Poster abstracts
Poster number 46 submitted by Vedud Purde
Reconstitution of diphtheria toxin catalytic domain in cancer cells
Vedud Purde (The Ohio State Biochemistry Program), Elena Kudryashova (Department of Chemistry and Biochemistry), David Heisler (Department of Chemistry and Biochemistry)
Abstract:
Diphtheria toxin (DT) is one of the deadliest compounds on earth. Toxicity of (DT) produced by Corynebacterium diphtheriae is defined by the catalytic domain (DTA), which transfers an ADP-ribosyl group from NAD+ to an unconventional diphthamide residue uniquely found in ribosomal elongation factor 2 (eEF2). ADP-ribosylation of diphthamide inactivates eEF2 and blocks ribosomal protein synthesis in the affected cell leading to cell death. DTA has been widely used in targeted therapeutics to construct immunotoxins - artificial proteins that contain a toxin conjugated to a targeting element with a purpose of delivering toxicity to cancer cells. A major limitation of this approach is a lack of true cancer-specific cell surface markers, leading to a non-specific side toxicity of immunotoxins towards non-transformed healthy cells.
We sought to employ split-inteins to develop a novel tool with increased selectivity towards cancer cells. Split-inteins are enzymes that are produced as part of different proteins, but can recognize and excise each other from a precursor protein resulting in the ligation of their flanking sequences. We designed, produced, and characterized different pairs of catalytically inactive fragments of DTA fused to split-intein domains (Split-DTA).
Our in vitro trans- splicing experiments showed that each of the five produced pairs is capable of reconstituting the full length DTA toxin. To deliver split toxins to target cells, they were fused to the N-terminal domain of the anthrax lethal factor (LFN). Products of in vitro trans-splicing reactions of 5 different split-DTA pairs resulted in protein synthesis inhibition in the treated mammalian cells confirming reconstitution of the catalytically active full length DTA toxin. To determine if trans- splicing and formation of active toxins occur in the cytosol, we sequentially delivered individual split constructs to HeLa cells, but inhibition in protein synthesis was not observed. However, when U2OS osteosarcoma cells were stably transfected with a gene encoding one part of a split pair while a corresponding counterpart was delivered as a protein, significant toxicity was observed. Particularly, a decrease in protein synthesis and the subsequent cell death were comparable to those caused by intact DTA
Keywords: Diphtheria toxin, cancer, split-inten