STATE-OF-THE-ART REVIEW
BH3-only proteins: a 20-year stock-take Marcel Doerflinger, Jason A. Glab and Hamsa Puthalakath Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Australia
Keywords activation; activationapoptosis; Bak; Bax; Bcl-2 family; BH3-only proteins; disease; mimetics; oligomerization; therapeutics Correspondence H. Puthalakath, Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Kingsbury Drive, Melbourne 3086, Australia Fax: +61 3 94791266 Tel: +61 3 94795226 E-mail: [email protected]
BH3-only proteins are the sentinels of cellular stress, and their activation commits cells to apoptosis. Since the discovery of the first BH3-only protein BAD almost 20 years ago, at least seven more BH3-only proteins have been identified in mammals. They are regulated by a variety of environmental stimuli or by developmental cues, and play a crucial role in cellular homeostasis. Some are considered to be tumor suppressors, and also play a significant role in other pathologies. Their non-apoptotic functions are controversial, but there is broad consensus emerging regarding their role in apoptosis, which may help in designing better therapeutic agents for treating a variety of human diseases.
(Received 19 November 2014, revised 24 December 2014, accepted 2 January 2015) doi:10.1111/febs.13190
Introduction Bcl-2 family members are the arbiters of the mitochondrial apoptotic pathway, which are conserved through evolution. This family of proteins is characterized by the presence of Bcl-2 homology domains (BH domain), the number of which may vary among family members. The stoichiometry of pro- versus anti-apoptotic Bcl-2 family members in the cell determines whether the cell lives or dies. The seminal discovery of the role of the prototypic member of this family, Bcl-2, in cell survival in mammals [1] and subsequently in Caenorhabditis elegans [2] heralded a new era in understanding the role of the cell survival pathway in mammalian physiology and development. Knowledge of this family has grown steadily to encompass a wide range of proteins broadly classified as anti-apoptotic (Bcl-2), proapoptotic (BH3-only) and adaptor (Bax/Bak) proteins (Fig. 1).
The ‘BH3-only’ proteins are considered to be essential initiators of the mitochondrial apoptotic pathway [3]. Genetic experiments have shown that these proteins are essential initiators of programmed cell death in species as distantly related as mice and C. elegans. Various BH3-only proteins share a homologous BH3 region comprising nine amino acids. Mutational analyses have demonstrated that this domain is required for the ability of the proteins to bind to Bcl2-like pro-survival proteins and to initiate apoptosis. In contrast to lower forms of eukaryotes such as C. elegans (which has only one BH3-only protein), mammals have at least eight BH3-only proteins that differ in their expression patterns and mode of activation. Studies in gene-targeted mice have indicated that different BH3-only proteins are required for the initiation of programmed cell death via distinct apoptotic
Abbreviations Bak, Acl-2 homologous antagonist killer; Bax, Bcl-2 associated X protein; Bcl-2, B cell lymphoma protein 2; BH domain, Bcl-2 homology domain; Bid, BH3 interacting-domain death agonist; BIM, Bcl-2 inhibitory molecule; CED-3,4,9, Cell death proteins 3, 4 and 9; EGL-1, egg laying abnormal-1; PUMA, p53 upregulated modifier of apoptosis.
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Fig. 1. Domain organization of various Bcl2 family members. Bcl-2 homology domains (BH domains) and transmembrane domains (TM) are indicated.
stimuli. These studies also revealed that such proteins are spatially and temporarily regulated [4], thus playing an important role in the development of multicellular organisms such as mammals. Analysis of patient-derived tissues/samples also revealed their role in the onset and development of diseases such as cancer [5] and sepsis [6]. Understanding the structural basis of how BH3-only proteins induce apoptosis has greatly helped in designing novel therapeutic agents. In this review, we provide a brief snapshot of the present state of knowledge, including some controversial issues and some suggestions on the future research direction for BH3-only proteins. However, this review does not place much emphasis on proteins with BH3-like domains that possess death-inducing ability upon over-expression or that are able to interact with anti-apoptotic Bcl-2 family proteins. Relatively little is known about their physiological relevance in apoptosis signaling to date. These include p193 [7], MAP-1 [8]; Spike [9], SphK2 [10], BRCC-2 [11], TG2 [12], Mule/Arf-Bp1 [13], ApoL6 [14], ApoL1 [15], ErbB4/Her4 [16] and ErbB4/Her2 [17].
BH3-only proteins: some are more equal than others Pioneering studies of the C. elegans apoptosis pathway [18] elucidated a simple linear pathway to cell death (i.e. EGL-1 ? CED-9 ? CED-4 ? CED-3). Elaboration of this process in higher-order eukaryotes (i.e. mammals) signifies divergent evolution. The vestigial form of this pathway, as exists in C. elegans, retains most of the salient features of this pathway. In a healthy cell, CED-9 (the equivalent of Bcl-2 in mammals) remains bound to CED-4 (the equivalent of Apaf1 in mammals) at the mitochonFEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
dria, and thereby prevents it from activating the caspase CED-3; caspases are cystein aspartyl proteases that, upon activation, cleave various cellular substrates [19]. Cells destined to die produce excess EGL-1 (the only BH3-only protein in C. elegans), which binds to CED-9 and displaces CED-4; CED-4 then moves to the nuclear envelope, presumably in association with CED-3 [19,20]. Unlike their mammalian counterparts, these molecules also have additional functions. For example, CED-9 acts as a psuedosubstrate to regulate CED-3 caspase activity [21], as well as playing a role in mitochondrial integrity, similar to Bax in mammals. However, the gene flow during evolution not only ensured division of labor amongst Bcl-2 family members, but also led to diversification of each group of Bcl-2 family proteins. In higher-order eukaryotes, developmental processes and the various micro-environmental niches in which cell types exist made it imperative to possess a range of pro- and anti-apoptotic genes that are subjected to spatial and temporal regulation. Thus, in mammals, there are at least five anti-apoptotic proteins, three multi-domain ‘Bax-like’ proteins and approximately eight BH3-only proteins (Fig. 1). The BH3-only proteins are considered to be the sentinels of cellular well-being [3]. They trigger apoptosis either by directly activating Bax-like proteins or by neutralizing anti-apoptotic Bcl-2 proteins (discussed below). There are structural similarities between various anti-apoptotic molecules, including viral homologs [22], in which BH domains 1, 2 and 3 form a hydrophobic cleft to which BH3-only proteins bind [23]. This led to the suggestion that BH3-only proteins are promiscuous in their binding to antiapoptotic Bcl-2 proteins, and that signaling specificity arises from diverse modes of regulation such as tran-
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scriptional and post-translational modifications [3]. Once activated, the BH3-only proteins target all prosurvival family members. However, subsequent studies using affinity measurement clearly showed hierarchal and selective binding between various BH3-only proteins and anti-apoptotic Bcl-2 family proteins [24,25]. Bim and Puma appeared to be bind to all prosurvival family members with equal affinity, and therefore are promiscuous binders, whereas the other members displayed differential affinity (up to 1000fold) towards various pro-survival proteins (Fig. 2). For example, while Noxa did not bind to Bcl-2, BclXL or Bcl-W, it bound to Mcl-1 and A1 with nanomolar affinities [13]. Furthermore, in vivo knock-in mouse studies in which the Bim BH3 domain was replaced with that of BAD, Noxa or Puma did not result in polycystic kidney disease in the Bcl-2–/– background, underscoring the differential affinity paradigm of BH3-only proteins [26]. These differential affinities are reflected in the profound phenotypes observed in both Bim and Puma knockout mice compared to other knockout mice. This information is also invaluable in designing tailor-made BH3 mimetic drugs against cancers that over-express various anti-apoptotic Bcl-2 members.
Bax/Bak activation: ‘hit and run’ versus the antipodean view In response to increased steady-state levels of BH3-only proteins inside the cell, the multi-domain pro-apoptotic proteins Bax and Bak are activated, causing the release of pro-apoptogenic factors such as cytochrome c [27].
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How these inactive molecules transform into potent killers has been the subject of intense study. The original hypothesis was put forth by Korsmeyer’s group [28], and was called the ‘hit and run’ model, whereby tBid (a truncated form of Bid cleaved by caspase 8) acted as a membrane-targeting death ligand for Bak, leading to cytochrome c release and apoptosis (Fig. 3A). Although tBid appeared to be required for Bak oligomerization, no tBid was found in the complex, prompting the authors to propose the ‘hit and run’ model whereby tBid is no longer bound after inducing a conformational change in Bak. As the majority of this study was performed in isolated mitochondria, the in vivo relevance of this model is difficult to establish. An alternative model put forward by Kuwana et al. [29] suggested two non-mutually exclusive modes of Bax/Bak activation. In one scenario, proteins such as Bid and Bim (and even Puma) activate Bax/Bak molecules directly, whereas in the other scenario, all the other BH3-only proteins act as de-repressors. The de-repressors act by preventing the interaction between activators and anti-apoptotic Bcl-2 members, thereby freeing the activators to directly activate Bax/Bak molecules. Implicit in this model is the possibility that the de-repressors have higher affinities for anti-apoptotic molecules (in contrast to previous results [24]) and/or that the expression levels of this group of proteins are higher than those of the activators. Direct activation also predicts that there will be increased binding between Bax/Bak and the activators in dying cells, but this is not the case [30–32]. If it were true, mice/cells deficient in Bim, Bid and Puma would be phenotypically similar to Bax/Bak knockout cells.
Fig. 2. Some BH3-only proteins are more equal than others. A subset of BH3-only proteins show promiscuous high-affinity binding to antiapoptotic Bcl-2 members, and therefore activate Bax/Bak more readily and induce apoptosis.
