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Control of mitochondrial apoptosis by the Bcl-2 family

Control of mitochondrial apoptosis by the Bcl-2 family Cell Science at a Glance 437 controlled first arose when it was observed apoptosis by controlling the Control of mitochondrial that certain mutants of the model organism permeabilization of the outer apoptosis by the Bcl-2 Caenorhabditis elegans caused failure of mitochondrial membrane. In response to apoptosis in cells that normally undergo many types of stress or damage, certain family this process during development members of the Bcl-2 family, known as Joslyn K. Brunelle and Anthony (Hengartner et al., 1992). Subsequently, it BH3-only proteins (see below), are Letai* was found that proteins that are encoded by activated. Certain BH3-only proteins cause Medical Oncology, Dana Farber Cancer Institute, the mutant genes discovered in C. elegans the activation of the pro-apoptotic proteins Boston, MA 02115, USA shared homology with mammalian Bcl-2-associated X protein (Bax) or Bcl-2 *Author for correspondence proteins, including B-cell CLL/lymphoma 2 antagonist/killer-1 (Bak) at the (e-mail: [email protected]) (Bcl-2) (Hengartner and Horvitz, 1994). mitochondrion. Activated Bax and Bak Journal of Cell Science 122, 437-441 Further study in mammals revealed that homo-oligomerize and participate in the Published by The Company of Biologists 2009 there is an intrinsic apoptotic pathway formation of pores in the outer doi:10.1242/jcs.031682 that involves the mitochondria and an mitochondrial membrane through which extrinsic apoptotic pathway that involves pro-apoptotic molecules escape, including Programmed cell death, or apoptosis, is death receptors. The mitochondrial pathway second mitochondria-derived activator of important for the development and of apoptosis in mammals, on which this caspase (Smac) (also known as Diablo) homeostasis of tissues. Too little cell death poster article is focused, is regulated by and cytochrome c. Release of cytochrome can result in autoimmune diseases or members of the Bcl-2 family of proteins. c leads to the activation of caspases, which cancer, whereas excessive cell death can are proteases that cleave key cellular lead to debilitating degenerative diseases Proteins of the Bcl-2 family have either proteins. This leads to many of the of the heart or nervous system. The pro- or anti-apoptotic activities and morphological characteristics of apoptosis, realization that apoptosis was genetically regulate the mitochondrial pathway of including condensed nuclei, DNA laddering Joslyn K. Brunelle and Anthony Letai Models of Bax or Bak activation Three possible classes of mitochondria Class A Death stimuli • Oncogene activation • DNA damage • Anoikis Bcl-2 Bax or Bak • Growth-factor withdrawal • Kinase inhibition • Microtubule perturbation BH3 profile Sensitizers Activators • Bid • p53? Puma? Others? • Bad • Noxa • Hrk • Bmf • Bim • Bik • BNIP3 • Puma? • MULE Activated Bax or Bak Pore formation and cytochrome c egress Functional Bax or Bak; MOMP occurs only in response to activators that overwhelm the Bax anti-apoptotic reserve or Bak Bid Bim Bad Noxa Puma Oligomerized Class B Bax or Bak BH3 profile Bcl-2 Mitochondrion Apoptosome Cytochrome c Effector caspase Low or absent Bax and Bak; MOMP does activation not occur in response to any BH3-only proteins Bid Bim Bad Noxa Puma Pore formation and cytochrome c egress Displacement of activator BH3 profile Class C Key Activator Bcl-2 BH3-only Activated Displacement Bcl-2 Activated Bax or Bak of activated Activator Bax or Bak Bax or Bak or sensitizer BH domains Bid Bim Bad Noxa Puma Anti-apoptotic BH4 BH3 BH1 BH2 Functional Bax and Bak; increased expression BH domains 1-4: Bcl-2, Bcl-XL, Bcl-w (Bfl-1 and Mcl-1 may lack BH4) of Bcl-2 or any anti-apoptotic proteins; MOMP occurs in response to displacement Pro-apoptotic Pro-apoptotic of activators or activated Bax or Bak by select BH3 BH3 BH1 BH2 BH3-only domains BH3 only: Bim, Bid, Bad, Bik, Puma, Noxa, Bmf, BNIP3, Bmf, Hrk, MULE BH domains 1-3: Bax and Bak Abbreviations: Bad, Bcl-2-associated death promoter; Bak, Bcl-2 antagonist/killer-1; Bcl-2-modifying factor; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting Bax, Bcl-2-associated X protein; Bcl-2, B-cell CLL/lymphoma 2; Bcl-w, Bcl-2-like 2; Bcl-XL, protein 3; Hrk, Harakiri; Mcl-1, myeloid cell leukemia-1; MOMP, mitochondrial outer membrane Bcl-2-like 1; Bfl-1, Bcl-2-related protein A1; BH, Bcl-2 homology; Bid, BH3-interacting-domain permeabilization; MULE, Mcl-1 ubiquitylation ligase E3; p53, protein 53; Puma, p53-upregulated death agonist; Bik, Bcl-2-interacting killer; Bim, Bcl-2-interacting mediator of cell death; Bmf, modulator of apoptosis. © Journal of Cell Science 2009 (122, pp. 437-441) (See poster insert) Journal of Cell Science MOMP MOMP MOMP 438 Journal of Cell Science 122 (4) and exposure of phosphatidylserine to the Box 1. The BH3 profiling technique outer leaflet of the plasma membrane. BH3 profiling is a technique that uses BH3 domains of BH3-only proteins to apply a standardized death signal to mitochondria. This allows for the comparison of how readily Expression of Bcl-2 or other related anti- different mitochondria, and hence cells, undergo apoptosis. Each anti-apoptotic protein of apoptotic proteins, including myeloid cell the Bcl-2 family has a distinct pattern of binding to certain BH3-only proteins. Peptides are leukemia-1 (Mcl-1), Bcl-2-like 1 (Bcl-XL), designed using the amino-acid sequence (approximately 20 amino acids) of the Bcl-2-like 2 (Bcl-w) and Bcl-2-related corresponding BH3-only protein. Mitochondria are isolated from the cell line or patient protein A1 (Bfl-1), block cell death in sample. Peptides are incubated