Mechanisms of Bcl-2 in Programmed Cell Death

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Mechanisms of Bcl-2 in Programmed Cell Death

• Laura Beth Hill
• St. Edwards University

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Apoptosis

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

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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

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What is Bcl-2?

• Family of proteins that includes promoters and
  inhibitors
• Proto-oncogene
• Localized to outer mitochondrial membrane

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Characteristics

• Bcl-2 family members can homo- and heterodimerize
• Participate in selective pore formation
• Expressed at different rates during development

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FOCUS OF SEMINARPossible 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)

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Cytochrome c Model

• Necessary for the initiation of apoptosis
• Found in the mitochondrial intermembrane space
• Localization suggests connection between Bcl-2
  and cytochrome c

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HL-60 cells
neo cells
bcl-2 cells
Isolation
Isolation
Staurosporine
Staurosporine
Immunoblot analysis
Immunoblot analysis
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Yangs 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

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Yangs 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

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Channel Formation Model

• Bcl-2 can regulate Ca2 fluxes and protein
  transport
• 3D structure of Bcl-xL is similar to the
  pore-forming domains of DT and the bacterial
  colicins

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Cells expressing Bcl-2
Wild-type
Mutant
Purification
Purification
Detection of single channels
Detection of single channels
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Schendels 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

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Schendels 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?

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Practical Importance

• Adjustment of apoptotic threshold
• Gene therapy to control neuronal death
• Protection of developing nervous system against
  neurotoxins (e.g. EtOH)

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Acknowledgements

• St. Edwards School of Natural Sciences faculty
  and staff