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Mechanisms of Bcl-2 in Programmed Cell Death

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Title: Mechanisms of Bcl-2 in Programmed Cell Death


1
Mechanisms of Bcl-2 in Programmed Cell Death
  • Laura Beth Hill
  • St. Edwards University

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

3
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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 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)

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 cells
bcl-2 cells
Isolation
Isolation
Staurosporine
Staurosporine
Immunoblot analysis
Immunoblot analysis
10
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
  • No significant amount of cytochrome c found in
    cytosol of control cells

11
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

12
Channel Formation Model
  • Bcl-2 can regulate ion fluxes and protein
    translocation
  • 3D structure of Bcl-xL 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
Cl- efflux assay
Detection of single channels
Detection of single channels
14
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 regulates channels?
  • How do pro-apoptotic proteins oppose
    anti-apoptotic proteins?

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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. Edwards School of Natural Sciences faculty
    and staff

20
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