with the mitochondria and mitochondrial outer-membrane response to many varieties of insult by permeabilization (MOMP) is measured. The resulting pattern of peptides that do or do not preventing the activation and homo- cause MOMP is the readout of the assay. BH3 profiling can also distinguish among three classes of apoptotic block that are used oligomerization of both Bax and Bak. by cancer cells to survive. A class A block indicates that functional activator BH3-only Anti-apoptotic proteins perform their anti- proteins are present at relatively low levels. In this case, the BH3-only protein activators death function by sequestering BH3-only Bid and Bim, but not any of the BH3-only protein sensitizer peptides, would cause MOMP. proteins or activated, monomeric Bax and In a class B block, the pro-apoptotic proteins Bax and/or Bak are absent or not functional. Bak. Cells that survive continuous, In this case, none of the BH3-only peptides would cause MOMP as Bax and/or Bak are permanent death signaling owing to the required for their effect. A class C block indicates that anti-apoptotic proteins are present presence of Bcl-2 are dependent on Bcl-2 and primed with BH3-only protein activators, or activated Bax or Bak. In this case, one can for their survival. It is known that certain compare the pattern of the BH3-only sensitizer peptides that cause MOMP with the binding cancer cells depend upon Bcl-2 and other code for the specific anti-apoptotic proteins to determine which anti-apoptotic proteins are anti-apoptotic proteins for survival. We primarily responsible for maintaining survival. have found that dependence on anti- apoptotic proteins can be identified in cancer cells using a strategy that we call understanding how certain insults and be activated by many noxious stimuli – BH3 profiling (see below and Box 1). In derangements commit selected cells to including DNA damage, growth-factor cancer cells that are dependent on Bcl-2, death, but spare others. withdrawal and oncogene activation – via the Bcl-2 protein binds pro-apoptotic BH3- mechanisms that include transcriptional only proteins such as Bcl-2-interacting Members of the Bcl-2 family upregulation, subcellular localization and/or mediator of cell death (Bim). We describe The Bcl-2 family can be divided into pro- post-translational modifications. For such cells as being ‘primed for death’. apoptotic and anti-apoptotic proteins. These example, p53-upregulated modulator of Molecular therapies that are targeted to proteins contain one or more Bcl-2 apoptosis (Puma) and Noxa (the latin word anti-apoptotic proteins such as Bcl-2 can homology (BH) domains, which share for damage; also known as PMAIP1) are induce apoptosis in primed cancer cells; sequence homology and are important for transcriptionally upregulated by p53 in one such Bcl-2 antagonist, Abbott (ABT)- heterodimeric interactions among members response to DNA damage (Nakano and 737, has shown impressive success in of the Bcl-2 family (Chittenden et al., 1995; Vousden, 2001; Oda et al., 2000). In some killing leukemia and lymphoma cells Danial and Korsmeyer, 2004). Most anti- cells, Bim can be sequestered within the (Table 1). apoptotic proteins contain BH domains 1-4. cytoskeleton, to be released only in response Pro-apoptotic proteins can be divided into to certain death stimuli (Puthalakath et al., In this brief Cell Science at a Glance poster two groups according to function and the 1999). BH3-interacting-domain death article, we discuss how proteins of the number of BH domains possessed. Bax and agonist (Bid) is activated by cleavage by Bcl-2 family control the crucial event Bak are pro-apoptotic proteins that contain caspase 8 to form truncated Bid (tBID) (Li in the commitment to apoptosis – the BH domains 1-3 and are known as et al., 1998), whereas Bcl-2-associated permeabilization of the mitochondrial multidomain pro-apoptotic or effector death promoter (Bad) is activated by outer membrane. In addition, we discuss proteins. The remaining pro-apoptotic dephosphorylation (Zha et al., 1996). The recently developed methods for probing proteins contain only the third BH domain relatively numerous pro- and anti-apoptotic how cancer cells manipulate members of and are known as BH3-only proteins. BH3- members of this family engage in complex the Bcl-2 family to block apoptosis. only proteins act as upstream sentinels of interactions with each other to ultimately Understanding these principles is key to cellular damage and derangement. They can decide whether a cell will commit to death Table 1. Current clinical strategies to target proteins of the Bcl-2 family Drug Company Clinical phase Function Additional comments ABT-263 Abbott Laboratories Phase I and I/II clinical trials in NHL, BH3-mimetic small molecule ABT-263 is an orally available CLL and SCLC targeting Bcl-2, Bcl-XL and Bcl-w compound that is closely related to ABT-737 Obatoclax Gemin X Multiple phase I and II clinical trials in BH3-mimetic small molecule Might be a pan-inhibitor of anti- (GX15-070) hematological malignancies and non- apoptotic proteins small-cell lung cancer Oblimersen Genta Many clinical trials including phase III in Antisense DNA targeting Bcl-2 Clinical activity marginal, but not clear melanoma and CLL that drug is reducing Bcl-2 levels in vivo AT-101 Ascenta Therapeutics Phase II clinical trials in a variety of BH3-mimetic small molecule AT-101 is the negative enantiomer of cancers gossypol CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin's lymphoma; SCLC, small-cell lung cancer. Journal of Cell Science Journal of Cell Science 122 (4) 439 by controlling permeabilization of the and the direct models. According to the cannot activate Bax and Bak directly, but mitochondrial