The N-Terminal Domains of Voltage Dependent Anion Channels as Novel Members of the BCL-2 Family: Their Roles and Interactions with the BH3 Domain of the BCL-2 Family Members
Date of Award
M.S. in Biological Science
Bradley W. Jones
Joshua E. Bloomekatz
University of Mississippi
VDACs are pore-forming proteins found within the mitochondria's outer membrane (MOM) whose function is to allow the exchange of metabolites such as ATP and ADP in and out of the mitochondria. In apoptotic cells, they allow the release of cytochrome c from the MOM into the cytosol to initiate apoptosis. The Bcl-2 family of proteins regulates mitochondrial-dependent apoptosis by inhibiting or promoting apoptosis through their BH3 domain. Recent studies have demonstrated that VDACs play essential roles in regulating mitochondrial apoptosis through their interactions with the Bcl-2 family. In this study, we proposed that VDACs are part of the Bcl-2 family members that interact with members of the Bcl-2 family through its putative N-terminal BH3 domain to promote apoptosis through Bax/Bak activation or inhibit apoptosis through mutual sequestration. To test whether VDAC BH3-like domains can function as BH3 domain, lentiviral vector plasmids containing mouse Bax domain swaps or Bcl-xL domain swaps cDNAs were constructed using In-Fusion assembly cloning. The plasmid vectors were combined with Lenti-X packaging single shots to produce pseudo-typed lentiviral particles in Lenti-X 293T cell lines. The supernatants were used to transfect Bax/Bak DKO mouse embryonic fibroblast cell lines seeded in media containing 4μg/ml polybrene. Stably expressing cell lines were selected two days post-infection by growing in 5.0μg/ml puromycin for 8-11 days. The stable cell lines were confirmed to express Bax construct genes through gene sequencing and RT-PCR. In a parallel experiment, pUAST-attB and pUBI-attB constructs containing the cDNAs of mouse VDAC 1 and 2, Bax, Bcl-xL, and their domain swaps were generated through restriction and insertion cloning. We have successfully established transgenic fly lines ubiquitously expressing VDAC 1 and 2 through the Ubiquitin promoter. Through the UAS/Gal4 binary systems, Bax, Bcl-xL, and their domain swaps have been expressed ectopically in the wings and eyes of fly lines of various mutant backgrounds using vg-Gal4 and GMR-Gal4 drivers, respectively.
Agyemang, Clement Nana, "The N-Terminal Domains of Voltage Dependent Anion Channels as Novel Members of the BCL-2 Family: Their Roles and Interactions with the BH3 Domain of the BCL-2 Family Members" (2022). Electronic Theses and Dissertations. 2415.
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