Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology


English (en)

Date of Award

January 2009

Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Emily Cheng


Mammals have evolved an intricate regulation of a genetically programmed apoptotic cell death that involves mitochondria. Diverse apoptotic signals converge on mitochondria, which causes the release of cytochrome c into the cytosol to activate Apaf-1. This initiates caspase activation, which results in irreversible cellular demise. The BCL-2 family proteins constitute a critical checkpoint in mitochondrion-dependent apoptosis. Multidomain proapoptotic BAX/BAK promotes mitochondrial outer membrane permeabilization, whereas anti-apoptotic BCL-2/BCL-XL/MCL-1 protects mitochondrial integrity and prevents cytochrome c release. The proapoptotic activity of BAX/BAK is triggered by BH3-only molecules: BH3s) which are upregulated by upstream death signals. However, how these subfamilies interact with one another to execute mitochondrial cell death remains unclear. Thus, this thesis aims at elucidating the mechanism regarding how the interplay between BCL-2 subfamilies determines cellular commitment to survival versus death and how BAX/BAK activation is triggered by BH3s. Our laboratory showed that BH3s can be further classified into two subclasses- `activator' tBID/BIM/PUMA that directly activates BAX/BAK to induce cytochrome c release and `inactivator' BH3s that antagonize the function of anti-apoptotic BCL-2 members. Here, a BAX/BAK mutagenesis study indicated that anti-apoptotic BCL-2 members prevent BAX/BAK activation by sequestering activator BH3s rather than by directly binding to BAX/BAK. I further demonstrated that inactivator BH3s are able to displace activator BH3s from anti-apoptotic BCL-2 members with unique specificity, thus preventing their sequestration of activator BH3s. Activator BH3s were shown to act downstream of inactivator BH3s to trigger BAX/BAK activation, establishing the hierarchy of BCL-2 subfamilies in regulating mitochondrial apoptosis. Then, I investigated the molecular mechanism whereby BAX/BAK is activated by activator BH3s. I demonstrated that BAX undergoes stepwise structural reorganization leading to two activation processes-mitochondrial targeting and homo-oligomerization. Activator BH3s initiate BAX activation by attacking and exposing α1 helix of BAX leading to the secondary disengagement of the α9 helix and mitochondrial translocation. Activator BH3s remain associated with BAX to drive homo-oligomerization at the mitochondria. BAK has bypassed the first activation step, but requires activator BH3s for homo-oligomerization. This study further emphasizes the direct engagement of activator BH3s in BAX/BAK-dependent mitochondrial apoptotic pathway. Lastly, our laboratory showed that BH3s trigger caspase-independent mitochondrial dysfunction only in the presence of BAX/BAK. I found that BAK exists as several distinct complexes at the mitochondria, one of which is functionally different from cytochrome c-releasing BAK oligomers but instead includes VDAC/ANT channels that regulate ATP/ADP transport to support ATP production by oxidative phosphorylation. tBID overexpression induces cell death in the absence of Apaf-1 by inhibiting VDAC-mediated ADP import into the mitochondria in a BAK-dependent manner, suggesting that activated BAK antagonizes VDAC activity to initiate mitochondrial dysfunction. This study provides novel insights into how BAK activation couples apoptosis and mitochondrial dysfunction to trigger cell death.


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