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A
B Fig. 3. The two alternative models for Bax/Bak activation. In the direct activation/ ’hit and run’ model (A), some BH3-only proteins (Bad) act as sensitizers, displacing activators (tBid) from the Bcl-2 complex. The free Bid molecules activate Bax/Bak to form oligomers. In the indirect activation model (B), BH3-only proteins such as Bim displace Bax/Bak from the Bcl-2 complex and thus are free to oligomerize. The cartoon is with an apology to Mark Anderson.
Furthermore, Bax is prone to detergent-induced conformational changes that may not reflect the physiological state. In our view, work performed by Huang’s group [24,33] (referred to here as the ‘antipodean view’) proposed a more plausible alternative model (the indirect activation model) whereby Bax/Bak molecules are held in check by anti-apoptotic Bcl-2 family proteins, similar to the situation in C. elegans. BH3-only proteins are able to displace Bax/Bak molecules from this complex, leading to their activation and eventually apoptosis (Fig. 3B). This is consistent with the observation that proteins such as Bim and Puma are potent killers and show promiscuous highaffinity binding of anti-apoptotic Bcl-2 family proteins, whereas less potent killers bind only a selected subset of pro-survival proteins. In addition, degradation of Mcl-1 by Noxa also supports an indirect activation model [34]. However, this proposition also has some drawbacks. Indirect activation of Bak appears to be possible by sequestration by Mcl-1 and Bcl-XL [25], but this does not appear to be the case for Bax. While Bak exists constitutively on the membrane, Bax is predominantly cytosolic, requiring some form of activation before it re-localizes to the FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
membrane. Therefore, it may be assumed that while indirect activation is the most likely mode of activation for Bak, weighted evidence suggests that direct activation is the most likely scenario for Bax activation [28,35,36]. Resolving these issues, i.e. how BH3only proteins kill, is a key step towards tailoring BH3 mimetics-based therapeutic agents.
Non-apoptotic function of BH3-only proteins: facts and fiction Notwithstanding the conundrum described above, Bax/Bak activation is one of the central tenets of the mitochondrial apoptotic pathway. The role of BH3only proteins in this process is undisputed. However, recent literature has also reported non-apoptotic roles for BH3-only proteins (Table 1). Non-apoptotic roles for transcription factors such as p53 [37] are well documented; however, a similar role for BH3-only proteins, which are highly unstructured with the exception of Bid [38] (which has an ordered structure formed only within the BH3 domain upon interaction with an anti-apoptotic protein), appears to be highly controversial. A secondary role in non-apoptotic functions (i.e. by regulating the non-apoptotic function of their
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Table 1. Non-apoptotic functions of BH3-only proteins.
Cellular function Autophagy Glucose metabolism Lipid transport NF-jB activation/inflammation
BH3-only protein/mimetic Noxa, ABT737 Bad Bid Bid
DNA damage response
Bid
Unfolded protein response
Bim/Puma
References Elgendy et al. [40]; Lindqvist et al. [41] Danial et al. [42,43]; Szlyk et al. [44] Esposti et al. [45] Luo et al. [46]; Yeretssian et al. [51]; Nachbur et al. [52] Kamer et al. [47]; Liu et al. [48]; Kaufmann et al. [49] Rodriguez et al. [55]; Herold et al. [56]
anti-apoptotic family members) is within the realms of possibility. For example, BAD may regulate the cell cycle through heterodimerization with Bcl-2 or Bcl-XL [39]. Another case point in this context is the regulation of autophagy, which is widely believed to be regulated by Beclin-1, which is a mammalian ortholog of yeast Atg6 with a BH3 domain. However, conventional BH3-only proteins such as Noxa may have an effect on autophagy by negating anti-apoptotic protein Mcl-1 [40]. Lindqvist et al. [41] recently reinforced this possibility by reporting that autophagy in mammalian cells is regulated by Bcl-2 family members in much the same way as they regulate apoptosis. Here, BH3-only proteins play a pivotal role in Bax/Bak activation, and the role for BH3-only proteins in autophagy is indirect. Here we discuss the literature that implies a direct role for BH3-only proteins in non-apoptotic cellular functions. One of the most compelling cases for the direct involvement of BH3-only proteins in non-apoptotic functions has been made by Korsmeyer’s group [42]. Their pioneering work demonstrated how BAD regulates glucokinase activity, respiration and ATP production, as well acting as a sentinel to sense glucose levels in pancreatic islets. Subsequent studies using mouse genetic models also demonstrated the therapeutic potential of BAD BH3 mimetics in restoring b cell function [43]. Furthermore, structural studies [44] also revealed that the BAD BH3 phosphomimetic binds a previously undescribed region near the enzyme’s active site without affecting allosteric regulation of the enzyme. This may pave the way for a new generation of glucokinase activators for treating type 2 diabetes. The possibility of a non-apoptotic role for Bid is highly controversial; one of the earliest reports suggested a role in lipid transport [45]. Bid was shown to 1010
have inherent lipid transferase activity, leading to augmented lipid transfer from ER membrane to the mitochondria. A potential caveat with respect to this study is that most of the experiments were performed using either isolated mitochondrial/ER membranes or liposomes, and the relevance of this study in vivo has yet to be established. Moreover, an altered lipid profile has not been reported in Bid knockout mouse organs. Bid has also been reported to contribute to mitochondrial generation of reactive oxygen species, which in turn leads to activation of the nuclear factor NF-jB upon exposure to a low dose of the carcinogen 5MCDE (5- methylchrysene-1,2-diol-3,4-epoxide). This has been attributed to its ‘anti-apoptotic’ activity [46]. Apart from being the antithesis to the common belief regarding the normal function of BH3-only proteins, the main problem with this proposition is that the low-dose carcinogen treatment did not lead to Bid activation, therefore could not have translocated to mitochondria. However, Bid was able to regulate mitochondrial generation of reactive oxygen species and therefore cellular survival. Bid has also been reported to play a role in the DNA damage response [47]. Bid was found to be phosphorylated by Atm kinase (at S61 and S78 in both mouse and human forms), and its role in cellcycle entry is dependent on these phosphorylation events. Accordingly, the topoisomerase II poison etoposide failed to induce S-phase blockage in Bid / cells compared to wild-type cells. A subsequent study [48] found that Bid functions at the level of the sensor complex in the DNA damage response directed by Atm and Rad3-related (Atr). Bid-deficient cells showed reduced accumulation of Atr and Atr-interacting protein (Atrip) on chromatin and at DNA damage foci. Thus, this study provided a possible mechanism for the role of Bid in the DNA damage response. However, Kaufman et al. [49] found no such link between Bid and the DNA damage response. Nine distinct cell types from Bid-deficient mice underwent cellcycle arrest and apoptosis in a manner indistinguishable from control wild-type cells in response to DNA damage or replicative stress. The mice did not show any increased risk of myeloid leukemia whereas the original study had reported an increased risk [47]. The anomalies in these studies were attributed to the mode of immortalization of the cell lines generated and the cell-cycle phase of the primary cells used [50]. However, the fact that consistent results were obtained from nine different cell types [49] is intriguing. Furthermore, the ATM-mediated phosphorylation at S61 and S78 suggests the interaction may not be mediated by the BH3 domain (residues 90–98). Understanding FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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the nuclear import dynamics of Bid, further analysis of the nuclear interaction partner(s), and understanding the structural basis of these interactions as for the BAD interaction with glucokinase [44] would help to resolve these discrepancies. In yet another context, a genome-wide RNA interference screen identified Bid as a regulator of the NOD1-mediated inflammatory response [51]. In this study, colonocytes lacking Bid or macrophages from Bid / mice were compromised in terms of their cytokine secretion in response to activators of nucleotidebinding and oligomerization domain (NOD) proteins. BID appeared to interact with NOD1, NOD2 and the IjB kinase complex, thus regulating the NF-jB pathway in cytokine secretion. Bid cleavage and the BH3 domain were redundant in this activation pathway, signifying that this process is independent of apoptosis. In the dextran-sulfate sodium-induced colitis model, the NOD agonist muramyl dipeptide afforded protection and prevented weight loss in wild-type mice, whereas Bid / mice exhibited both weight loss and crypt loss in the colon. This was attributed to lack of NOD-dependent cytokine signaling and the tissue repair response in Bid / mice. Intriguingly, using the same strain of mice, Nachbur et al. [52] found no such relationship between Bid and the NOD-dependent inflammatory response and cytokine secretion. The reasons for the discrepancies in these observations have not yet been determined. The unfolded protein response (UPR) is another scenario in which BH3-only proteins are known to play an important role. Although most studies suggest that BH3-only proteins such as Bim and Puma are induced in response to the UPR [53,54], leading to the mitochondrial apoptotic response, a recent study by Rodriguez et al. [55] reported a role for Bim and Puma in the initiation phase of the UPR. Both Bim and Puma appeared to bind the ER kinase/nuclease IRE1a in a BH3 domain-dependent manner, thus regulating Xbp-1 splicing and cytokine secretion in lipopolysaccharide-stimulated B cells. However, a follow-up study [56] found no such relationship between Bim or Puma and Xbp-1 splicing. Lipopolysaccharide-induced cytokine secretion from B cells from Bim / and Puma / mice was similar to that from wild-type B cells. This study was consistent with the original finding that Bim / mice have abnormally increased serum levels of IgM and IgG due to increased accumulation of antibody-secreting plasma cells; Xbp-1 splicing is absolutely necessary for plasma cell differentiation [57]. Furthermore, the reported interaction between Bim, Puma and IRE1a was BH3 domain-dependent [55], and this interaction FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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was augmented by the presence of Bcl-2, suggesting that a single BH3 domain may engage two proteins simultaneously. In light of these observations, and based on the well-established essential roles of Bim and Puma in initiation of ER stress-induced apoptosis [53,54], it may be concluded that these BH3-only proteins function exclusively downstream but not upstream in the UPR.
