1. Mechanisms of Bcl-2 in Programmed Cell Death Laura Beth Hill St. Edward’s University

2. Apoptosis  Essential for normal embryonic development  Natural and pathological  Morphologic characteristics  Regulated by proteins in Bcl-2 family

5. The Apoptotic Process  Cell receives death signal  Mitochondrial membrane potential decreases  Transport of cytochrome c through membrane into cytosol  Cytochrome c binds to Apaf-1  Caspase activity initiated  Cell degradation

6. What is Bcl-2?  Family of proteins that includes promoters and inhibitors  Proto-oncogene  Localized to outer mitochondrial membrane  Can form homo- and heterodimers  Exhibits biphasic expression

7. FOCUS OF SEMINAR Possible Mechanisms  Bcl-2 blocks release of cytochrome c from mitochondrial membrane (Yang, et al., 1997)  Bcl-2 forms channels in lipid membranes (Schendel, et al., 1997)

8. Cytochrome c Model  Necessary for the initiation of apoptosis  Found in the mitochondrial intermembrane space  Localization suggests connection between Bcl-2 and cytochrome c

9. HL-60 cells neo cellsbcl-2 cells Isolation Staurosporine Immunoblot analysis

10. Yang’s Results  Cytochrome c in neo cells showed cytosol increase, with corresponding decrease in mitochondria  No significant change of cytochrome c in mitochondria or cytosol of Bcl-2 cells  No significant amount of cytochrome c found in cytosol of control cells

11. Yang’s Conclusion  Bcl-2 prevents the release of cytochrome c  Mechanism by which Bcl-2 blocks release unknown  Structural similarity to bacterial toxins suggests pore-forming ability

12. Channel Formation Model  Bcl-2 can regulate ion fluxes and protein translocation  3D structure of Bcl-x L is similar to the pore-forming domains of DT and the bacterial colicins

13. Protein Production Bcl-2 (  5,6) mutant Bcl-2 wild-type Cl - efflux assay Detection of single channels Detection of single channels

14. Schendel’s Results  Bcl-2 formed ion-conducting pores in a manner similar to that of bacterial toxins  Bcl-2 mutant produced only non-specific Cl - efflux  Bcl-2 in planar lipid bilayers formed discrete cation-selective channels  Bcl-2 mutant did not form channels here

16. Schendel’s Conclusions  Biophysical evidence proves that Bcl-2 forms channels in membranes  Channels reside in closed state  What regulates channels?  How do pro-apoptotic proteins oppose anti-apoptotic proteins?

18. Practical Importance  Association of disease with inappropriate apoptosis  Adjustment of apoptotic threshold  Gene therapy to control cell death  Protection of developing nervous system against neurotoxins (e.g. EtOH)

19. Acknowledgements St. Edward’s School of Natural Sciences faculty and staff