outer membrane. indirect model, Bax and Bak must always can bind anti-apoptotic proteins and cause be bound by anti-apoptotic proteins to the release of activator BH3-only proteins, Mitochondrial permeabilization prevent their activation. BH3-only proteins leading to activation of Bax and Bak. A Mitochondrial outer membrane permeabili - provoke death solely by binding to anti- prediction of this model is that the deletion zation (MOMP), which releases numerous apoptotic proteins, causing release and of all activators would result in a profound pro-apoptotic proteins into the cytosol, is the activation of Bax and Bak (Willis et al., block in apoptosis that is equivalent to the pivotal event in the intrinsic apoptotic 2007). A prediction of this model is that, for loss of Bax and Bak. By contrast, however, pathway. Bax and Bak double-knockout a cell to survive, all Bax and Bak proteins a combined knockout of Bid and Bim cells fail to undergo MOMP in response to must be bound to an anti-apoptotic protein results in relatively minor defects in many different death stimuli, including such as Bcl-2 and/or Mcl-1. However, co- apoptosis (Willis et al., 2007), but it is likely staurosporine, ultraviolet (UV) radiation, immunoprecipitation of anti-apoptotic that additional factors other than Bid and growth-factor deprivation, DNA damage proteins with Bax and Bak demonstrates Bim can act as activators. In fact, there are and endoplasmic reticulum stress (Wei that usually only a small minority of data that support the role of Puma, p53 and et al., 2001). Apoptosis that is caused by Bak is so bound. Furthermore, co- heat as activators of Bax and Bak (Chipuk BH3-only proteins absolutely requires Bax immunoprecipitation is often carried out in et al., 2004; Kim et al., 2006; Pagliari et al., and Bak (Kuwana et al., 2005; Lindsten the presence of detergents such as Triton 2005), and possibly others remain et al., 2000; Wei et al., 2000; Wei et al., X-100 or Tergitol-type nonyl undiscovered. The important point is that 2001). Bax proteins can be found as phenoxylpolyethoxylethanol-40 (NP-40). the activation of Bax and Bak might be monomers in the cytosol or loosely These detergents can artificially induce Bax affected by factors outside of the Bcl-2 associated with the outer mitochondrial and Bak conformations that mimic an family of proteins. Very recent results have membrane when not activated. Bax activated state and stimulate artifactual demonstrated a structure of a complex of the translocates to and inserts into the binding to anti-apoptotic proteins (Hsu and BH3 domain of Bim with Bax (Gavathiotis mitochondrial outer membrane during the Youle, 1997). This can result in an et al., 2008). Surprisingly, the interaction activation process (Billen et al., 2008; Hsu overestimation of the amount of Bax that is takes place on the Bax surface distal from et al., 1997; Wolter et al., 1997). Bak is actually bound to anti-apoptotic proteins. the hydrophobic pocket formed by the BH1, inserted into the outer mitochondrial Artificial activation of Bax and Bak does BH2 and BH3 domains. The analogous membrane even when not activated (Wei not occur in buffers that contain detergents pocket is used by anti-apoptotic proteins to et al., 2000). such as 3-[3-(Cholamidopropyl)dimethyl - bind BH3 domains. ammonio]-1-propanesulfonate (CHAPS). One of the steps that is involved in the However, it might be that Bax and Bak can The gulf between the two models is not activation of Bax and Bak is a alternatively interact with proteins that unbridgeable. As long as one is willing to conformational change that exposes the remove the requirement for sequestration by accept that there are activated subsets of N-terminus of the proteins, which is anti-apoptotic proteins (Cheng et al., 2003). Bax and Bak that are required to kill, and otherwise hidden in the inactive state More recent iterations of this model include that must be sequestered by anti-apoptotic (Yethon et al., 2003). This activated the concept that the sequestered forms of proteins to maintain survival, a unifying conformation can be recognized by Bax and Bak are those fractions of the model can be constructed. In this model, conformation-specific antibodies, such as total Bax and Bak population that are activated Bax and Bak are responsible for 6A7, which is specific for Bax (Hsu and already activated, perhaps spontaneously, or the permeabilization of membranes. They Youle, 1997). Following activation, Bax perhaps by other unspecified means achieve the activated state either by and Bak form homo-oligomers that can be (Fletcher et al., 2008). interacting with activator proteins, visualized via western blotting following by spontaneously activating, or via other chemical crosslinking (Wei et al., 2000). According to the direct model of Bax and unknown means. Anti-apoptotic proteins Bax and Bak oligomers participate in Bak activation, activator proteins (which inhibit death by sequestering activator forming pores in and cause permeabilization include Bid, Bim and possibly others such proteins or activated Bax and Bak. In of the outer mitochondrial membrane, as Puma and p53) directly interact with and addition to activating Bax and Bak (a leading to the release of the contents of induce conformational changes in Bax property possessed by only a subset of the mitochondrial intermembrane space, and Bak. Studies using full-length proteins, BH3-only proteins), BH3-only proteins including cytochrome c and Smac, into the lipid membranes and real-time fluorescence cause death by displacing activators and cytosol (Wang, 2001). These contents drive resonance energy transfer (FRET) analyses Bax and Bak from anti-apoptotic proteins, the activation of caspases, which are