Analysis of endogenous Bax complexes during apoptosis using blue native PAGE: implications for Bax activation and oligomerization
| Autor: | Andrew P. Gilmore, Anthony J. Valentijn, John-Paul Upton |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Protein Conformation
bcl-X Protein Apoptosis Mitochondrion Biochemistry Article Cell Line Epitopes Mice 03 medical and health sciences 0302 clinical medicine Bcl-2-associated X protein Cell Adhesion Animals Humans Anoikis Molecular Biology Caspase bcl-2-Associated X Protein 030304 developmental biology 0303 health sciences biology Cytochrome c Epithelial Cells Cell Biology Mitochondria Cell biology Enzyme Activation Molecular Weight Cytosol Cross-Linking Reagents bcl-2 Homologous Antagonist-Killer Protein Caspases Multiprotein Complexes 030220 oncology & carcinogenesis biology.protein Electrophoresis Polyacrylamide Gel Bcl-2 Homologous Antagonist-Killer Protein BH3 Interacting Domain Death Agonist Protein |
| Zdroj: | Biochemical Journal. 412:347-357 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20071548 |
| Popis: | Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore. |
| Databáze: | OpenAIRE |
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