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A novel ubiquitin fusion system bypasses the mitochondria and generates biologically active Smac/DIABLO.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2003 Feb 28; Vol. 278 (9), pp. 7494-9. Date of Electronic Publication: 2003 Jan 02. - Publication Year :
- 2003
-
Abstract
- 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.
- Subjects :
- Animals
Apoptosis
Apoptosis Regulatory Proteins
Blotting, Western
Caspase 3
Caspase 7
Caspase 9
Caspase Inhibitors
Cell Death
Cell Line
Cytochrome c Group metabolism
Cytoplasm metabolism
DNA metabolism
Electrophoresis, Polyacrylamide Gel
Etoposide pharmacology
Glutathione Transferase metabolism
HeLa Cells
Humans
Intracellular Signaling Peptides and Proteins
Mice
Microscopy, Fluorescence
Mitochondria metabolism
Peptides chemistry
Plasmids metabolism
Precipitin Tests
Protein Binding
Protein Structure, Tertiary
Subcellular Fractions
Transfection
Carrier Proteins metabolism
Mitochondrial Proteins metabolism
Recombinant Fusion Proteins metabolism
Ubiquitin metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 278
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12511567
- Full Text :
- https://doi.org/10.1074/jbc.C200695200