has provided convincing evidence of such permitting progression of the death signal. proteases that cleave and disable crucial an interaction and for its role in membrane This model is summarized in the poster proteins throughout the cell. permeabilization (Lovell et al., 2008). Anti- accompanying this article. apoptotic proteins prevent death by binding Bax and Bak activation and sequestering such pro-apoptotic Specificity in the interaction Given the lethal consequences of Bax and activator BH3-only proteins, and also by between anti-apoptotic and Bak activation, understanding how their binding any monomeric, activated Bax and BH3-only proteins activation is controlled is key to Bak proteins that might be present. In this Mammalian anti-apoptotic proteins understanding how a cell makes the model, BH3-only proteins are further include Bcl-2, Bcl-XL, Mcl-1, Bcl-w decision to undergo apoptosis. Two models divided into activator and sensitizer and Bfl-1. Anti-apoptotic proteins bind and of Bax and Bak activation exist: the indirect categories (Letai et al., 2002). Sensitizers sequester pro-apoptotic proteins, including Journal of Cell Science 440 Journal of Cell Science 122 (4) Fletcher, J. I., Meusburger, S., Hawkins, C. J., Riglar, activator BH3-only proteins and Bax and limited results in normal tissues suggests D. T., Lee, E. F., Fairlie, W. D., Huang, D. C. and Bak, to prevent apoptosis. Sensitizer that this is less likely to be the case in Adams, J. M. (2008). Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proc. Natl. proteins provoke apoptosis by normal tissues. A strategy of Bcl-2 Acad. Sci. USA 105, 18081-18087. competitively inhibiting this interaction. antagonism is being employed in clinical Gavathiotis, E., Suzuki, M., Davis, M. L., Pitter, K., Bird, G. H., Katz, S. G., Tu, H. C., Kim, H., Cheng, E. Each anti-apoptotic protein has its own trials of agents such as ABT-263, as some H., Tjandra, N. et al. (2008). BAX activation is initiated pattern of interaction with sensitizer cancers are dependent on Bcl-2 for survival at a novel interaction site. Nature 455, 1076-1081. proteins (Certo et al., 2006; Chen et al., (Oltersdorf et al., 2005; Tse et al., 2008). Hengartner, M. O. and Horvitz, H. R. (1994). C. elegans cell survival gene ced-9 encodes a functional homolog of 2005; Kuwana et al., 2005; Opferman the mammalian proto-oncogene bcl-2. Cell 76, 665-676. et al., 2003). For example, Bad selectively Future directions Hengartner, M. O., Ellis, R. E. and Horvitz, H. R. binds Bcl-2, Bcl-XL and Bcl-w; Noxa (1992). Caenorhabditis elegans gene ced-9 protects cells Although many important discoveries have from programmed cell death. Nature 356, 494-499. specifically binds Mcl-1; and Harakiri been made regarding the roles of members Hsu, Y. T. and Youle, R. J. (1997). Nonionic detergents (Hrk) is specific for Bcl-XL. Bim, Bid and of the Bcl-2 family in the mitochondrial induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272, 13829-13834. Puma can bind to all the anti-apoptotic apoptosis pathway, important questions Hsu, Y. T., Wolter, K. G. and Youle, R. J. (1997). Cytosol- proteins. Mcl-1 has additional unique remain. Are there other important activator to-membrane redistribution of Bax and Bcl-X(L) during characteristics. Mcl-1 ubiquitylation ligase apoptosis. Proc. Natl. Acad. Sci. USA 94, 3668-3672. and sensitizer proteins? Are there Kim, H., Rafiuddin-Shah, M., Tu, H. C., Jeffers, J. R., E3 (MULE) contains a BH3 domain that undiscovered mechanisms for holding Bax Zambetti, G. P., Hsieh, J. J. and Cheng, E. H. (2006). specifically binds Mcl-1, which leads to and Bak at bay? How do the noxious Hierarchical regulation of mitochondrion-dependent Mcl-1 ubiquitylation and degradation apoptosis by BCL-2 subfamilies. Nat. Cell Biol. 8, 1348- stimuli we routinely use to kill cells in (Zhong et al., 2005). This interaction, in culture and in vivo interact with proteins of Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P. and addition to the numerous PEST sequences Craig, R. W. (1993). MCL1, a gene expressed in the Bcl-2 family to cause apoptosis? programmed myeloid cell differentiation, has sequence [which have many proline (P), glutamic Finally, can direct inhibition of anti- similarity to BCL2. Proc. Natl. Acad. Sci. USA 90, 3516- acid (E), serine (S) and threonine (T) apoptotic proteins be an effective tool in residues (Kozopas et al., 1993)] possessed Kuwana, T., Bouchier-Hayes, L., Chipuk, J. E., treating cancer? We await the results of Bonzon, C., Sullivan, B. A., Green, D. R. and by Mcl-1, might account for the short half- clinical trials of drugs such as ABT-263 Newmeyer, D. D. (2005). BH3 domains of BH3-only life of the Mcl-1 protein. The specificity of (Table 1) to answer this key question. proteins differentially regulate Bax-mediated these interactions can be exploited to mitochondrial membrane permeabilization both directly The authors gratefully acknowledge support from and indirectly. Mol. Cell 17, 525-535. deduce important elements of the control NIH grants R01 CA129974 and P01 CA068484. A.L. Letai, A., Bassik, M. C., Walensky, L. D., Sorcinelli, M. of apoptosis by individual cell types using D., Weiler, S. and Korsmeyer, S. J. (2002). Distinct BH3 is a Leukemia and Lymphoma Society Scholar. a procedure called BH3 profiling. domains either sensitize or activate mitochondrial Deposited in PMC for release after 12 months. apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183-192. BH3 profiling References Li, H., Zhu, H., Xu, C. J. and Yuan, J. (1998). Cleavage of BID by caspase 8 mediates the mitochondrial damage The dependence of a cell or mitochondrion Billen, L. P., Kokoski, C. L., Lovell, J. F., Leber, B. and in the Fas pathway of apoptosis. Cell 94, 491-501. Andrews, D. W. (2008). Bcl-XL inhibits membrane on any one of the anti-apoptotic proteins Lindsten, T., Ross, A. J., King, A., Zong, W. X., permeabilization by competing with Bax. 