BH3-Only Proteins and Their Roles in Programmed Cell Death

Oncogene (2009) 27, S128–S136 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc REVIEW BH3-only proteins and their roles in programmed cell death M Giam1,2, DCS Huang1 and P Bouillet1 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia and 2Department of Medical Biology, The University of Melbourne, Melbourne, Australia The Bcl-2 family of proteins controls the mitochondrial space is required for the activation of the initiator pathway to apoptosis. It consists of pro-survival and pro- caspase-9 by the apoptotic protease-activating factor-1. apoptotic members, and their interactions decide whether Proteins of the Bcl-2 family control the integrity of the apoptogenic factor confined to the mitochondrial inter- mitochondrial membrane in healthy cells, and its membrane space can leak to the cytosol. Despite the permeabilization in response to apoptotic stimuli (Cory intense efforts to understand the molecular mechanisms and Adams, 2002). that lead to the permeabilization of the mitochondrial membrane, this particular issue remains a matter of The Bcl-2 family intense controversy. It is well accepted that pro-apoptotic Bax and Bak are directly responsible for the damage to All members of the Bcl-2 family share a close homology the mitochondria, but pro-survival family members pre- in up to four characteristic regions termed the BH (Bcl-2 vent them from doing so. It is also accepted that stress homology) domains (BH1–4) (Figure 1). They have signals activate selected Bcl-2 homology (BH)3-only either a pro-survival or a pro-apoptotic function. proteins. But do these BH3-only proteins bind and activate Bax and Bak directly, or do they inhibit the The Pro-survival proteins pro-survival family members? The pro-survival proteins of the Bcl-2 family include Oncogene (2009) 27, S128–S136; doi:10.1038/onc.2009.50 Bcl-2, Bcl-w, Bcl-xL, Mcl-1 and A1. They possess up to four BH domains and all have similar 3D structures Keywords: Bcl-2 family; BH3-only proteins; Bax/Bak (Strasser, 2005). The BH1, BH2 and BH3 domains activation; direct/indirect models; mitochondria; cyto- interact to form a hydrophobic groove on the surface of chrome c release the protein. This groove accommodates the BH3 domain of its pro-apoptotic partners and is critical for the pro-survival function of these proteins. Overexpres- sion of any of the pro-survival Bcl-2 family members Introduction prevents the death induced by many apoptotic stimuli, indicating a significant functional redundancy between Apoptosis is essential for the development and main- these proteins. In physiological conditions, however, it is tenance of tissue homeostasis. It allows the safe rare that a single pro-survival protein ensures the elimination of cells that are no longer needed, for survival of a cell population. As an example, T cells example, at the end of an immune response. Apoptosis successively use Bcl-2 and Mcl-1, then A1 and Bcl-xL, occurs as the result of the activation of cysteine then Mcl-1 and Bcl-xL, and finally Bcl-2 again to survive proteases called caspases, which dismantle the cells by the different stages of their development (Strasser et al., degrading essential proteins. Two apoptotic pathways 2008). This may reflect either a selective expression of converge on the activation of caspases:the ‘extrinsic these genes in response to survival signals that may be pathway’, also called the ‘death receptor pathway’, different at each stage of maturation, or an adaptation involves the activation of membrane receptors of the toward the need to counter apoptotic signals that come tumor necrosis factor receptor family and the activation in different forms at each of these stages. Genetic studies of initiator caspases, 8 and 10. The ‘intrinsic pathway’, have helped to identify the physiological role of the pro- also called ‘mitochondrial’ or ‘Bcl-2-regulated pathway’, survival proteins in mice. The various phenotypes is activated as a result of cellular stresses such as growth illustrate the different requirements for these proteins factor deprivation, DNA damage or exposure to in different cell types and at various stages of develop- cytotoxic substances. Mitochondria play a major role ment. Bcl-2-deficient mice develop polycystic kidney in the Bcl-2-regulated pathway. Indeed, release of disease and profound lymphopenia and turn gray as a cytochrome c from the mitochondrial intermembrane result of the loss of melanocyte stem cells (Veis et al., 1993; Nakayama et al., 1994). These mice fail to thrive and die early of renal failure. Loss of Bcl-xL causes Correspondence:Dr P Bouillet, The Walter and Eliza Hall Institute of Medical Research, Molecular Genetics of Cancer Division, 1G Royal massive apoptosis in the nervous and hematopoietic Parade, Parkville, 3050 Victoria, Australia. systems and embryos die at mid-gestation (Motoyama E-mail:[email protected] et al., 1995). Embryos lacking Mcl-1 die at the time of BH3-only proteins and their