A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO.
Smac/DIABLO is a mitochondrial protein that is proteolytically processed and released during apoptosis along with cytochrome c and other proapoptotic factors. Once in the cytosol, Smac protein binds to inhibitors of apoptosis (IAP) proteins and disrupts the ability of the IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial processing and release has complicated efforts to delineate the effect of Smac overexpression and IAP inhibition on cell death processes. In this report, we document a novel expression system using ubiquitin fusions to express mature, biologically active Smac in the cytosol of transfected cells. Processing of the ubiquitin-Smac fusions is rapid and complete and generates mature Smac protein initiating correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is required for proper function. The biological activity of this exogenous protein was demonstrated by its interaction with X-linked IAP, one of the most potent of the IAPs. The presence of mature Smac was not sufficient to trigger apoptosis of healthy cells. However, cells with excess Smac protein were greatly sensitized to apoptotic triggers such as etoposide exposure. Cancer cells typically display deregulated apoptotic pathways, including Bcl2 overexpression, thereby suppressing the release of cytochrome c and Smac. The ability to circumvent the requirement for mitochondrial processing and release is critical to developing Smac as a possible gene therapy payload in cancer chemosensitization.
Hunter
Allison M
AM
Solange Gauthier Karsh Molecular Genetics Laboratory, Children's Hospital of Eastern Ontario, Research Institute, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada.
Kottachchi
Dan
D
Lewis
Jennifer
J
Duckett
Colin S
CS
Korneluk
Robert G
RG
Liston
Peter
P
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D061945:Caspase Inhibitors; D016923:Cell Death; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005047:Etoposide / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013347:Subcellular Fractions; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; C412403:DIABLO protein, human; C499605:Diablo protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D005047:Etoposide; D004247:DNA; D005982:Glutathione Transferase; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9