Bax, apoptosis, bioenergetics, cancer therapy, ct20p
Bax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells, MitoTracker staining patterns differed, suggesting the existence of Bax -dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in Bax -deficient cells, while glycolysis was increased. These results suggest that cells lacking Bax have a deficiency in the ability to generate ATP through cellular respiration, supported by detection of reduced citrate synthase activity in Bax -deficient cells. Expression of either full length or C-terminal truncated Bax in Bax -deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of Bax was not dependent upon its C-terminal helix. Expression of BCL-2 in Bax-containing cells resulted in a subsequent loss of ATP measured, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. Bax is composed of nine alpha-helices. While three of these helices have features of a trans-membrane region, the contribution of each domain to the apoptotic or non-apoptotic functions of Bax remains unknown. To examine this, we focused on the C-terminal alpha-9 helix, an amphipathic domain with putative membrane binding iv properties and discovered that it has an inherent membrane-binding and cytotoxic capacity. A peptide based on the last twenty amino acids (CT20p) of the alpha-9 helix was synthesized and proved a potent inducer of cell death independent of any apoptotic stimuli. The solubility of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs), and these CT20p-NPs caused the death of colon and breast cancer cells in vitro and induced tumor regression in vivo, using a murine breast cancer tumor model. CT20p caused increased mitochondrial membrane potential followed by cell death via membrane rupture, without the characteristic membrane asymmetry associated with apoptosis. Hence, while CT20p is based on Bax, its innate cytotoxic activity is unlike the parent protein and could be a powerful anti-cancer agent that bypasses drug resistance, can be encapsulated in tumor-targeted nanoparticles and has potential application in combination therapies to activate multiple death pathways in cancer cells. While previous work revealed novel aspects of the biology of Bax that were unrecognized, new structural information is needed to fully elucidate the complexity of Bax’s function. One approach is to use computational modeling to assess the solved structure of Bax and provide insight into the structural components involved in the activity of the protein. Use of molecular dynamics simulators such as GROMACS, as well as other computational tools provides a powerful means by which to test the feasibility of certain modifications in defined parameters. Such work revealed that the removal of the C-terminal alpha-9 helix of Bax, which normally resides within a hydrophobic pocket, significantly destabilized the protein, perhaps explaining how the protein transitions from soluble to membrane-bound form and maintain energy v production via aerobic respiration or, conversely, how the C-terminus helix conveys cytotoxicity. Collectively, this work reveals that Bax is more than an inducer of cell death but has complex activities yet to be determined.
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Doctor of Philosophy (Ph.D.)
College of Medicine
Molecular Biology and Microbiology
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Dissertations, Academic -- Medicine, Medicine -- Dissertations, Academic
Boohaker, Rebecca, "The Dynamic Functions Of Bax Are Dependent On Key Structural And Regulatory Features" (2012). Electronic Theses and Dissertations. 2292.