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Science Zha, J., Harada, H., Yang, E., Jockel, J. and slides. 292, 727-730. Korsmeyer, S. J. (1996). Serine phosphorylation of death Commentaries and Cell Science at a Glance JCS Commentaries highlight and critically discuss recent and exciting findings that will interest those who work in cell biology, molecular biology, genetics and related disciplines, whereas Cell Science at a Glance poster articles are short primers that act as an introduction to an area of cell biology, and include a large poster and accompanying text. Both of these article types, designed to appeal to specialists and nonspecialists alike, are commissioned from leading figures in the field and are subject to rigorous peer-review and in-house editorial appraisal. Each issue of the journal usually contains at least one of each article type. JCS thus provides readers with more than 50 topical pieces each year, which cover the complete spectrum of cell science. 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Control of mitochondrial apoptosis by the Bcl-2 family

K., Brunelle, Joslyn; Anthony, Letai,
Journal of Cell Science , Volume 122 (4) – Feb 15, 2009
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The Company of Biologists
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0021-9533
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10.1242/jcs.031682
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Abstract

Cell Science at a Glance 437 controlled first arose when it was observed apoptosis by controlling the Control of mitochondrial that certain mutants of the model organism permeabilization of the outer apoptosis by the Bcl-2 Caenorhabditis elegans caused failure of mitochondrial membrane. In response to apoptosis in cells that normally undergo many types of stress or damage, certain family this process during development members of the Bcl-2 family, known as Joslyn K. Brunelle and Anthony (Hengartner et al., 1992). Subsequently, it BH3-only proteins (see below), are Letai* was found that proteins that are encoded by activated. Certain BH3-only proteins cause Medical Oncology, Dana Farber Cancer Institute, the mutant genes discovered in C. elegans the activation of the pro-apoptotic proteins Boston, MA 02115, USA shared homology with mammalian Bcl-2-associated X protein (Bax) or Bcl-2 *Author for correspondence proteins, including B-cell CLL/lymphoma 2 antagonist/killer-1 (Bak) at the (e-mail: [email protected]) (Bcl-2) (Hengartner and Horvitz, 1994). mitochondrion. Activated Bax and Bak Journal of Cell Science 122, 437-441 Further study in mammals revealed that homo-oligomerize and participate in the Published by The Company of Biologists 2009 there is an intrinsic apoptotic pathway formation of pores in the outer doi:10.1242/jcs.031682 that involves the mitochondria and an mitochondrial membrane through which extrinsic apoptotic pathway that involves pro-apoptotic molecules escape, including Programmed cell death, or apoptosis, is death receptors. The mitochondrial pathway second mitochondria-derived activator of important for the development and of apoptosis in mammals, on which this caspase (Smac) (also known as Diablo) homeostasis of tissues. Too little cell death poster article is focused, is regulated by and cytochrome c. Release of cytochrome can result in autoimmune diseases or members of the Bcl-2 family of proteins. c leads to the activation of caspases, which cancer, whereas excessive cell death can are proteases that cleave key cellular lead to debilitating degenerative diseases Proteins of the Bcl-2 family have either proteins. This leads to many of the of the heart or nervous system. The pro- or anti-apoptotic activities and morphological characteristics of apoptosis, realization that apoptosis was genetically regulate the mitochondrial pathway of including condensed nuclei, DNA laddering Joslyn K. Brunelle and Anthony Letai Models of Bax or Bak activation Three possible classes of mitochondria Class A Death stimuli • Oncogene activation • DNA damage • Anoikis Bcl-2 Bax or Bak • Growth-factor withdrawal • Kinase inhibition • Microtubule perturbation BH3 profile Sensitizers Activators • Bid • p53? Puma? Others? • Bad • Noxa • Hrk • Bmf • Bim • Bik • BNIP3 • Puma? • MULE Activated Bax or Bak Pore formation and cytochrome c egress Functional Bax or Bak; MOMP occurs only in response to activators that overwhelm the Bax anti-apoptotic reserve or Bak Bid Bim Bad Noxa Puma Oligomerized Class B Bax or Bak BH3 profile Bcl-2 Mitochondrion Apoptosome Cytochrome c Effector caspase Low or absent Bax and Bak; MOMP does activation not occur in response to any BH3-only proteins Bid Bim Bad Noxa Puma Pore formation and cytochrome c egress Displacement of activator BH3 profile Class C Key Activator Bcl-2 BH3-only Activated Displacement Bcl-2 Activated Bax or Bak of activated Activator Bax or Bak Bax or Bak or sensitizer BH domains Bid Bim Bad Noxa Puma Anti-apoptotic BH4 BH3 BH1 BH2 Functional Bax and Bak; increased expression BH domains 1-4: Bcl-2, Bcl-XL, Bcl-w (Bfl-1 and Mcl-1 may lack BH4) of Bcl-2 or any anti-apoptotic proteins; MOMP occurs in response to displacement Pro-apoptotic Pro-apoptotic of activators or activated Bax or Bak by select BH3 BH3 BH1 BH2 BH3-only domains BH3 only: Bim, Bid, Bad, Bik, Puma, Noxa, Bmf, BNIP3, Bmf, Hrk, MULE BH domains 1-3: Bax and Bak Abbreviations: Bad, Bcl-2-associated death promoter; Bak, Bcl-2 antagonist/killer-1; Bcl-2-modifying factor; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting