Apoptosis is essential for cellular homeostasis and is also a pathologic feature of various diseases. The balance between Bcl-2 family proteins determines whether a cell will live or die. Bax, a member of the BCL-2 family proteins, is a pro-apoptotic protein that exists in both a soluble, cytoplasmic form and a membrane-bound form. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the mitochondria, initiating apoptotic events. However, little is known about whether Bax is involved in the regulation of mitochondrial function under non-apoptotic conditions, and how Bax binds to mitochondria to exert its activity. Here, we investigate the role of Bax in the regulation of mitochondrial function under non-apoptotic conditions and explore the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli. Using Bax-containing and Bax-deficient (Bax⁻/⁻) HCT-116 cells, we examined Bax cellular localization and its effects on mitochondria bioenergetics, and also tested whether over-expression of full-length Bax in Bax⁻/⁻ cells would recover mitochondrial metabolic activity. To determine the effects of Bax localization upon mitochondrial function, we measured citrate synthase activity and ATP generation. We showed that Bax localized to the outer and inner mitochondrial membranes in non-apoptotic cells, enabling the activity of citrate synthase and the generation of ATP. Loss of Bax led to impairment of respiring mitochondria morphology and reduced oxidative capacity, all of which was restored by expression of full-length or C-terminal-deleted Bax. These findings indicate that under non-apoptotic conditions, the constitutive expression of Bax is necessary for mitochondrial bioenergetics. To determine the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli, we previously performed in silico-mutagenesis and predicted that Lysines 189/190, in the C-terminal [alpha]9 helix, could regulate Bax binding to mitochondria. We demonstrated here that these lysines are the structural elements responsible for controlling how Bax interacts with the mitochondrial membrane. Expression of full-length Bax led to mitochondrial translocation and apoptosis, whereas deletion of the [alpha]9 helix resulted in cytosolic retention and dramatically reduced cell death. Mutation of the two lysine residues changed how Bax bound to mitochondrial membranes. We replicated the results achieved with full-length Bax by attaching the [alpha]9 helix of Bax to GFP or to a regulatory element, the degradation domain (DD), and induced apoptosis upon expression in cells. We demonstrated that the [alpha]9 helix alone promoted the mitochondrial translocation of Bax and increased apoptosis. These results indicate that the C-terminal [alpha]9 helix could be further studied for use in cancer therapies. Overall, we have demonstrated that the constitutive expression of the inactive form of Bax in non-apoptotic cells is necessary for mitochondrial bioenergetics, and have identified the C-terminal [alpha]9 helix of Bax as the effector domain of apoptotic function.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-7653 |
| Date | 01 January 2011 |
| Creators | Zhang, Ge |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
Page generated in 0.0023 seconds