Abstract
Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.
Highlights
The C2 toxin from Clostridium botulinum is the prototype of binary actin-ADP-ribosylating toxins [1] and consists of the enzyme component C2I, which mono-ADP-ribosylates G-actin and the separate binding/translocation component C2II
The present study relies upon a genetically engineered fusion protein based on non-toxic C2 toxin which delivers the p53 tumor suppressor protein into the cytosol of various cancer cell lines
P53 was cloned in frame to the adaptor domain C2IN that mediates the interaction with the C2IIa binding/translocation unit, leading to its subsequent cellular uptake
Summary
The C2 toxin from Clostridium botulinum is the prototype of binary actin-ADP-ribosylating toxins [1] and consists of the enzyme component C2I, which mono-ADP-ribosylates G-actin and the separate binding/translocation component C2II. In response to the acidification occurring in early endosomes, C2IIa is subjected to a conformational switch, triggering its insertion into the endosomal membrane where it forms a trans-membrane pore [5]. This allows for the translocation of C2I into the cytosol, which is promoted by host cell chaperones such as Hsp and peptidyl prolyl cis/trans isomerases [6,7]. C2I catalyzes the covalent transfer of ADP-ribose onto G-actin using NAD+ as cosubstrate [8] This covalent modification results in a collapse of the actin cytoskeleton and triggers caspase-dependent cell death [9]
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