Bax, Bcl-2-associated X protein; Bcl-2, B-cell CLL/lymphoma 2; Bcl-w, Bcl-2-like 2; Bcl-XL, protein 3; Hrk, Harakiri; Mcl-1, myeloid cell leukemia-1; MOMP, mitochondrial outer membrane Bcl-2-like 1; Bfl-1, Bcl-2-related protein A1; BH, Bcl-2 homology; Bid, BH3-interacting-domain permeabilization; MULE, Mcl-1 ubiquitylation ligase E3; p53, protein 53; Puma, p53-upregulated death agonist; Bik, Bcl-2-interacting killer; Bim, Bcl-2-interacting mediator of cell death; Bmf, modulator of apoptosis. © Journal of Cell Science 2009 (122, pp. 437-441) (See poster insert) Journal of Cell Science MOMP MOMP MOMP 438 Journal of Cell Science 122 (4) and exposure of phosphatidylserine to the Box 1. The BH3 profiling technique outer leaflet of the plasma membrane. BH3 profiling is a technique that uses BH3 domains of BH3-only proteins to apply a standardized death signal to mitochondria. This allows for the comparison of how readily Expression of Bcl-2 or other related anti- different mitochondria, and hence cells, undergo apoptosis. Each anti-apoptotic protein of apoptotic proteins, including myeloid cell the Bcl-2 family has a distinct pattern of binding to certain BH3-only proteins. Peptides are leukemia-1 (Mcl-1), Bcl-2-like 1 (Bcl-XL), designed using the amino-acid sequence (approximately 20 amino acids) of the Bcl-2-like 2 (Bcl-w) and Bcl-2-related corresponding BH3-only protein. Mitochondria are isolated from the cell line or patient protein A1 (Bfl-1), block cell death in sample. Peptides are incubated with the mitochondria and mitochondrial outer-membrane response to many varieties of insult by permeabilization (MOMP) is measured. The resulting pattern of peptides that do or do not preventing the activation and homo- cause MOMP is the readout of the assay. BH3 profiling can also distinguish among three classes of apoptotic block that are used oligomerization of both Bax and Bak. by cancer cells to survive. A class A block indicates that functional activator BH3-only Anti-apoptotic proteins perform their anti- proteins are present at relatively low levels. In this case, the BH3-only protein activators death function by sequestering BH3-only Bid and Bim, but not any of the BH3-only protein sensitizer peptides, would cause MOMP. proteins or activated, monomeric Bax and In a class B block, the pro-apoptotic proteins Bax and/or Bak are absent or not functional. Bak. Cells that survive continuous, In this case, none of the BH3-only peptides would cause MOMP as Bax and/or Bak are permanent death signaling owing to the required for their effect. A class C block indicates that anti-apoptotic proteins are present presence of Bcl-2 are dependent on Bcl-2 and primed with BH3-only protein activators, or activated Bax or Bak. In this case, one can for their survival. It is known that certain compare the pattern of the BH3-only sensitizer peptides that cause MOMP with the binding cancer cells depend upon Bcl-2 and other code for the specific anti-apoptotic proteins to determine which anti-apoptotic proteins are anti-apoptotic proteins for survival. We primarily responsible for maintaining survival. have found that dependence on anti- apoptotic proteins can be identified in cancer cells using a strategy that we call understanding how certain insults and be activated by many noxious stimuli – BH3 profiling (see below and Box 1). In derangements commit selected cells to including DNA damage, growth-factor cancer cells that are dependent on Bcl-2, death, but spare others. withdrawal and oncogene activation – via the Bcl-2 protein binds pro-apoptotic BH3- mechanisms that include transcriptional only proteins such as Bcl-2-interacting Members of the Bcl-2 family upregulation, subcellular localization and/or mediator of cell death (Bim). We describe The Bcl-2 family can be divided into pro- post-translational modifications. For such cells as being ‘primed for death’. apoptotic and anti-apoptotic proteins. These example, p53-upregulated modulator of Molecular therapies that are targeted to proteins contain one or more Bcl-2 apoptosis (Puma) and Noxa (the latin word anti-apoptotic proteins such as Bcl-2 can homology (BH) domains, which share for damage; also known as PMAIP1) are induce apoptosis in primed cancer cells; sequence homology and are important for transcriptionally upregulated by p53 in one such Bcl-2 antagonist, Abbott (ABT)- heterodimeric interactions among members response to DNA damage (Nakano and 737, has shown impressive success in of the Bcl-2 family (Chittenden et al., 1995; Vousden, 2001; Oda et al., 2000). In some killing leukemia and lymphoma cells Danial and Korsmeyer, 2004). Most anti- cells, Bim can be sequestered within the (Table 1). apoptotic proteins contain BH domains 1-4. cytoskeleton, to be released only in response Pro-apoptotic proteins can be divided into to certain death stimuli (Puthalakath et al., In this brief Cell Science at a Glance poster two groups according to function and the 1999). BH3-interacting-domain death article, we discuss how proteins of the number of BH domains possessed. Bax and agonist (Bid) is activated by cleavage by Bcl-2 family control the crucial event Bak are pro-apoptotic proteins that contain caspase 8 to form truncated Bid (tBID) (Li in the commitment to apoptosis – the BH domains 1-3 and are known as et al., 1998), whereas Bcl-2-associated permeabilization of the mitochondrial multidomain pro-apoptotic or effector death promoter (Bad) is activated by outer membrane. In addition, we discuss proteins. The remaining pro-apoptotic dephosphorylation (Zha et al., 1996). The recently developed methods for probing proteins contain only the third BH domain relatively numerous pro- and anti-apoptotic how cancer cells manipulate members of and are known as