Mitochondrial outer membrane permeabilization (MOMP) is regulated by protein-protein and protein-membrane interactions between Bcl-2 family proteins. These interactions are governed by the concentrations and relative binding affinities of the proteins for each other. These affinities are altered by conformation changes of Bcl-2 family proteins resulting from interactions with each other and with membranes. How Bcl-2 proteins transition into and out of the conformations that controls their functions, and ultimately the fate of the cell, is not well understood. Here, kinetic analysis of the pore-forming Bcl-2 family member, Bax, revealed that Bax undergoes a conformational rearrangement through at least one structurally distinct intermediate that is a necessary precursor to pore formation. We discover that four cancer-associated Bax point mutants are trapped in the intermediate state, suggesting that transitions into and out of this intermediate can be modulated independently with consequences for the execution of apoptosis. Furthermore we report that the conformation changes Bax undergoes can be regulated by phosphorylation of Bax on residue S184 by the pro-survival kinase, Akt. Phosphorylation converts Bax into an anti-apoptotic protein that functions in a dominant-negative fashion. Bioinformatics revealed that in human cancers, higher levels of Bax are positively associated with high levels of PI3K/AKT pathway genes representing an added mechanism for cancer cells to evade apoptosis. Additionally we studied the interactions between Bax, the anti-apoptotic protein Bcl-XL, the sensitizer BH3 protein Bad and the BH3 activator protein Bid. We uncover a new mechanism of apoptosis regulation whereby Bad binds to one monomer of a Bcl-XL dimer eliciting an activating conformation change in a tBid bound to the other monomer of the Bcl-XL dimer. This allows Bad to function as a non-competitive inhibitor of Bcl-XL, and represents a novel mechanism that significantly enhances the potency of Bad to elicit apoptosis. / Thesis / Doctor of Philosophy (PhD) / Every day the human body creates billions of cells replacing damaged or unwanted cells. The death of these cells is tightly controlled and can result in disease when misregulated. Cancers arise when there is too little cell death and neurodegenerative diseases, such as Alzheimer’s, arise from too much cell death. Much research, including this thesis, is focused on understanding how cells die because once understood, cell death can be manipulated to treat disease. Cell death ironically occurs at the mitochondria, a cellular organ normally responsible for creating the energy required for the cell to live. When cell death is initiated, the mitochondria get holes poked into them, releasing pro-death factors that irreversibly commit the cell to dying. The work presented here uncovers new information about the regulation of the hole poking process, how it is blocked in breast cancer and how the process may be modulated to treat cancers.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21485 |
| Date | January 2017 |
| Creators | Kale, Justin |
| Contributors | Andrews, David, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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