BH3-only proteins in therapeutics: not all roads lead to perdition Bcl-2 family proteins are implicated in a variety of pathophysiologies associated with altered cellular homeostasis. The prototypic member of the family, Bcl-2, was first identified in B-cell lymphoma [1]. Since then, pro-survival Bcl-2 family proteins have been implicated in a variety of cancers, including chronic lymphocytic leukemia, acute lymphoblastic leukemia, small cell lung cancer, prostate cancer and non-Hodgkin’s lymphoma. Similarly, Mcl-1 expression is associated with multiple myeloma, chronic myeloid leukemia, pancreatic cancer and melanoma. High levels of expression of pro-survival proteins are often associated with increased chemoresistance, and are therefore attractive targets for cancer therapy. One of the earliest anti-Bcl-2 drugs was an antisense RNAbased 18-base phosphorothioate oligonucleotide that was complementary to the first six codons of bcl-2 mRNA [58]. Initial in vitro studies using patientderived tumor cell lines and in vivo pre-clinical trials using mouse models offered promising results, leading to phase I/II clinical trials under the trade name Oblimersen (Genta Inc., Berkeley Heights, NJ, USA). However, mixed results, particularly failure in a melanoma trial, led to its non-approval by the US Food and Drug Administration. RNA oligonucleotide-based drugs are extremely sequence-specific for the target molecule, and therefore expected to be have limited spectrum of activity. In addition, their phosphorothioate backbone shows nonspecific binding to proteins such as fibronectin, fibroblast growth factor and human immunodeficiency virus reverse transcriptase etc, giving rise to misleading results [59]. The new-generation drugs termed ‘BH3 mimetics’ that were developed based on the interaction between BH3-only proteins and their anti-apoptotic counterparts offer great promise in treating neoplastic disorders [60]. These small molecules mimic the BH3 domain of BH3-only proteins, are selective towards anti-apoptotic Bcl-2 family members, and target multiple members of the family. The prototype BH3 mimetic ABT737, developed by Abbott Laboratories
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Table 2. Dysregulated BH3-only proteins as potential therapeutic targets in diseases. Disease Reduced function Various cancers Autoimmune diseases Rheumatoid arthritis Excessive function Cardiomyopathy Sepsis (and other severe infections, e.g. Ebola, anthrax) Neurodegenerative diseases Diabetes Immunedeficiency after chemo- and radiotherapy
(North Chicago, IL, USA), was based on the BAD BH3 domain and was able to bind multiple Bcl-2 family members including Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1 [61]. However, initial studies showed that this drug led to thrombocytopenia owing to the death of platelets mediated by Bcl-XL inhibition. A new derivative of this drug (ABT199) has high specificity towards Bcl-2, with greatly reduced binding of BclXL, and thus has anti-tumor activity while sparing platelets [62]. The fact that these drugs mostly target cancers with a high level of Bcl-2 expression, whereas in most malignancies Mcl-1 over expression leads to drug resistance. This has led to the development of BH3 mimetics specifically targeting Mcl-1. These drugs are at various stages of clinical assessment, and a comprehensive review of these various drugs has been published recently [63]. Inducing cell death through use of a BH3-only protein or its derivatives as a form of treatment has wider potential applications than just oncology. There are other pathophysiologies in which BH3 mimetics may be useful. For example, increased eosinophil survival (due to the presence of pro-survival Bcl-2 family proteins) is correlated with disease severity in mouse models and in childhood acute asthma [64,65]. However, as cancer is a high-profile disease, due to the potential market size and the efforts of high-profile cancer survivors and effective lobby groups, the BH3-only research is significantly skewed towards eliciting an apoptotic response in cancer cells. However, the economic and health impacts of cardiovascular diseases and neurodegenerative disorders are equally significant as those of cancer. Sepsis alone kills more people than breast cancer, prostate cancer and HIV/AIDS combined. An increased rate of apoptosis by BH3-only proteins is implicated in all these morbidities (Table 2) [66–68]. Understanding the molecular pathways involved in upregulation of BH3-only protein expression [67] will 1012
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References
Bim, Puma, Noxa, Bmf, Bad Bim, Bmf Bim, Bid
Happo et al. [70]
Bim, Bnip3 Bim, Bid, Puma
Lee et al. [67]; Diwan et al. [74] Chang et al. [68]; Weber et al. [6]; Parrino et al. [75] Ghosh et al. [76] Wali et al. [77] Villunger et al. [78], [79]
Bim, Puma Bim, Puma Bim, Puma, Noxa
Bouillet et al. [57]; Labi et al. [71] Scatizzi et al. [72]; Scatizzi et al. [73]
undoubtedly result in development of therapeutic agents targeting these diseases in which preventing apoptosis is a desirable outcome.
Conclusion and perspectives Since the discovery of the first BH3-only protein nearly 20 years ago [69], great strides have been made in understanding their structure, function, functional interactions and role in pathogenesis, which has eventually led to the development of novel therapeutic agents. We also know that each member has differential affinity for their anti-apoptotic counterparts, and this has helped to design tailor-made therapeutic agents that offer great promise in countering chemoresistance in cancer therapy. Notwithstanding these observations, there is a huge gap in our understanding of BH3 biology. While there is consensus among groups reharding the differential affinity of BH3-only proteins towards their anti-apoptotic counterparts, there is some debate on their role in activating Bax/ Bak adaptor molecules. In this review, we have provided a balanced view of both models, and in our opinion, both modes of activation may exist in a cell type- or stimulus-dependent manner. There appears to be a great deal of confusion regarding the role of BH3-only proteins in non-apoptotic function, especially with respect to their role in the DNA damage response, inflammation and initiation of the UPR. Only by identifying the key molecules with which BH3-only proteins interact in these pathways and analyzing these interactions at structural level will these issues be resolved. Finally, in our view, a greater research emphasis should also be placed on harnessing the benefits of our knowledge on regulation of BH3-only proteins so that therapeutic agents may be developed to treat diseases such as neurodegenerative disorders, diabetes, cardiomyopathy and sepsis. FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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Acknowledgements We would like to thank all the H.P. lab members for their support and helpful discussions. We also would like to thank Andrew Coley, Department of Biochemistry, La Trobe University, Melbourne, Australia for reviewing this manuscript. H.P. is supported by an Australian Research Council Future Fellowship (FT0990683) and by an Australian Research Council project grant (DP110100417).
Author contribution HP: Discussed the subject matter with MD and JAG and wrote the manuscript and drew some of the figures. MD: Wrote parts of the manuscript. JAG: Created the cartoons.
References 1 Vaux DL, Cory S & Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440–442. 2 Vaux DL, Weissman IL & Kim SK (1992) Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955–1957. 3 Puthalakath H & Strasser A (2002) Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ 9, 505–512. 4 Wong WW & Puthalakath H (2008) Bcl-2 family proteins: the sentinels of the mitochondrial apoptosis pathway. IUBMB Life 60, 390–397. 5 Ng KP, Hillmer AM, Chuah CT, Juan WC, Ko TK, Teo AS, Ariyaratne PN, Takahashi N, Sawada K, Fei Y et al. (2012) A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nat Med 18, 521–528. 6 Weber SU, Schewe JC, Lehmann LE, Muller S, Book M, Klaschik S, Hoeft A & Stuber F (2008) Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis. Crit Care 12, R128. 7 Tsai SC, Pasumarthi KB, Pajak L, Franklin M, Patton B, Wang H, Henzel WJ, Stults JT & Field LJ (2000) Simian virus 40 large T antigen binds a novel Bcl-2 homology domain 3-containing proapoptosis protein in the cytoplasm. J Biol Chem 275, 3239–3246. 8 Tan KO, Tan KM, Chan SL, Yee KS, Bevort M, Ang KC & Yu VC (2001) MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains. J Biol Chem 276, 2802–2807.
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9 Mund T, Gewies A, Schoenfeld N, Bauer MK & Grimm S (2003) Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum. FASEB J 17, 696–698. 10 Liu H, Toman RE, Goparaju SK, Maceyka M, Nava VE, Sankala H, Payne SG, Bektas M, Ishii I, Chun J et al. (2003) Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis. J Biol Chem 278, 40330–40336. 11 Broustas CG, Gokhale PC, Rahman A, Dritschilo A, Ahmad I & Kasid U (2004) BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner. J Biol Chem 279, 26780–26788. 12 Rodolfo C, Mormone E, Matarrese P, Ciccosanti F, Farrace MG, Garofano E, Piredda L, Fimia GM, Malorni W & Piacentini M (2004) Tissue transglutaminase is a multifunctional BH3-only protein. J Biol Chem 279, 54783–54792. 13 Zhong Q, Gao W, Du F & Wang X (2005) Mule/ARFBP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell 121, 1085–1095. 14 Liu Z, Lu H, Jiang Z, Pastuszyn A & Hu CA (2005) Apolipoprotein l6, a novel proapoptotic Bcl-2 homology 3-only protein, induces mitochondriamediated apoptosis in cancer cells. Mol Cancer Res 3, 21–31. 15 Wan G, Zhaorigetu S, Liu Z, Kaini R, Jiang Z & Hu CA (2008) Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. J Biol Chem 283, 21540–21549. 16 Naresh A, Long W, Vidal GA, Wimley WC, Marrero L, Sartor CI, Tovey S, Cooke TG, Bartlett JM & Jones FE (2006) The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells. Cancer Res 66, 6412–6420. 17 Strohecker AM, Yehiely F, Chen F & Cryns VL (2008) Caspase cleavage of HER-2 releases a Bad-like cell death effector. J Biol Chem 283, 18269–18282. 18 Horvitz HR (1999) Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res 59, 1701s–1706s. 19 Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y & Horvitz HR (2000) Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287, 1485–1489. 20 Conradt B & Horvitz HR (1998) The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl–2-like protein CED-9. Cell 93, 519–529. 21 Xue D & Horvitz HR (1997) Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature 390, 305–308.