BH3-only proteins. BH3- members of this family engage in complex the Bcl-2 family to block apoptosis. only proteins act as upstream sentinels of interactions with each other to ultimately Understanding these principles is key to cellular damage and derangement. They can decide whether a cell will commit to death Table 1. Current clinical strategies to target proteins of the Bcl-2 family Drug Company Clinical phase Function Additional comments ABT-263 Abbott Laboratories Phase I and I/II clinical trials in NHL, BH3-mimetic small molecule ABT-263 is an orally available CLL and SCLC targeting Bcl-2, Bcl-XL and Bcl-w compound that is closely related to ABT-737 Obatoclax Gemin X Multiple phase I and II clinical trials in BH3-mimetic small molecule Might be a pan-inhibitor of anti- (GX15-070) hematological malignancies and non- apoptotic proteins small-cell lung cancer Oblimersen Genta Many clinical trials including phase III in Antisense DNA targeting Bcl-2 Clinical activity marginal, but not clear melanoma and CLL that drug is reducing Bcl-2 levels in vivo AT-101 Ascenta Therapeutics Phase II clinical trials in a variety of BH3-mimetic small molecule AT-101 is the negative enantiomer of cancers gossypol CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin's lymphoma; SCLC, small-cell lung cancer. Journal of Cell Science Journal of Cell Science 122 (4) 439 by controlling permeabilization of the and the direct models. According to the cannot activate Bax and Bak directly, but mitochondrial outer membrane. indirect model, Bax and Bak must always can bind anti-apoptotic proteins and cause be bound by anti-apoptotic proteins to the release of activator BH3-only proteins, Mitochondrial permeabilization prevent their activation. BH3-only proteins leading to activation of Bax and Bak. A Mitochondrial outer membrane permeabili - provoke death solely by binding to anti- prediction of this model is that the deletion zation (MOMP), which releases numerous apoptotic proteins, causing release and of all activators would result in a profound pro-apoptotic proteins into the cytosol, is the activation of Bax and Bak (Willis et al., block in apoptosis that is equivalent to the pivotal event in the intrinsic apoptotic 2007). A prediction of this model is that, for loss of Bax and Bak. By contrast, however, pathway. Bax and Bak double-knockout a cell to survive, all Bax and Bak proteins a combined knockout of Bid and Bim cells fail to undergo MOMP in response to must be bound to an anti-apoptotic protein results in relatively minor defects in many different death stimuli, including such as Bcl-2 and/or Mcl-1. However, co- apoptosis (Willis et al., 2007), but it is likely staurosporine, ultraviolet (UV) radiation, immunoprecipitation of anti-apoptotic that additional factors other than Bid and growth-factor deprivation, DNA damage proteins with Bax and Bak demonstrates Bim can act as activators. In fact, there are and endoplasmic reticulum stress (Wei that usually only a small minority of data that support the role of Puma, p53 and et al., 2001). Apoptosis that is caused by Bak is so bound. Furthermore, co- heat as activators of Bax and Bak (Chipuk BH3-only proteins absolutely requires Bax immunoprecipitation is often carried out in et al., 2004; Kim et al., 2006; Pagliari et al., and Bak (Kuwana et al., 2005; Lindsten the presence of detergents such as Triton 2005), and possibly others remain et al., 2000; Wei et al., 2000; Wei et al., X-100 or Tergitol-type nonyl undiscovered. The important point is that 2001). Bax proteins can be found as phenoxylpolyethoxylethanol-40 (NP-40). the activation of Bax and Bak might be monomers in the cytosol or loosely These detergents can artificially induce Bax affected by factors outside of the Bcl-2 associated with the outer mitochondrial and Bak conformations that mimic an family of proteins. Very recent results have membrane when not activated. Bax activated state and stimulate artifactual demonstrated a structure of a complex of the translocates to and inserts into the binding to anti-apoptotic proteins (Hsu and BH3 domain of Bim with Bax (Gavathiotis mitochondrial outer membrane during the Youle, 1997). This can result in an et al., 2008). Surprisingly, the interaction activation process (Billen et al., 2008; Hsu overestimation of the amount of Bax that is takes place on the Bax surface distal from et al., 1997; Wolter et al., 1997). Bak is actually bound to anti-apoptotic proteins. the hydrophobic pocket formed by the BH1, inserted into the outer mitochondrial Artificial activation of Bax and Bak does BH2 and BH3 domains. The analogous membrane even when not activated (Wei not occur in buffers that contain detergents pocket is used by anti-apoptotic proteins to et al., 2000). such as 3-[3-(Cholamidopropyl)dimethyl - bind BH3 domains. ammonio]-1-propanesulfonate (CHAPS). One of the steps that is involved in the However, it might be that Bax and Bak can The gulf between the two models is not activation of Bax and Bak is a alternatively interact with proteins that unbridgeable. As long as one is willing to conformational change that exposes the remove the requirement for sequestration by accept that there are activated subsets of N-terminus of the proteins, which is anti-apoptotic proteins (Cheng et al., 2003). Bax and Bak that are required to kill, and otherwise hidden in the inactive state More recent iterations of this model include that must be sequestered by anti-apoptotic (Yethon et al., 2003). This activated the concept that the sequestered forms of proteins to maintain survival, a unifying conformation can be recognized by Bax and Bak are those fractions of the model can be constructed. In this model, conformation-specific antibodies, such as total Bax