Structure-Function Analysis of a Novel Inhibitory Mechanism for Pro-Apoptotic BAK

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Project Summary Mitochondrial apoptosis is a form of programmed cell death regulated by BCL-2 family proteins. BAX and BAK are the executioner proteins of the pathway and, in response to cellular stress, transform from inactive monomers to toxic oligomers that porate the mitochondrial outer membrane, leading to cell death. Anti-apoptotic members of the BCL-2 family bind and block BAX and BAK to prevent unwanted cell death, and the delicate balance between BAX/BAK activation and inhibition is required for homeostasis. Cancer cells usurp the anti-apoptotic suppression pathway to ensure their immortality. The classic mechanism of suppression involves sequestration of the BH3 “killer domain” of pro-apoptotic members in a surface groove located at the C-terminal face of anti- apoptotic members. The BCL-2 inhibitor ABT-199, which is showing clinical efficacy in BCL-2 dependent cancers, blocks the anti-apoptotic groove and thereby restores apoptosis. This year, the Walensky lab discovered a distinct mechanism for apoptotic suppression by direct interaction between the BH4 domain of anti- apoptotic BCL-2 and a novel inhibitory interaction site on BAX. Whether or not BAK, the mitochondrial resident analog of BAX, is also subject to BH4 regulation is unknown. Using newly generated stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after the BH4 domains of anti-apoptotic BCL-2 family proteins, I recently detected specific BH4/BAK binding interactions. Thus, I hypothesize that negative regulation by the BH4 domain regions of anti-apoptotic proteins may be a general phenomenon and represents a previously unappreciated mechanism for pathologic suppression of BAK-mediated apoptosis. To elucidate this novel structure-function mechanism, I will apply multidisciplinary approaches in pursuit of the following aims: (1) Generate a library of stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after BH4 motifs to characterize their functional interactions with full-length BAK, and (2) Apply diverse structural methods to both determine the effect of BH4 engagement on BAK conformational activation and define the BH4/BAK binding interface. Thus, the goal of my research proposal is to apply a unique constellation of chemical, biochemical, and structural approaches to characterize a novel mechanism for suppression of pro-apoptotic BAK activation by BH4 domain interaction. Given the oncogenic role of anti-apoptotic blockade of BAK activation, my results could inform a new strategy to restore BAK-mediated apoptosis in human cancer. I look forward to pursuing a comprehensive interdisciplinary training program for my graduate studies at Harvard Medical School and the Dana-Farber Cancer Institute, in preparation for a scientific career as an independent investigator who can operate at the interface of chemical biology, cancer research, and drug development.