Expression of an anti apoptotic recombinant short peptide in mammalian cells.
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.
Matza-Porges
S
S
Department of Virology, The Hebrew University-Hadassah Medical School, The Hebrew University of Jerusalem, P.O. Box 12272, Jerusalem 91120, Israel.
Horresh
I
I
Tavor
E
E
Panet
A
A
Honigman
A
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D053222:Fas Ligand Protein; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D048069:Tumor Necrosis Factors / Q000494:pharmacology; D014764:Viral Proteins / Q000096:biosynthesis / Q000494:pharmacology
D061945:Caspase Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D048069:Tumor Necrosis Factors; D014764:Viral Proteins; C492234:p35 protein, Baculovirus; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases