1β treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1β; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1β has an important role in the regulation of global protein synthesis.

Ökaryotik hücrelerde mikrofilament yapısının ana bileşeni olan aktin, sinyal yolaklarını aktin bağlayan proteinlerle etkileşerek düzenler. Daha önceki çalışmalarımızda difteri toksini ve mutant difteri toksini (CRM-197) ile enfekte olan endotel hücrelerinde toksinin A fragmenti ile etkileşen filamentöz aktinin depolimerleştiği saptanmıştır. Bu çalışmada endotel hücrelerinde F-aktin stabilizasyonu sağlanarak difteri toksininin hücre içi trafiğinin belirlenmesi amaçlanmıştır. Gereç ve Yöntem: Hücre kültüründe endotel hücreleri çoğaltıldı ve jasplakinolid (0.1 µmol/ml) ile sırası ile 30 ve 60 dakika uygulandı. Diferi toksini (0.75 nmol/ml) uygulaması için jasplakinolid ile inkübasyon süresi 15 dakika ile sınırlandırıldı. Hücre içi F-aktin stabilizasyonu ve difteri toksini trafiği immünofloresan mikroskopisi ile görüntülendi. Bulgular: Bekletme süresine bağlı olarak stres liflerinin jasplakinolid uygulanan endotel hücrelerinin hücre zarında belirginleştiği belirlendi. F-aktin stabilizasyonu sağlanan endotel hücrelerinde difteri toksini trafiğinin engellenmediği ve A fragmenti'nin perinükleer alana yöneldiği görüntülendi. Sonuç: Hücre içinde F-aktin stabilizasyonu ile G-aktin/F-aktin dönüşümünün engellenmesi difteri toksini trafiğini durdurmamaktadır. Bu sonuç toksin trafiğinde filamentöz aktinin önemini gösteren çalışmaları desteklemektedir.