Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology


English (en)

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul Schlesinger


The BCL2 protein family is the primary gatekeeper of mitochondrial apoptosis and governs integrity of the organelles' outer membranes. Permeabilization of mitochondrial outer membranes permits egress of cytochrome c and other apoptogenic factors, resulting in apoptosome formation, caspase activation, and subsequent proteolytic demolition of cells. Proapoptotic BAX & BAK effect the release of cytochrome c while their antiapoptotic counterparts like BCL-2, BCL-XL, & MCL-1 oppose this permeabilization. A third class of the BCL2 family, the prodeath BH3-only proteins, act as sentinels of cell stress and exert their influences by occupying antiapoptotic BCL2 members and/or activating BAX/BAK. Cell-free reconstitution assays have revealed that BAX/BAK undergo significant conformational changes to oligomerize and form pores in membranes. Previously unresolved was the basis for the outer cell membranes' escape from BAX poration during apoptosis. Unlike outer cell membranes, which are roughly 40% cholesterol, mitochondrial outer membranes are only 5-10% cholesterol. Vesicle leakage assays demonstrated that BAX pore activation is severely inhibited by the sterol. Inclusion of the total enantiomer of cholesterol in our assays uncovered that this BAX functional suppression was due to bilayer structure alteration rather than a stereospecific protein-cholesterol interaction. Real-time observation of BAX-vesicle binding showed that cholesterol curbs membrane integration by the protein, thus suppressing oligomerization and pore formation. Oxysterols and bile acids are physiological derivatives of cholesterol. Further employment of our vesicle leakage regime revealed that 25-hydroxycholesterol at low micromolar concentrations accelerates BAX pore formation, suggesting a compensatory mechanism for BAX inhibition by cholesterol. Bile acids lithocholic and chenodeoxycholic acids are toxic and induce apoptosis at high concentrations, thus we reasoned that the physiological detergents may directly activate BAX. While truncated BAX: ΔC) was effectively activated by monomeric detergent, the full-length protein required micellar bile acids, implying that bile acids could play at most only an amplification role in BAX-mediated apoptosis. In nonstressed cells, BAX exists as a soluble, cytosolic monomer or loosely affiliated with mitochondria while cellular recognition of death signals induces BAX transition to a membrane-integral state. The physical basis of this translocation and locale of BAX activation are poorly characterized. Assemblage of a cell-free scheme comprising full-length BAX, antiapoptotic BCL-XL, and BH3-only activators cBID & BIMS, and synthetic vesicles revealed that BIMS more effectively activates BAX and is less suppressible by BCL-XL. Each of these four proteins can independently adsorb to membranes and that BCL-XL "outraces" BAX to their in-membrane functional sites. Membrane-bound cBID and BIMS robustly accelerate the bilayer integrations of BAX & BCL-XL; the two activators recruit equivalently at the membrane surface, however, suggesting that BIMS, unlike cBID, can activate BAX prior to interaction on a bilayer scaffold.


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