Mechanisms of Bcl-2 in Programmed Cell Death Laura Beth Hill St. Edward’s University
Apoptosis Essential for normal embryonic development Natural and pathological Morphologic characteristics Regulated by proteins in Bcl-2 family
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
What is Bcl-2? Family of proteins that includes promoters and inhibitors Proto-oncogene Localized to outer mitochondrial membrane
Characteristics Bcl-2 family members can homo- and heterodimerize Participate in selective pore formation Expressed at different rates during development
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)
Cytochrome c Model Necessary for the initiation of apoptosis Found in the mitochondrial intermembrane space Localization suggests connection between Bcl-2 and cytochrome c
HL-60 cells neo cellsbcl-2 cells Isolation Staurosporine Immunoblot analysis
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
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
Channel Formation Model Bcl-2 can regulate Ca 2+ fluxes and protein transport 3D structure of Bcl-x L is similar to the pore-forming domains of DT and the bacterial colicins
Cells expressing Bcl-2 Mutant Wild-type Purification Detection of single channels Detection of single channels
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
Schendel’s Conclusions Biophysical evidence proves that Bcl-2 forms channels in membranes Channels reside in closed state What controls opening and closing? What does Bcl-2 transport? How do pro-apoptotic proteins oppose anti-apoptotic proteins?
Practical Importance Adjustment of apoptotic threshold Gene therapy to control neuronal death Protection of developing nervous system against neurotoxins (e.g. EtOH)
Acknowledgements St. Edward’s School of Natural Sciences faculty and staff