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22 Huang Q, Petros AM, Virgin HW, Fesik SW & Olejniczak ET (2002) Solution structure of a Bcl-2 homolog from Kaposi sarcoma virus. Proc Natl Acad Sci USA 99, 3428–3433. 23 Kelekar A & Thompson CB (1998) Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 8, 324–330. 24 Chen LWS, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL, Adams JM & Huang DC (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17, 393–403. 25 Willis SNCL, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM & Huang DC (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19, 1294–1305. 26 Merino D, Giam M, Hughes PD, Siggs OM, Heger K, O’Reilly LA, Adams JM, Strasser A, Lee EF, Fairlie WD et al. (2009) The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins. J Cell Biol 186, 355–362. 27 Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D & Reed JC (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95, 4997–5002. 28 Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB & Korsmeyer SJ (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14, 2060–2071. 29 Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR & Newmeyer DD (2005) BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 17, 525–535. 30 Nechushtan A, Smith CL, Lamensdorf I, Yoon SH & Youle RJ (2001) Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 153, 1265–1276. 31 Antonsson B, Montessuit S, Sanchez B & Martinou JC (2001) Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276, 11615–11623. 32 Zhu Y, Swanson BJ, Wang M, Hildeman DA, Schaefer BC, Liu X, Suzuki H, Mihara K, Kappler J & Marrack P (2004) Constitutive association of the proapoptotic protein Bim with Bcl-2-related proteins on mitochondria in T cells. Proc Natl Acad Sci USA 101, 7681–7686. 33 Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE, Ierino H, Lee EF, Fairlie WD, Bouillet P et al. (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856–859.
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34 Czabotar PE, Lee EF, van Delft MF, Day CL, Smith BJ, Huang DC, Fairlie WD, Hinds MG & Colman PM (2007) Structural insights into the degradation of Mcl-1 induced by BH3 domains. Proc Natl Acad Sci USA 104, 6217–6222. 35 Wang K, Yin XM, Chao DT, Milliman CL & Korsmeyer SJ (1996) BID: a novel BH3 domain-only death agonist. Genes Dev 10, 2859–2869. 36 Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B & Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144, 891–901. 37 Montero J, Dutta C, van Bodegom D, Weinstock D & Letai A (2013) p53 regulates a non-apoptotic death induced by ROS. Cell Death Differ 20, 1465–1474. 38 Hinds MG, Smits C, Fredericks-Short R, Risk JM, Bailey M, Huang DC & Day CL (2007) Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death Differ 14, 128–136. 39 Chattopadhyay A, Chiang CW & Yang E (2001) BAD/BCL-[X(L)] heterodimerization leads to bypass of G0/G1 arrest. Oncogene 20, 4507–4518. 40 Elgendy M, Sheridan C, Brumatti G & Martin SJ (2011) Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol Cell 42, 23–35. 41 Lindqvist LM, Heinlein M, Huang DC & Vaux DL (2014) Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc Natl Acad Sci USA 111, 8512–8517. 42 Danial NN, Gramm CF, Scorrano L, Zhang CY, Krauss S, Ranger AM, Datta SR, Greenberg ME, Licklider LJ, Lowell BB et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952–956. 43 Danial NN, Walensky LD, Zhang CY, Choi CS, Fisher JK, Molina AJ, Datta SR, Pitter KL, Bird GH, Wikstrom JD et al. (2008) Dual role of proapoptotic BAD in insulin secretion and beta cell survival. Nat Med 14, 144–153. 44 Szlyk B, Braun CR, Ljubicic S, Patton E, Bird GH, Osundiji MA, Matschinsky FM, Walensky LD & Danial NN (2014) A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators. Nat Struct Mol Biol 21, 36–42. 45 Esposti MD, Erler JT, Hickman JA & Dive C (2001) Bid, a widely expressed proapoptotic protein of the Bcl2 family, displays lipid transfer activity. Mol Cell Biol 21, 7268–7276.
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46 Luo W, Li J, Zhang D, Cai T, Song L, Yin XM, Desai D, Amin S, Chen J & Huang C (2010) Bid mediates anti-apoptotic COX-2 induction through the IKKbeta/NFkappaB pathway due to 5-MCDE exposure. Curr Cancer Drug Targets 10, 96–106. 47 Kamer I, Sarig R, Zaltsman Y, Niv H, Oberkovitz G, Regev L, Haimovich G, Lerenthal Y, Marcellus RC & Gross A (2005) Proapoptotic BID is an ATM effector in the DNA-damage response. Cell 122, 593–603. 48 Liu Y, Bertram CC, Shi Q & Zinkel SS (2011) Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress. Cell Death Differ 18, 841–852. 49 Kaufmann T, Tai L, Ekert PG, Huang DC, Norris F, Lindemann RK, Johnstone RW, Dixit VM & Strasser A (2007) The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest. Cell 129, 423–433. 50 Zinkel SS, Hurov KE & Gross A (2007) Bid plays a role in the DNA damage response. Cell 130, 9–10. 51 Yeretssian G, Correa RG, Doiron K, Fitzgerald P, Dillon CP, Green DR, Reed JC & Saleh M (2011) Non-apoptotic role of BID in inflammation and innate immunity. Nature 474, 96–99. 52 Nachbur U, Vince JE, O’Reilly LA, Strasser A & Silke J (2012) Is BID required for NOD signalling? Nature 488, E4–6; discussion E6-8. 53 Puthalakath H, O’Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, Hughes PD, Michalak EM, McKimm-Breschkin J, Motoyama N et al. (2007) ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129, 1337–1349. 54 Reimertz C, Kogel D, Rami A, Chittenden T & Prehn JH (2003) Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway. J Cell Biol 162, 587–597. 55 Rodriguez DA, Zamorano S, Lisbona F, Rojas-Rivera D, Urra H, Cubillos-Ruiz JR, Armisen R, Henriquez DR, Cheng EH, Letek M et al. (2012) BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1alpha. EMBO J 31, 2322–2335. 56 Herold MJ, O’Reilly LA, Lin A, Srivastava R, Doerflinger M, Bouillet P, Strasser A & Puthalakath H (2014) Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA. Cell Death Dis 5, e1354. 57 Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM & Strasser A (1999) Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735–1738.
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58 Kitada S, Miyashita T, Tanaka S & Reed JC (1993) Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 3, 157–169. 59 Dias N & Stein CA (2002) Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl2 antisense oligonucleotides. Eur J Pharm Biopharm 54, 263–269. 60 Kang MH & Reynolds CP (2009) Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res 15, 1126–1132. 61 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ et al. (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681. 62 Touzeau C, Dousset C, Le Gouill S, Sampath D, Leverson JD, Souers AJ, Maiga S, Bene MC, Moreau P, Pellat-Deceunynck C et al. (2014) The Bcl2 specific BH3 mimetic ABT-199: a promising targeted therapy for t(11;14) multiple myeloma. Leukemia 28, 210–212. 63 Billard C (2013) BH3 mimetics: status of the field and new developments. Mol Cancer Ther 12, 1691–1700. 64 Tumes DJ, Wong AC, Sewell WA, McColl SR, Connolly A & Dent LA (2008) Differential rates of apoptosis and recruitment limit eosinophil accumulation in the lungs of asthma-resistant CBA/Ca mice. Mol Immunol 45, 3609–3617. 65 El-Gamal Y, Heshmat N, Mahran M & El-Gabbas Z (2004) Expression of the apoptosis inhibitor Bcl-2 in sputum eosinophils from children with acute asthma. Clin Exp Allergy 34, 1701–1706. 66 Akhter R, Sanphui P & Biswas SC (2014) The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in beta-amyloid-induced neuron death. J Biol Chem 289, 10812–10822. 67 Lee YY, Moujalled D, Doerflinger M, Gangoda L, Weston R, Rahimi A, de Alboran I, Herold M, Bouillet P, Xu Q et al. (2013) CREB-binding protein (CBP) regulates beta-adrenoceptor (beta-AR)-mediated apoptosis. Cell Death Differ 20, 941–952. 68 Chang KC, Unsinger J, Davis CG, Schwulst SJ, Muenzer JT, Strasser A & Hotchkiss RS (2007) Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis. FASEB J 21, 708–719. 69 Yang E, Zha J, Jockel J, Boise LH, Thompson CB & Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80, 285–291. 70 Happo L, Strasser A & Cory S (2012) BH3-only proteins in apoptosis at a glance. J Cell Sci 125, 1081–1087. 71 Labi V, Woess C, Tuzlak S, Erlacher M, Bouillet P, Strasser A, Tzankov A, Villunger A (2014) Deregulated
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cell death and lymphocyte homeostasis cause premature lethality in mice lacking BH3 only proteins Bim and Bmf. Blood 123, 2652–2662. Scatizzi JC, Bickel E, Hutcheson J, Haines GK & Perlman H (2006) Bim deficiency leads to exacerbation and prolongation of joint inflammation in experimental arthritis. Arthritis Rheum 54, 3182–3193. Scatizzi JC, Hutcheson J, Bickel E, Haines GK & Perlman H (2007) Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis. Arthritis Res Ther 9, R49. Diwan A, Krenz M, Syed FM, Wansapura J, Ren X, Koesters AG, Li H, Kirshenbaum LA, Hahn HS, Robbins J (2007) Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice. J Clin Investig 117, 2825–2833. Parrino J, Hotchkiss RS & Bray M (2007) Prevention of immune cell apoptosis as potential therapeutic strategy for severe infections. Emerg Infect Dis 13, 191–198. Ghosh AP, Klocke BJ, Ballestas ME & Roth KA (2012) CHOP potentially co-operates with FOXO3a in neuronal cells to regulate PUMA and BIM expression in response to ER stress. PLoS One 7, e39568.