roles M Giam et al S129 Pro-survival members BH4 BH3 BH1 BH2 TM Bcl-2, Bcl-xL, Bcl-w, Mcl-1,A1 Pro-apoptotic members Multidomain effectors BH3 BH1 BH2 TM Bax, Bak, Bok BH3-only proteins BH3 TM Bim, Puma, Noxa, Bik, Bmf, Bad, Hrk, Bid Figure 1 The Bcl-2 family is made up of proteins that contain conserved functional Bcl-2 Homology domains. The family can be subdivided into pro-survival and pro-apoptotic proteins. The latter can be further divided into multidomain effectors and BH3-only proteins. The transmembrane domain (TM) is not found in some of the BH3–only proteins. implantation (Rinkenberger et al., 2000), whereas BaxÀ/ÀBakÀ/À cells, showing the strict requirement for conditional deletion of Mcl-1 in the hematopoietic Bax or Bak downstream of the BH3-only proteins. system only showed a requirement of Mcl-1 for the However, it is surprising that 10% BaxÀ/ÀBakÀ/À mice survival of hematopoietic progenitors (Opferman et al., survived to adulthood and could live for several months 2003). The loss of A1a resulted in accelerated neutrophil (Lindsten et al., 2000). This shows that some develop- apoptosis, but this mild phenotype may be because of mental cell death can still proceed in the absence of these the existence of three additional A1 genes that may have two pro-apoptotic molecules. Bok, a third multi-domain substituted for the missing A1a (Hamasaki et al., 1998). pro-apoptotic protein resembling Bax and Bak, could It has been known for a long time that overexpressed potentially fulfill the same function. Unfortunately, this pro-survival proteins prevent the release of cytochrome protein has been poorly studied so far, and it is not at all c to the cytosol and thereby also the subsequent initiator clear whether it can compensate for the absence of Bax caspase activation. However, the molecular mechanisms or Bak in certain cell types. In vivo, Bax and Bak appear that they use to protect mitochondrial integrity are still to be mostly redundant, as the presence of a single allele debated. of either of them was sufficient to prevent all the defects observed in BaxÀ/ÀBakÀ/À mice (Lindsten et al., 2000). However, recent reports suggest that this redundancy Bax, Bak and Bok may be more limited, because some apoptotic signals Bcl-2-regulated apoptosis requires Bax or Bak for seem to rely specifically on one of them for their mitochondrial damage. These two pro-apoptotic pro- execution. For example, the BH3-only protein, Bik/ teins contain BH1-3 domains, and have a 3D structure Nbk, was reported to kill in the presence of Bax only, very similar to that of the pro-survival members of but to be inert in the presence of Bak only (Theodorakis the family (Sattler et al., 1997; Suzuki et al., 2000; et al., 2002; Gillissen et al., 2003). Moldoveanu et al., 2006). Mice lacking Bax show mild Bax and Bak are the main effectors of the Bcl-2- lymphoid hyperplasia and male sterility because of regulated pathway. When activated, they permeabilize sperm-cell differentiation defects (Knudson et al., 1995). the mitochondria to allow the release of apoptogenic Mice lacking Bak have no documented defect so far. proteins into the cytosol. This is thought to occur Most mice lacking both Bax and Bak complete through the formation of pores in the outer mitochon- embryonic development, but die around birth because drial membrane. The pore theory appeared with the they fail to nurse (Lindsten et al., 2000). These mice realization that the 3D structures of Bcl-xL (and later, of show several developmental defects including the Bax) had significant similarity with the pore-forming persistence of interdigital webs, imperforated vaginal translocation domain of the diphteria toxin (Suzuki canal and accumulation of cells in the central nervous et al., 2000). Despite intense efforts to show the and hematopoietic systems (Lindsten et al., 2000; Wei existence of this pore, its molecular composition remains et al., 2001). Many cell types derived from these mice elusive. The presence of Bax or Bak is required to allow proved to be completely resistant to a large array of cytochrome c out of the mitochondria or large macro- apoptotic stimuli such as UV-irradiation, DNA-dama- molecules out of synthetic lipidic vesicles, but how they ging drugs and cytokine deprivation, but were still achieve this not completely understood. Although Bax sensitive to death by tumor necrosis factor family and Bak seem to have identical functions, one striking ligands (which trigger the extrinsic pathway) (Wei difference between them is their subcellular localization et al., 2001). Of particular interest, death induced by in healthy cells (Reed, 2006). Bak is an integral overexpression of pro-apoptotic BH3-only members of membrane protein on the outer mitochondrial mem- the Bcl-2 family seemed to be completely blocked in brane, whereas inactive Bax is a largely cytosolic protein Oncogene BH3-only proteins and their roles M Giam et al S130 (Wolter et al., 1997).

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