and Bak population that are activated Bax and Bak are responsible for 6A7, which is specific for Bax (Hsu and already activated, perhaps spontaneously, or the permeabilization of membranes. They Youle, 1997). Following activation, Bax perhaps by other unspecified means achieve the activated state either by and Bak form homo-oligomers that can be (Fletcher et al., 2008). interacting with activator proteins, visualized via western blotting following by spontaneously activating, or via other chemical crosslinking (Wei et al., 2000). According to the direct model of Bax and unknown means. Anti-apoptotic proteins Bax and Bak oligomers participate in Bak activation, activator proteins (which inhibit death by sequestering activator forming pores in and cause permeabilization include Bid, Bim and possibly others such proteins or activated Bax and Bak. In of the outer mitochondrial membrane, as Puma and p53) directly interact with and addition to activating Bax and Bak (a leading to the release of the contents of induce conformational changes in Bax property possessed by only a subset of the mitochondrial intermembrane space, and Bak. Studies using full-length proteins, BH3-only proteins), BH3-only proteins including cytochrome c and Smac, into the lipid membranes and real-time fluorescence cause death by displacing activators and cytosol (Wang, 2001). These contents drive resonance energy transfer (FRET) analyses Bax and Bak from anti-apoptotic proteins, the activation of caspases, which are has provided convincing evidence of such permitting progression of the death signal. proteases that cleave and disable crucial an interaction and for its role in membrane This model is summarized in the poster proteins throughout the cell. permeabilization (Lovell et al., 2008). Anti- accompanying this article. apoptotic proteins prevent death by binding Bax and Bak activation and sequestering such pro-apoptotic Specificity in the interaction Given the lethal consequences of Bax and activator BH3-only proteins, and also by between anti-apoptotic and Bak activation, understanding how their binding any monomeric, activated Bax and BH3-only proteins activation is controlled is key to Bak proteins that might be present. In this Mammalian anti-apoptotic proteins understanding how a cell makes the model, BH3-only proteins are further include Bcl-2, Bcl-XL, Mcl-1, Bcl-w decision to undergo apoptosis. Two models divided into activator and sensitizer and Bfl-1. Anti-apoptotic proteins bind and of Bax and Bak activation exist: the indirect categories (Letai et al., 2002). Sensitizers sequester pro-apoptotic proteins, including Journal of Cell Science 440 Journal of Cell Science 122 (4) Fletcher, J. I., Meusburger, S., Hawkins, C. J., Riglar, activator BH3-only proteins and Bax and limited results in normal tissues suggests D. T., Lee, E. F., Fairlie, W. D., Huang, D. C. and Bak, to prevent apoptosis. Sensitizer that this is less likely to be the case in Adams, J. M. (2008). Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proc. Natl. proteins provoke apoptosis by normal tissues. A strategy of Bcl-2 Acad. Sci. USA 105, 18081-18087. competitively inhibiting this interaction. antagonism is being employed in clinical Gavathiotis, E., Suzuki, M., Davis, M. L., Pitter, K., Bird, G. H., Katz, S. G., Tu, H. C., Kim, H., Cheng, E. Each anti-apoptotic protein has its own trials of agents such as ABT-263, as some H., Tjandra, N. et al. (2008). BAX activation is initiated pattern of interaction with sensitizer cancers are dependent on Bcl-2 for survival at a novel interaction site. Nature 455, 1076-1081. proteins (Certo et al., 2006; Chen et al., (Oltersdorf et al., 2005; Tse et al., 2008). 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Are there other important activator to-membrane redistribution of Bax and Bcl-X(L) during characteristics. Mcl-1 ubiquitylation ligase apoptosis. Proc. Natl. Acad. Sci. USA 94, 3668-3672. and sensitizer proteins? Are there Kim, H., Rafiuddin-Shah, M., Tu, H. C., Jeffers, J. R., E3 (MULE) contains a BH3 domain that undiscovered mechanisms for holding Bax Zambetti, G. P., Hsieh, J. J. and Cheng, E. H. (2006). specifically binds Mcl-1, which leads to and Bak at bay? How do the noxious Hierarchical regulation of mitochondrion-dependent Mcl-1 ubiquitylation and degradation apoptosis by BCL-2 subfamilies. Nat. Cell Biol. 8, 1348- stimuli we routinely use to kill cells in (Zhong et al., 2005). This interaction, in culture and in vivo interact with proteins of Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P. and addition to the numerous PEST sequences Craig, R. W. (1993). 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Science Zha, J., Harada, H., Yang, E., Jockel, J. and slides. 292, 727-730. Korsmeyer, S. J. (1996). Serine phosphorylation of death Commentaries and Cell Science at a Glance JCS Commentaries highlight and critically discuss recent and exciting findings that will interest those who work in cell biology, molecular biology, genetics and related disciplines, whereas Cell Science at a Glance poster articles are short primers that act as an introduction to an area of cell biology, and include a large poster and accompanying text. Both of these article types, designed to appeal to specialists and nonspecialists alike, are commissioned from leading figures in the field and are subject to rigorous peer-review and in-house editorial appraisal. Each issue of the journal usually contains at least one of each article type. JCS thus provides readers with more than 50 topical pieces each year, which cover the complete spectrum of cell science. 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Published: Feb 15, 2009

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