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77 Wali JA, Rondas D, McKenzie MD, Zhao Y, Elkerbout L, Fynch S, Gurzov EN, Akira S, Mathieu C, Kay TM (2014) The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity. Cell Death Dis 5, e1124. 78 Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM & Strasser A (2003) p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036–1038. 79 Villunger A, Scott C, Bouillet P & Strasser A (2003) Essential role for the BH3-only protein Bim but redundant roles for Bax, Bcl-2, and Bcl-w in the control of granulocyte survival. Blood 101, 2393–2400.
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FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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BH3-only proteins: a 20-year stock-take Marcel Doerflinger, Jason A. Glab and Hamsa Puthalakath Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Melbourne, Australia
Keywords activation; activationapoptosis; Bak; Bax; Bcl-2 family; BH3-only proteins; disease; mimetics; oligomerization; therapeutics Correspondence H. Puthalakath, Department of Biochemistry, La Trobe Institute of Molecular Science, La Trobe University, Kingsbury Drive, Melbourne 3086, Australia Fax: +61 3 94791266 Tel: +61 3 94795226 E-mail: [email protected]
BH3-only proteins are the sentinels of cellular stress, and their activation commits cells to apoptosis. Since the discovery of the first BH3-only protein BAD almost 20 years ago, at least seven more BH3-only proteins have been identified in mammals. They are regulated by a variety of environmental stimuli or by developmental cues, and play a crucial role in cellular homeostasis. Some are considered to be tumor suppressors, and also play a significant role in other pathologies. Their non-apoptotic functions are controversial, but there is broad consensus emerging regarding their role in apoptosis, which may help in designing better therapeutic agents for treating a variety of human diseases.
(Received 19 November 2014, revised 24 December 2014, accepted 2 January 2015) doi:10.1111/febs.13190
Introduction Bcl-2 family members are the arbiters of the mitochondrial apoptotic pathway, which are conserved through evolution. This family of proteins is characterized by the presence of Bcl-2 homology domains (BH domain), the number of which may vary among family members. The stoichiometry of pro- versus anti-apoptotic Bcl-2 family members in the cell determines whether the cell lives or dies. The seminal discovery of the role of the prototypic member of this family, Bcl-2, in cell survival in mammals [1] and subsequently in Caenorhabditis elegans [2] heralded a new era in understanding the role of the cell survival pathway in mammalian physiology and development. Knowledge of this family has grown steadily to encompass a wide range of proteins broadly classified as anti-apoptotic (Bcl-2), proapoptotic (BH3-only) and adaptor (Bax/Bak) proteins (Fig. 1).
The ‘BH3-only’ proteins are considered to be essential initiators of the mitochondrial apoptotic pathway [3]. Genetic experiments have shown that these proteins are essential initiators of programmed cell death in species as distantly related as mice and C. elegans. Various BH3-only proteins share a homologous BH3 region comprising nine amino acids. Mutational analyses have demonstrated that this domain is required for the ability of the proteins to bind to Bcl2-like pro-survival proteins and to initiate apoptosis. In contrast to lower forms of eukaryotes such as C. elegans (which has only one BH3-only protein), mammals have at least eight BH3-only proteins that differ in their expression patterns and mode of activation. Studies in gene-targeted mice have indicated that different BH3-only proteins are required for the initiation of programmed cell death via distinct apoptotic
Abbreviations Bak, Acl-2 homologous antagonist killer; Bax, Bcl-2 associated X protein; Bcl-2, B cell lymphoma protein 2; BH domain, Bcl-2 homology domain; Bid, BH3 interacting-domain death agonist; BIM, Bcl-2 inhibitory molecule; CED-3,4,9, Cell death proteins 3, 4 and 9; EGL-1, egg laying abnormal-1; PUMA, p53 upregulated modifier of apoptosis.
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Fig. 1. Domain organization of various Bcl2 family members. Bcl-2 homology domains (BH domains) and transmembrane domains (TM) are indicated.
stimuli. These studies also revealed that such proteins are spatially and temporarily regulated [4], thus playing an important role in the development of multicellular organisms such as mammals. Analysis of patient-derived tissues/samples also revealed their role in the onset and development of diseases such as cancer [5] and sepsis [6]. Understanding the structural basis of how BH3-only proteins induce apoptosis has greatly helped in designing novel therapeutic agents. In this review, we provide a brief snapshot of the present state of knowledge, including some controversial issues and some suggestions on the future research direction for BH3-only proteins. However, this review does not place much emphasis on proteins with BH3-like domains that possess death-inducing ability upon over-expression or that are able to interact with anti-apoptotic Bcl-2 family proteins. Relatively little is known about their physiological relevance in apoptosis signaling to date. These include p193 [7], MAP-1 [8]; Spike [9], SphK2 [10], BRCC-2 [11], TG2 [12], Mule/Arf-Bp1 [13], ApoL6 [14], ApoL1 [15], ErbB4/Her4 [16] and ErbB4/Her2 [17].
BH3-only proteins: some are more equal than others Pioneering studies of the C. elegans apoptosis pathway [18] elucidated a simple linear pathway to cell death (i.e. EGL-1 ? CED-9 ? CED-4 ? CED-3). Elaboration of this process in higher-order eukaryotes (i.e. mammals) signifies divergent evolution. The vestigial form of this pathway, as exists in C. elegans, retains most of the salient features of this pathway. In a healthy cell, CED-9 (the equivalent of Bcl-2 in mammals) remains bound to CED-4 (the equivalent of Apaf1 in mammals) at the mitochonFEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
dria, and thereby prevents it from activating the caspase CED-3; caspases are cystein aspartyl proteases that, upon activation, cleave various cellular substrates [19]. Cells destined to die produce excess EGL-1 (the only BH3-only protein in C. elegans), which binds to CED-9 and displaces CED-4; CED-4 then moves to the nuclear envelope, presumably in association with CED-3 [19,20]. Unlike their mammalian counterparts, these molecules also have additional functions. For example, CED-9 acts as a psuedosubstrate to regulate CED-3 caspase activity [21], as well as playing a role in mitochondrial integrity, similar to Bax in mammals. However, the gene flow during evolution not only ensured division of labor amongst Bcl-2 family members, but also led to diversification of each group of Bcl-2 family proteins. In higher-order eukaryotes, developmental processes and the various micro-environmental niches in which cell types exist made it imperative to possess a range of pro- and anti-apoptotic genes that are subjected to spatial and temporal regulation. Thus, in mammals, there are at least five anti-apoptotic proteins, three multi-domain ‘Bax-like’ proteins and approximately eight BH3-only proteins (Fig. 1). The BH3-only proteins are considered to be the sentinels of cellular well-being [3]. They trigger apoptosis either by directly activating Bax-like proteins or by neutralizing anti-apoptotic Bcl-2 proteins (discussed below). There are structural similarities between various anti-apoptotic molecules, including viral homologs [22], in which BH domains 1, 2 and 3 form a hydrophobic cleft to which BH3-only proteins bind [23]. This led to the suggestion that BH3-only proteins are promiscuous in their binding to antiapoptotic Bcl-2 proteins, and that signaling specificity arises from diverse modes of regulation such as tran-
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scriptional and post-translational modifications [3]. Once activated, the BH3-only proteins target all prosurvival family members. However, subsequent studies using affinity measurement clearly showed hierarchal and selective binding between various BH3-only proteins and anti-apoptotic Bcl-2 family proteins [24,25]. Bim and Puma appeared to be bind to all prosurvival family members with equal affinity, and therefore are promiscuous binders, whereas the other members displayed differential affinity (up to 1000fold) towards various pro-survival proteins (Fig. 2). For example, while Noxa did not bind to Bcl-2, BclXL or Bcl-W, it bound to Mcl-1 and A1 with nanomolar affinities [13]. Furthermore, in vivo knock-in mouse studies in which the Bim BH3 domain was replaced with that of BAD, Noxa or Puma did not result in polycystic kidney disease in the Bcl-2–/– background, underscoring the differential affinity paradigm of BH3-only proteins [26]. These differential affinities are reflected in the profound phenotypes observed in both Bim and Puma knockout mice compared to other knockout mice. This information is also invaluable in designing tailor-made BH3 mimetic drugs against cancers that over-express various anti-apoptotic Bcl-2 members.
Bax/Bak activation: ‘hit and run’ versus the antipodean view In response to increased steady-state levels of BH3-only proteins inside the cell, the multi-domain pro-apoptotic proteins Bax and Bak are activated, causing the release of pro-apoptogenic factors such as cytochrome c [27].
