Expression of an anti apoptotic recombinant short peptide in mammalian cells

Abstract

Understanding the mechanisms of the apoptotic and anti apoptotic processes may lead to a better way to control these cascades. Here we demonstrated for the first time the feasibility to express a short functional peptide in mammalian cells that abrogates the apoptosis cascade through interference with the proteolytic activity of the initiator caspase 9 and the executing caspase 3 enzymes. The expression of a short peptide that includes the pseudo-substrate motif of the apoptosis inhibitor protein P35 (Asp-Gln-Met-Asp) leads to the abrogation of cell death induced through either the mitochondrial or the death receptors pathways. Short open reading frames have been detected in several mammalian mRNAs, primarily upstream of the main long reading frame (uORFs), however, direct evidence for de-novo peptides translation has not been provided. Utilizing biochemical and imaging techniques we demonstrate here that the functional recombinant peptide was localized to the cytpoplasmic fraction of the cell. In conclusion, this work demonstrates that ribosomes recognize short ORFs to translate stable short recombinant peptides in mammalian cells. Expression of these intracellular peptides results in the knock down of apoptotic processes to generate apoptosis resistant stable cells.

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides

“…In addition, uPEPs may not be synthesized at high enough levels to be functionally relevant, either because of low translation efficiencies or because their encoding uORFs are only present in rare transcript variants. However, translated uPEPs have been identified in human cells by mass spectrometry [ 41 ] and recently trans activity has been demonstrated for at least two small peptides in mammals, a 43 residue naturally occurring uPEP [ 42 ] and a recombinant 15 residue peptide [ 43 ]. Interestingly, in the former case, biological activity was found even though the peptide itself could not be detected.…”

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

“…It was proposed that such a peptide can be encoded by some uORFs, but evidence for its de novo synthesis has not been provided yet. (59) It is widely considered that biologically active peptides are frequently produced through the post-translational processing of larger precursors. However, it seems likely that many small protein and peptide-encoding genes are simply underpredicted in the eukaryotic genomes (e.g.…”

Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors

“…It should be noted that constitutive caspase-3 activity was consistently higher in the mock nontumorigenic MRC5 cells, as compared to the mock A549 tumor cells, as expected. 18 Two positive controls were included in the experiment: (i) ETO, a well-known cytotoxic drug, 32,33 caused, as expected, a stronger activation of caspase-3 in dividing nontumorigenic cells as compared to the tumor cells; (ii) Addition of caspase-3-specific inhibitor, Z-VAD-FMK, to the ETO-treated cells, blocked the enzymatic activity, indicating that indeed this assay specifically measures caspase-3 enzyme in the cell extracts (data not shown). Taken together, these results indicate that although both the virus and ETO-induced apoptosis, the relative specificity of these agents toward the two cells appears to be at variance.…”