Bax-induced apoptotic cell death.

A is an ordered cascade of enzymatic events that culminates in cell death and the cleavage of DNA into characteristic nucleosomal fragments. It is operative during embryonic development and, during adult life, plays a key role in various physiological processes, such as tissue remodeling and execution and regulation of the immune response. The consequences of inappropriate apoptotic responses are profound; for example, the failure of cells to initiate apoptosis in response to DNA damage has been implicated in the development and progression of cancer, whereas inappropriate activation of apoptosis is thought to be a contributing factor in Alzheimer’s disease and Parkinson’s disease. Rigorous signaling requirements and a complex network of inhibitory molecules maintain tight control of the apoptotic process, while permitting rapid and effective responses to diverse extracellular and intracellular signals. Growth-factor withdrawal, specific peptide hormones (e.g., tumor necrosis factor a, Fas ligand, and TRAIL), changes in intracellular or extracellular calcium, drugs (e.g., staurosporine and phorbol esters), DNA damage, and mitochondrial poisons can all induce apoptosis, with each of these triggers activating a different portion of the pathway. As our knowledge of the apoptotic cell death cascade increases, it is becoming apparent that the system is more complex than first thought. In a recent issue of PNAS, Khaled and colleagues (1) have addressed how cells might accomplish this fine-tuning of the apoptosis cascade by analyzing the regulation of the proapoptotic molecule Bax in response to the withdrawal of growth factors. The marked similarities in apoptosis among all metazoans have contributed greatly to our understanding of the mammalian pathway. In the nematode Caenorhabditis elegans, three gene products are central to the regulation of the death cascade, CED-3, CED-4, and CED-9. CED-4 binds to CED-3, thereby regulating the activation of CED-3 and the death cascade, whereas CED-9 blocks the activity of CED-4 (2). In the mammalian system, caspase 8, a cysteine protease, shares homology with the C. elegans protein CED-3. Caspase 8 is an initiator caspase that activates effector caspases, including caspase 6, caspase 3, and caspase 7, through selective cleavage (Fig. 1). Caspase 8 contains a death effector domain, which is capable of interacting with the death effector domains contained in the adaptor molecules that link it to specific extracellular and intracellular signals. For example, the peptide hormone Fas ligand binds to a specific transmembrane receptor, Fas. On trimerization, Fas interacts with a specific adaptor protein FADD, which contains a death effector domain, and activates caspase 8. Similar initiation pathways have been established for tumor necrosis factor a and TRAIL. The effector caspases 3 and 7 also are regulated by the APAF-1 protein, a homolog of CED-4, in a manner analogous to that of the nematode CED-3–CED-4 interaction. APAF-1 contains an Nterminal caspase recruitment domain. When the APAF-1 protein is homodimerized in the presence of dATP and cytochrome C released from mitochondria, APAF-1 brings two caspase 9 molecules together, thereby activating this initiator caspase. The activated caspase 9 then induces the cleavage of the effector proteases, caspase 3 and 7, activating their protease activity (Fig. 1). Interestingly, caspase 3 and 7 also may cleave the initiator caspase 8, which, in its position at the head of this pathway, can activate a positive feedback loop for this cascade. The effector caspases are responsible for the controlled degradation process that is characteristic of apoptotic cell death. In addition to degrading specific proteins, such as laminin, the effector caspases target the complex containing the nuclease responsible for DNA cleavage (CAD). CAD resides in the cytoplasm in a complex with its inhibitor (ICAD). During apoptosis, the effector caspases cleave ICAD, thereby allowing the active CAD to migrate from the cytoplasm into the nucleus and to initiate DNA degradation. Because the cellular consequences of errant activation of the apoptotic cascade are considerable, the enzymes that play an important role in regulating this cascade must be under strict inhibitory controls. The caspases cannot be activated in the absence of either trimerization of a receptor or dimerization of APAF-1. In addition, mammalian cells make use of a set of regulatory proteins, the bcl-2 family, which show similarities to the nematode protein CED-9. Consistent with its role in blocking cell death, Bcl-2 was identified initially through analysis of chromosomal translocations in follicular and diffuse lymphomas. The Bcl-2 family of prosurvival proteins includes proteins Bcl-XL, Bcl-w, Mcl-1, etc., which share multiple amino acid domains, notably, the BH1, BH2, BH3, and BH4 domains and a hydrophobic membrane anchor. Bcl-2 family members are located in the outer membrane of the mitochondria and function, at least in part, by blocking the release of cytochrome C from the mitochondria (Fig. 1). The cloning of these Bcl-2 prosurvival proteins revealed related proteins that, in contrast to the prototype Bcl-2 family members, are proapoptotic when overexpressed. One such proapoptotic protein, Bax, contains BH1, BH2, and BH3 domains and a hydrophobic membrane anchor but lacks the BH4 domain. Bax has similarities to Egl-1, a protein of C. elegans that inhibits CED-9. Other Bax family members, including Bak, Bok, Bik, Bad, Bid, etc., all contain BH3 domains, although the majority lack the BH1 and BH2 domains and the hydrophobic membrane domain. In contrast to the Bcl-2 family members, insertion of Bax family members into the mitochondrial membrane induces the release of cytochrome C and the induction of apoptotic cell death. The regulation of this diverse proapoptotic Bax family seems to be quite complex. The assorted mechanisms used by this family of proteins to achieve activation suggest that this system may permit the triggering of the apoptotic pathway in response to specific sets of multiple weak signals rather than to sufficiently strong individual stimuli. Members of the Bax family of proteins are activated variously by cleavage by caspases, inhibition of protein kinases andyor activation of phospha-