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How these inactive molecules transform into potent killers has been the subject of intense study. The original hypothesis was put forth by Korsmeyer’s group [28], and was called the ‘hit and run’ model, whereby tBid (a truncated form of Bid cleaved by caspase 8) acted as a membrane-targeting death ligand for Bak, leading to cytochrome c release and apoptosis (Fig. 3A). Although tBid appeared to be required for Bak oligomerization, no tBid was found in the complex, prompting the authors to propose the ‘hit and run’ model whereby tBid is no longer bound after inducing a conformational change in Bak. As the majority of this study was performed in isolated mitochondria, the in vivo relevance of this model is difficult to establish. An alternative model put forward by Kuwana et al. [29] suggested two non-mutually exclusive modes of Bax/Bak activation. In one scenario, proteins such as Bid and Bim (and even Puma) activate Bax/Bak molecules directly, whereas in the other scenario, all the other BH3-only proteins act as de-repressors. The de-repressors act by preventing the interaction between activators and anti-apoptotic Bcl-2 members, thereby freeing the activators to directly activate Bax/Bak molecules. Implicit in this model is the possibility that the de-repressors have higher affinities for anti-apoptotic molecules (in contrast to previous results [24]) and/or that the expression levels of this group of proteins are higher than those of the activators. Direct activation also predicts that there will be increased binding between Bax/Bak and the activators in dying cells, but this is not the case [30–32]. If it were true, mice/cells deficient in Bim, Bid and Puma would be phenotypically similar to Bax/Bak knockout cells.
Fig. 2. Some BH3-only proteins are more equal than others. A subset of BH3-only proteins show promiscuous high-affinity binding to antiapoptotic Bcl-2 members, and therefore activate Bax/Bak more readily and induce apoptosis.
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A
B Fig. 3. The two alternative models for Bax/Bak activation. In the direct activation/ ’hit and run’ model (A), some BH3-only proteins (Bad) act as sensitizers, displacing activators (tBid) from the Bcl-2 complex. The free Bid molecules activate Bax/Bak to form oligomers. In the indirect activation model (B), BH3-only proteins such as Bim displace Bax/Bak from the Bcl-2 complex and thus are free to oligomerize. The cartoon is with an apology to Mark Anderson.
Furthermore, Bax is prone to detergent-induced conformational changes that may not reflect the physiological state. In our view, work performed by Huang’s group [24,33] (referred to here as the ‘antipodean view’) proposed a more plausible alternative model (the indirect activation model) whereby Bax/Bak molecules are held in check by anti-apoptotic Bcl-2 family proteins, similar to the situation in C. elegans. BH3-only proteins are able to displace Bax/Bak molecules from this complex, leading to their activation and eventually apoptosis (Fig. 3B). This is consistent with the observation that proteins such as Bim and Puma are potent killers and show promiscuous highaffinity binding of anti-apoptotic Bcl-2 family proteins, whereas less potent killers bind only a selected subset of pro-survival proteins. In addition, degradation of Mcl-1 by Noxa also supports an indirect activation model [34]. However, this proposition also has some drawbacks. Indirect activation of Bak appears to be possible by sequestration by Mcl-1 and Bcl-XL [25], but this does not appear to be the case for Bax. While Bak exists constitutively on the membrane, Bax is predominantly cytosolic, requiring some form of activation before it re-localizes to the FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
membrane. Therefore, it may be assumed that while indirect activation is the most likely mode of activation for Bak, weighted evidence suggests that direct activation is the most likely scenario for Bax activation [28,35,36]. Resolving these issues, i.e. how BH3only proteins kill, is a key step towards tailoring BH3 mimetics-based therapeutic agents.
Non-apoptotic function of BH3-only proteins: facts and fiction Notwithstanding the conundrum described above, Bax/Bak activation is one of the central tenets of the mitochondrial apoptotic pathway. The role of BH3only proteins in this process is undisputed. However, recent literature has also reported non-apoptotic roles for BH3-only proteins (Table 1). Non-apoptotic roles for transcription factors such as p53 [37] are well documented; however, a similar role for BH3-only proteins, which are highly unstructured with the exception of Bid [38] (which has an ordered structure formed only within the BH3 domain upon interaction with an anti-apoptotic protein), appears to be highly controversial. A secondary role in non-apoptotic functions (i.e. by regulating the non-apoptotic function of their
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Table 1. Non-apoptotic functions of BH3-only proteins.
Cellular function Autophagy Glucose metabolism Lipid transport NF-jB activation/inflammation
BH3-only protein/mimetic Noxa, ABT737 Bad Bid Bid
DNA damage response
Bid
Unfolded protein response
Bim/Puma
References Elgendy et al. [40]; Lindqvist et al. [41] Danial et al. [42,43]; Szlyk et al. [44] Esposti et al. [45] Luo et al. [46]; Yeretssian et al. [51]; Nachbur et al. [52] Kamer et al. [47]; Liu et al. [48]; Kaufmann et al. [49] Rodriguez et al. [55]; Herold et al. [56]
anti-apoptotic family members) is within the realms of possibility. For example, BAD may regulate the cell cycle through heterodimerization with Bcl-2 or Bcl-XL [39]. Another case point in this context is the regulation of autophagy, which is widely believed to be regulated by Beclin-1, which is a mammalian ortholog of yeast Atg6 with a BH3 domain. However, conventional BH3-only proteins such as Noxa may have an effect on autophagy by negating anti-apoptotic protein Mcl-1 [40]. Lindqvist et al. [41] recently reinforced this possibility by reporting that autophagy in mammalian cells is regulated by Bcl-2 family members in much the same way as they regulate apoptosis. Here, BH3-only proteins play a pivotal role in Bax/Bak activation, and the role for BH3-only proteins in autophagy is indirect. Here we discuss the literature that implies a direct role for BH3-only proteins in non-apoptotic cellular functions. One of the most compelling cases for the direct involvement of BH3-only proteins in non-apoptotic functions has been made by Korsmeyer’s group [42]. Their pioneering work demonstrated how BAD regulates glucokinase activity, respiration and ATP production, as well acting as a sentinel to sense glucose levels in pancreatic islets. Subsequent studies using mouse genetic models also demonstrated the therapeutic potential of BAD BH3 mimetics in restoring b cell function [43]. Furthermore, structural studies [44] also revealed that the BAD BH3 phosphomimetic binds a previously undescribed region near the enzyme’s active site without affecting allosteric regulation of the enzyme. This may pave the way for a new generation of glucokinase activators for treating type 2 diabetes. The possibility of a non-apoptotic role for Bid is highly controversial; one of the earliest reports suggested a role in lipid transport [45]. Bid was shown to 1010
have inherent lipid transferase activity, leading to augmented lipid transfer from ER membrane to the mitochondria. A potential caveat with respect to this study is that most of the experiments were performed using either isolated mitochondrial/ER membranes or liposomes, and the relevance of this study in vivo has yet to be established. Moreover, an altered lipid profile has not been reported in Bid knockout mouse organs. Bid has also been reported to contribute to mitochondrial generation of reactive oxygen species, which in turn leads to activation of the nuclear factor NF-jB upon exposure to a low dose of the carcinogen 5MCDE (5- methylchrysene-1,2-diol-3,4-epoxide). This has been attributed to its ‘anti-apoptotic’ activity [46]. Apart from being the antithesis to the common belief regarding the normal function of BH3-only proteins, the main problem with this proposition is that the low-dose carcinogen treatment did not lead to Bid activation, therefore could not have translocated to mitochondria. However, Bid was able to regulate mitochondrial generation of reactive oxygen species and therefore cellular survival. Bid has also been reported to play a role in the DNA damage response [47]. Bid was found to be phosphorylated by Atm kinase (at S61 and S78 in both mouse and human forms), and its role in cellcycle entry is dependent on these phosphorylation events. Accordingly, the topoisomerase II poison etoposide failed to induce S-phase blockage in Bid / cells compared to wild-type cells. A subsequent study [48] found that Bid functions at the level of the sensor complex in the DNA damage response directed by Atm and Rad3-related (Atr). Bid-deficient cells showed reduced accumulation of Atr and Atr-interacting protein (Atrip) on chromatin and at DNA damage foci. Thus, this study provided a possible mechanism for the role of Bid in the DNA damage response. However, Kaufman et al. [49] found no such link between Bid and the DNA damage response. Nine distinct cell types from Bid-deficient mice underwent cellcycle arrest and apoptosis in a manner indistinguishable from control wild-type cells in response to DNA damage or replicative stress. The mice did not show any increased risk of myeloid leukemia whereas the original study had reported an increased risk [47]. The anomalies in these studies were attributed to the mode of immortalization of the cell lines generated and the cell-cycle phase of the primary cells used [50]. However, the fact that consistent results were obtained from nine different cell types [49] is intriguing. Furthermore, the ATM-mediated phosphorylation at S61 and S78 suggests the interaction may not be mediated by the BH3 domain (residues 90–98). Understanding FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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the nuclear import dynamics of Bid, further analysis of the nuclear interaction partner(s), and understanding the structural basis of these interactions as for the BAD interaction with glucokinase [44] would help to resolve these discrepancies. In yet another context, a genome-wide RNA interference screen identified Bid as a regulator of the NOD1-mediated inflammatory response [51]. In this study, colonocytes lacking Bid or macrophages from Bid / mice were compromised in terms of their cytokine secretion in response to activators of nucleotidebinding and oligomerization domain (NOD) proteins. BID appeared to interact with NOD1, NOD2 and the IjB kinase complex, thus regulating the NF-jB pathway in cytokine secretion. Bid cleavage and the BH3 domain were redundant in this activation pathway, signifying that this process is independent of apoptosis. In the dextran-sulfate sodium-induced colitis model, the NOD agonist muramyl dipeptide afforded protection and prevented weight loss in wild-type mice, whereas Bid / mice exhibited both weight loss and crypt loss in the colon. This was attributed to lack of NOD-dependent cytokine signaling and the tissue repair response in Bid / mice. Intriguingly, using the same strain of mice, Nachbur et al. [52] found no such relationship between Bid and the NOD-dependent inflammatory response and cytokine secretion. The reasons for the discrepancies in these observations have not yet been determined. The unfolded protein response (UPR) is another scenario in which BH3-only proteins are known to play an important role. Although most studies suggest that BH3-only proteins such as Bim and Puma are induced in response to the UPR [53,54], leading to the mitochondrial apoptotic response, a recent study by Rodriguez et al. [55] reported a role for Bim and Puma in the initiation phase of the UPR. Both Bim and Puma appeared to bind the ER kinase/nuclease IRE1a in a BH3 domain-dependent manner, thus regulating Xbp-1 splicing and cytokine secretion in lipopolysaccharide-stimulated B cells. However, a follow-up study [56] found no such relationship between Bim or Puma and Xbp-1 splicing. Lipopolysaccharide-induced cytokine secretion from B cells from Bim / and Puma / mice was similar to that from wild-type B cells. This study was consistent with the original finding that Bim / mice have abnormally increased serum levels of IgM and IgG due to increased accumulation of antibody-secreting plasma cells; Xbp-1 splicing is absolutely necessary for plasma cell differentiation [57]. Furthermore, the reported interaction between Bim, Puma and IRE1a was BH3 domain-dependent [55], and this interaction FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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was augmented by the presence of Bcl-2, suggesting that a single BH3 domain may engage two proteins simultaneously. In light of these observations, and based on the well-established essential roles of Bim and Puma in initiation of ER stress-induced apoptosis [53,54], it may be concluded that these BH3-only proteins function exclusively downstream but not upstream in the UPR.
