All good things come to an end - including life. In the case of cells, death often comes in the form of apoptosis, a tightly regulated form of cell death where cellular components are membrane bound and rapidly cleared. Apoptosis is a normal process that occurs in development, tissue homeostasis, and the immune response. Careful monitoring of the cell population through detection of major histocompatibility complex (MHC) class I molecules facilitates rapid removal of virus infected and cancer cells. Successful cancer cells frequently acquire two mutations - one that promotes cell growth and another that inhibits the apoptosis that will be induced by unchecked cell growth. For example, overexpression of BCL2 and MYC occur in the development of lymphomas and other kinds of cancers.
Apoptosis can be divided into two pathways: the intrinsic and extrinsic pathways (see figure Apoptotic pathways
). The intrinsic pathway is regulated by BCL2 family members and effector function is mediated through release of proteins such as cytochrome c through the process of mitochondrial outer membrane permeabilization (MOMP). MOMP results in activation of caspase 9, which activates caspase 3. Caspase 3 mediates cleavage of all vital cellular proteins and packaging of cellular components for easy removal (1).
The extrinsic pathway of apoptosis, also called the death receptor pathway, is triggered by ligation of death receptors belonging to the tumor necrosis factor (TNF) family. TNF receptors contain an intracellular death domain and can recruit and activate caspase 8. This results in the activation of caspases 3, 6 and 7, and subsequent apoptotic steps. Note that the extrinsic pathway occurs independently of the BCL2 family. The intrinsic and extrinsic pathways rarely overlap, except in hepatocytes, where caspase 8 cleavage mediates the activation of BID, a proapoptotic member of the BCL2 family (1–3).
There are three classes of BCL2 proteins that differ in how they regulate apoptosis: 1. inhibitors (BCL2, BCLXL, etc), 2. promoters (BAX, BAK, etc), and 3. proteins that regulate the antiapoptotic BCL2 proteins (proteins sharing the BH3 motif such as BAD, BID, BIM, etc). These proteins can act as activators or inhibitors (1, 4).
The promoters of apoptosis, BAX and BAK, function in promoting caspase activation via induction of MOMP. It is thought that BAX interacts with an activator such as BID, which induces a conformational change in BAX, resulting in oligomerization and insertion into the MOM, to form a pore. Release of proteins from the mitochondrial intermembrane space results in fragmentation of mitochondria into small subunits. Cytochrome c is released and binds apoptotic peptidase activating factor 1 (APAF1). This leads to the assembly of a heptameric protein ring called the apoptosome, which binds and activates caspase 9. Caspase 9 stimulates the caspase cascade, committing the cell to apoptosis (1, 4).
The prosurvival members of the BCL2 family (BCL2, BCLXL etc) inhibit the proapoptitic family members (BAX and BAK). However, until recently the mechanism of this inhibition was unclear. There were two major questions in the field. How are proapoptotic members activated? And how do antiapoptotic members inhibit the activity of the proapoptotic proteins?
The "rheostat" model was developed in the 1990's to address the question of regulation in the BCL2 family of proteins (5). The basic concepts were that the proapoptotic and antiapoptotic proteins balance each other with the more abundant protein dominating, pushing the cell towards apoptosis or survival. BAX was shown to interact with BCL2 and this was believed to be the mechanism of inhibition of BAX activity - sequestration.
Over time, the model has evolved. It was shown that BAX existed as a monomer in the cytosol, suggesting that inhibition did not occur through direct interaction with BCL2 (6). In another version of the model, activator BH3-only proteins were proposed to act as receptors, recruiting proapoptotic proteins to the mitochondrial outer membrane and BH3-only proteins with inhibitor function were proposed to bind proapoptotic proteins, preventing them from accumulating on the MOM (7). Willis et al demonstrated that BH3-only proteins promote apoptosis by inhibition the BCL2 inhibitors of apoptosis (8).
A mechanism for BAX regulation remained elusive since BAX did not form a stable complex with prosurvival BCL2 proteins, nor was there evidence of colocalization. Now, Edlich et al proposes a new model for regulation of Bax activity (9). Through addition of disulfide bridges, Edlich et al engineered a cytosolic version of BAX that could not interact with BCLXL. The engineered BAX accumulates on the mitochondrial surface but did not induce apoptosis. They examined translocation of BAX to the mitochondrial surface using fluorescence loss in photobleaching, and found that GFP-BAX regularly cycled on and off the MOM in healthy cells. This translocation was mediated by the presence of antiapoptotic proteins on the MOM. Overexpression of the prosurvival protein, BCLXL, significantly increased the rate of translocation to the cytosol. Translocation to the cytosol required BAX and BCLXL interaction. Once in the cytosol, BAX no longer associated with BCLXL, allowing translocation back to the MOM. Inhibition of BCL2 and BCLXL reduced BAX retrotranslocation. The authors propose a new model for regulation of BAX activity where BCLXL maintains BAX in the cytosol by inducing constant retrotranslocation (see figure Regulation of mitochondral apoptosis
This retrotranslocation model provides a new understanding of BAX regulation, however questions still remain. How do different categories of BH3-only proteins fit into this model? In transformed cells, inactive BAX has been reported to associate with the intracellular membranes of the ER and mitochondria. What are the effects of transformation on BAX translocation? How does this affect apoptosis in cancer cells? Are components of this pathway affected in cancer? Is expression of BCLXL upregulated in cancer? Does this result in defective BAX induced apoptosis? What is the purpose of the constant cycling of BAX between the MOM and cytosol? This suggests that BAX may have an additional function (10).
- Youle, Richard J., and Andreas Strasser, (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47-59.
- Yin, Xiao-Ming, et al., (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886-891.
- Kaufmann, Thomas, et al., (2007) The BH3-Only Protein Bid Is Dispensable for DNA Damage- and Replicative Stress-Induced Apoptosis or Cell-Cycle Arrest. Cell 129, 423-433.
- Chipuk, J. E., T. Moldoveanu, F. Llambi, M. J. Parsons, and D. R. Green, (2010) The BCL-2 family reunion. Mol. Cell 37, 299-310.
- Korsmeyer, S. J., J. R. Shutter, D. J. Veis, D. E. Merry, and Z. N. Oltvai, (1993) Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death. Semin. Cancer. Biol 4, 327-32.
- Hsu, Y. T., and R. J. Youle, (1998) Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J. Biol. Chem 273, 10777-83.
- Lovell, J. F., et al., (2008) Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 135, 1074-84.
- Willis, S. N., et al., (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856-9.
- Edlich, Frank, et al., (2011) Bcl-xL Retrotranslocates Bax from the Mitochondria into the Cytosol. Cell 145, 104-116.
- Soriano, M. E., and L. Scorrano, (2011) Traveling bax and forth from mitochondria to control apoptosis. Cell 145, 15-7.