BH3-only proteins in therapeutics: not all roads lead to perdition Bcl-2 family proteins are implicated in a variety of pathophysiologies associated with altered cellular homeostasis. The prototypic member of the family, Bcl-2, was first identified in B-cell lymphoma [1]. Since then, pro-survival Bcl-2 family proteins have been implicated in a variety of cancers, including chronic lymphocytic leukemia, acute lymphoblastic leukemia, small cell lung cancer, prostate cancer and non-Hodgkin’s lymphoma. Similarly, Mcl-1 expression is associated with multiple myeloma, chronic myeloid leukemia, pancreatic cancer and melanoma. High levels of expression of pro-survival proteins are often associated with increased chemoresistance, and are therefore attractive targets for cancer therapy. One of the earliest anti-Bcl-2 drugs was an antisense RNAbased 18-base phosphorothioate oligonucleotide that was complementary to the first six codons of bcl-2 mRNA [58]. Initial in vitro studies using patientderived tumor cell lines and in vivo pre-clinical trials using mouse models offered promising results, leading to phase I/II clinical trials under the trade name Oblimersen (Genta Inc., Berkeley Heights, NJ, USA). However, mixed results, particularly failure in a melanoma trial, led to its non-approval by the US Food and Drug Administration. RNA oligonucleotide-based drugs are extremely sequence-specific for the target molecule, and therefore expected to be have limited spectrum of activity. In addition, their phosphorothioate backbone shows nonspecific binding to proteins such as fibronectin, fibroblast growth factor and human immunodeficiency virus reverse transcriptase etc, giving rise to misleading results [59]. The new-generation drugs termed ‘BH3 mimetics’ that were developed based on the interaction between BH3-only proteins and their anti-apoptotic counterparts offer great promise in treating neoplastic disorders [60]. These small molecules mimic the BH3 domain of BH3-only proteins, are selective towards anti-apoptotic Bcl-2 family members, and target multiple members of the family. The prototype BH3 mimetic ABT737, developed by Abbott Laboratories
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Table 2. Dysregulated BH3-only proteins as potential therapeutic targets in diseases. Disease Reduced function Various cancers Autoimmune diseases Rheumatoid arthritis Excessive function Cardiomyopathy Sepsis (and other severe infections, e.g. Ebola, anthrax) Neurodegenerative diseases Diabetes Immunedeficiency after chemo- and radiotherapy
(North Chicago, IL, USA), was based on the BAD BH3 domain and was able to bind multiple Bcl-2 family members including Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1 [61]. However, initial studies showed that this drug led to thrombocytopenia owing to the death of platelets mediated by Bcl-XL inhibition. A new derivative of this drug (ABT199) has high specificity towards Bcl-2, with greatly reduced binding of BclXL, and thus has anti-tumor activity while sparing platelets [62]. The fact that these drugs mostly target cancers with a high level of Bcl-2 expression, whereas in most malignancies Mcl-1 over expression leads to drug resistance. This has led to the development of BH3 mimetics specifically targeting Mcl-1. These drugs are at various stages of clinical assessment, and a comprehensive review of these various drugs has been published recently [63]. Inducing cell death through use of a BH3-only protein or its derivatives as a form of treatment has wider potential applications than just oncology. There are other pathophysiologies in which BH3 mimetics may be useful. For example, increased eosinophil survival (due to the presence of pro-survival Bcl-2 family proteins) is correlated with disease severity in mouse models and in childhood acute asthma [64,65]. However, as cancer is a high-profile disease, due to the potential market size and the efforts of high-profile cancer survivors and effective lobby groups, the BH3-only research is significantly skewed towards eliciting an apoptotic response in cancer cells. However, the economic and health impacts of cardiovascular diseases and neurodegenerative disorders are equally significant as those of cancer. Sepsis alone kills more people than breast cancer, prostate cancer and HIV/AIDS combined. An increased rate of apoptosis by BH3-only proteins is implicated in all these morbidities (Table 2) [66–68]. Understanding the molecular pathways involved in upregulation of BH3-only protein expression [67] will 1012
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References
Bim, Puma, Noxa, Bmf, Bad Bim, Bmf Bim, Bid
Happo et al. [70]
Bim, Bnip3 Bim, Bid, Puma
Lee et al. [67]; Diwan et al. [74] Chang et al. [68]; Weber et al. [6]; Parrino et al. [75] Ghosh et al. [76] Wali et al. [77] Villunger et al. [78], [79]
Bim, Puma Bim, Puma Bim, Puma, Noxa
Bouillet et al. [57]; Labi et al. [71] Scatizzi et al. [72]; Scatizzi et al. [73]
undoubtedly result in development of therapeutic agents targeting these diseases in which preventing apoptosis is a desirable outcome.
Conclusion and perspectives Since the discovery of the first BH3-only protein nearly 20 years ago [69], great strides have been made in understanding their structure, function, functional interactions and role in pathogenesis, which has eventually led to the development of novel therapeutic agents. We also know that each member has differential affinity for their anti-apoptotic counterparts, and this has helped to design tailor-made therapeutic agents that offer great promise in countering chemoresistance in cancer therapy. Notwithstanding these observations, there is a huge gap in our understanding of BH3 biology. While there is consensus among groups reharding the differential affinity of BH3-only proteins towards their anti-apoptotic counterparts, there is some debate on their role in activating Bax/ Bak adaptor molecules. In this review, we have provided a balanced view of both models, and in our opinion, both modes of activation may exist in a cell type- or stimulus-dependent manner. There appears to be a great deal of confusion regarding the role of BH3-only proteins in non-apoptotic function, especially with respect to their role in the DNA damage response, inflammation and initiation of the UPR. Only by identifying the key molecules with which BH3-only proteins interact in these pathways and analyzing these interactions at structural level will these issues be resolved. Finally, in our view, a greater research emphasis should also be placed on harnessing the benefits of our knowledge on regulation of BH3-only proteins so that therapeutic agents may be developed to treat diseases such as neurodegenerative disorders, diabetes, cardiomyopathy and sepsis. FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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Acknowledgements We would like to thank all the H.P. lab members for their support and helpful discussions. We also would like to thank Andrew Coley, Department of Biochemistry, La Trobe University, Melbourne, Australia for reviewing this manuscript. H.P. is supported by an Australian Research Council Future Fellowship (FT0990683) and by an Australian Research Council project grant (DP110100417).
Author contribution HP: Discussed the subject matter with MD and JAG and wrote the manuscript and drew some of the figures. MD: Wrote parts of the manuscript. JAG: Created the cartoons.
References 1 Vaux DL, Cory S & Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440–442. 2 Vaux DL, Weissman IL & Kim SK (1992) Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955–1957. 3 Puthalakath H & Strasser A (2002) Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ 9, 505–512. 4 Wong WW & Puthalakath H (2008) Bcl-2 family proteins: the sentinels of the mitochondrial apoptosis pathway. IUBMB Life 60, 390–397. 5 Ng KP, Hillmer AM, Chuah CT, Juan WC, Ko TK, Teo AS, Ariyaratne PN, Takahashi N, Sawada K, Fei Y et al. (2012) A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nat Med 18, 521–528. 6 Weber SU, Schewe JC, Lehmann LE, Muller S, Book M, Klaschik S, Hoeft A & Stuber F (2008) Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis. Crit Care 12, R128. 7 Tsai SC, Pasumarthi KB, Pajak L, Franklin M, Patton B, Wang H, Henzel WJ, Stults JT & Field LJ (2000) Simian virus 40 large T antigen binds a novel Bcl-2 homology domain 3-containing proapoptosis protein in the cytoplasm. J Biol Chem 275, 3239–3246. 8 Tan KO, Tan KM, Chan SL, Yee KS, Bevort M, Ang KC & Yu VC (2001) MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains. J Biol Chem 276, 2802–2807.
FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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9 Mund T, Gewies A, Schoenfeld N, Bauer MK & Grimm S (2003) Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum. FASEB J 17, 696–698. 10 Liu H, Toman RE, Goparaju SK, Maceyka M, Nava VE, Sankala H, Payne SG, Bektas M, Ishii I, Chun J et al. (2003) Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis. J Biol Chem 278, 40330–40336. 11 Broustas CG, Gokhale PC, Rahman A, Dritschilo A, Ahmad I & Kasid U (2004) BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner. J Biol Chem 279, 26780–26788. 12 Rodolfo C, Mormone E, Matarrese P, Ciccosanti F, Farrace MG, Garofano E, Piredda L, Fimia GM, Malorni W & Piacentini M (2004) Tissue transglutaminase is a multifunctional BH3-only protein. J Biol Chem 279, 54783–54792. 13 Zhong Q, Gao W, Du F & Wang X (2005) Mule/ARFBP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell 121, 1085–1095. 14 Liu Z, Lu H, Jiang Z, Pastuszyn A & Hu CA (2005) Apolipoprotein l6, a novel proapoptotic Bcl-2 homology 3-only protein, induces mitochondriamediated apoptosis in cancer cells. Mol Cancer Res 3, 21–31. 15 Wan G, Zhaorigetu S, Liu Z, Kaini R, Jiang Z & Hu CA (2008) Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. J Biol Chem 283, 21540–21549. 16 Naresh A, Long W, Vidal GA, Wimley WC, Marrero L, Sartor CI, Tovey S, Cooke TG, Bartlett JM & Jones FE (2006) The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells. Cancer Res 66, 6412–6420. 17 Strohecker AM, Yehiely F, Chen F & Cryns VL (2008) Caspase cleavage of HER-2 releases a Bad-like cell death effector. J Biol Chem 283, 18269–18282. 18 Horvitz HR (1999) Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res 59, 1701s–1706s. 19 Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y & Horvitz HR (2000) Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287, 1485–1489. 20 Conradt B & Horvitz HR (1998) The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl–2-like protein CED-9. Cell 93, 519–529. 21 Xue D & Horvitz HR (1997) Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature 390, 305–308.
1013
BH3-only proteins
22 Huang Q, Petros AM, Virgin HW, Fesik SW & Olejniczak ET (2002) Solution structure of a Bcl-2 homolog from Kaposi sarcoma virus. Proc Natl Acad Sci USA 99, 3428–3433. 23 Kelekar A & Thompson CB (1998) Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 8, 324–330. 24 Chen LWS, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL, Adams JM & Huang DC (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17, 393–403. 25 Willis SNCL, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM & Huang DC (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19, 1294–1305. 26 Merino D, Giam M, Hughes PD, Siggs OM, Heger K, O’Reilly LA, Adams JM, Strasser A, Lee EF, Fairlie WD et al. (2009) The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins. J Cell Biol 186, 355–362. 27 Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D & Reed JC (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95, 4997–5002. 28 Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB & Korsmeyer SJ (2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14, 2060–2071. 29 Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR & Newmeyer DD (2005) BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 17, 525–535. 30 Nechushtan A, Smith CL, Lamensdorf I, Yoon SH & Youle RJ (2001) Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 153, 1265–1276. 31 Antonsson B, Montessuit S, Sanchez B & Martinou JC (2001) Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276, 11615–11623. 32 Zhu Y, Swanson BJ, Wang M, Hildeman DA, Schaefer BC, Liu X, Suzuki H, Mihara K, Kappler J & Marrack P (2004) Constitutive association of the proapoptotic protein Bim with Bcl-2-related proteins on mitochondria in T cells. Proc Natl Acad Sci USA 101, 7681–7686. 33 Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE, Ierino H, Lee EF, Fairlie WD, Bouillet P et al. (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856–859.
1014
M. Doerflinger et al.
34 Czabotar PE, Lee EF, van Delft MF, Day CL, Smith BJ, Huang DC, Fairlie WD, Hinds MG & Colman PM (2007) Structural insights into the degradation of Mcl-1 induced by BH3 domains. Proc Natl Acad Sci USA 104, 6217–6222. 35 Wang K, Yin XM, Chao DT, Milliman CL & Korsmeyer SJ (1996) BID: a novel BH3 domain-only death agonist. Genes Dev 10, 2859–2869. 36 Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B & Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144, 891–901. 37 Montero J, Dutta C, van Bodegom D, Weinstock D & Letai A (2013) p53 regulates a non-apoptotic death induced by ROS. Cell Death Differ 20, 1465–1474. 38 Hinds MG, Smits C, Fredericks-Short R, Risk JM, Bailey M, Huang DC & Day CL (2007) Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death Differ 14, 128–136. 39 Chattopadhyay A, Chiang CW & Yang E (2001) BAD/BCL-[X(L)] heterodimerization leads to bypass of G0/G1 arrest. Oncogene 20, 4507–4518. 40 Elgendy M, Sheridan C, Brumatti G & Martin SJ (2011) Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol Cell 42, 23–35. 41 Lindqvist LM, Heinlein M, Huang DC & Vaux DL (2014) Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc Natl Acad Sci USA 111, 8512–8517. 42 Danial NN, Gramm CF, Scorrano L, Zhang CY, Krauss S, Ranger AM, Datta SR, Greenberg ME, Licklider LJ, Lowell BB et al. (2003) BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis. Nature 424, 952–956. 43 Danial NN, Walensky LD, Zhang CY, Choi CS, Fisher JK, Molina AJ, Datta SR, Pitter KL, Bird GH, Wikstrom JD et al. (2008) Dual role of proapoptotic BAD in insulin secretion and beta cell survival. Nat Med 14, 144–153. 44 Szlyk B, Braun CR, Ljubicic S, Patton E, Bird GH, Osundiji MA, Matschinsky FM, Walensky LD & Danial NN (2014) A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators. Nat Struct Mol Biol 21, 36–42. 45 Esposti MD, Erler JT, Hickman JA & Dive C (2001) Bid, a widely expressed proapoptotic protein of the Bcl2 family, displays lipid transfer activity. Mol Cell Biol 21, 7268–7276.
FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
M. Doerflinger et al.
46 Luo W, Li J, Zhang D, Cai T, Song L, Yin XM, Desai D, Amin S, Chen J & Huang C (2010) Bid mediates anti-apoptotic COX-2 induction through the IKKbeta/NFkappaB pathway due to 5-MCDE exposure. Curr Cancer Drug Targets 10, 96–106. 47 Kamer I, Sarig R, Zaltsman Y, Niv H, Oberkovitz G, Regev L, Haimovich G, Lerenthal Y, Marcellus RC & Gross A (2005) Proapoptotic BID is an ATM effector in the DNA-damage response. Cell 122, 593–603. 48 Liu Y, Bertram CC, Shi Q & Zinkel SS (2011) Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress. Cell Death Differ 18, 841–852. 49 Kaufmann T, Tai L, Ekert PG, Huang DC, Norris F, Lindemann RK, Johnstone RW, Dixit VM & Strasser A (2007) The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest. Cell 129, 423–433. 50 Zinkel SS, Hurov KE & Gross A (2007) Bid plays a role in the DNA damage response. Cell 130, 9–10. 51 Yeretssian G, Correa RG, Doiron K, Fitzgerald P, Dillon CP, Green DR, Reed JC & Saleh M (2011) Non-apoptotic role of BID in inflammation and innate immunity. Nature 474, 96–99. 52 Nachbur U, Vince JE, O’Reilly LA, Strasser A & Silke J (2012) Is BID required for NOD signalling? Nature 488, E4–6; discussion E6-8. 53 Puthalakath H, O’Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, Hughes PD, Michalak EM, McKimm-Breschkin J, Motoyama N et al. (2007) ER stress triggers apoptosis by activating BH3-only protein Bim. Cell 129, 1337–1349. 54 Reimertz C, Kogel D, Rami A, Chittenden T & Prehn JH (2003) Gene expression during ER stress-induced apoptosis in neurons: induction of the BH3-only protein Bbc3/PUMA and activation of the mitochondrial apoptosis pathway. J Cell Biol 162, 587–597. 55 Rodriguez DA, Zamorano S, Lisbona F, Rojas-Rivera D, Urra H, Cubillos-Ruiz JR, Armisen R, Henriquez DR, Cheng EH, Letek M et al. (2012) BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1alpha. EMBO J 31, 2322–2335. 56 Herold MJ, O’Reilly LA, Lin A, Srivastava R, Doerflinger M, Bouillet P, Strasser A & Puthalakath H (2014) Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA. Cell Death Dis 5, e1354. 57 Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM & Strasser A (1999) Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735–1738.
FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
BH3-only proteins
58 Kitada S, Miyashita T, Tanaka S & Reed JC (1993) Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 3, 157–169. 59 Dias N & Stein CA (2002) Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl2 antisense oligonucleotides. Eur J Pharm Biopharm 54, 263–269. 60 Kang MH & Reynolds CP (2009) Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res 15, 1126–1132. 61 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ et al. (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681. 62 Touzeau C, Dousset C, Le Gouill S, Sampath D, Leverson JD, Souers AJ, Maiga S, Bene MC, Moreau P, Pellat-Deceunynck C et al. (2014) The Bcl2 specific BH3 mimetic ABT-199: a promising targeted therapy for t(11;14) multiple myeloma. Leukemia 28, 210–212. 63 Billard C (2013) BH3 mimetics: status of the field and new developments. Mol Cancer Ther 12, 1691–1700. 64 Tumes DJ, Wong AC, Sewell WA, McColl SR, Connolly A & Dent LA (2008) Differential rates of apoptosis and recruitment limit eosinophil accumulation in the lungs of asthma-resistant CBA/Ca mice. Mol Immunol 45, 3609–3617. 65 El-Gamal Y, Heshmat N, Mahran M & El-Gabbas Z (2004) Expression of the apoptosis inhibitor Bcl-2 in sputum eosinophils from children with acute asthma. Clin Exp Allergy 34, 1701–1706. 66 Akhter R, Sanphui P & Biswas SC (2014) The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in beta-amyloid-induced neuron death. J Biol Chem 289, 10812–10822. 67 Lee YY, Moujalled D, Doerflinger M, Gangoda L, Weston R, Rahimi A, de Alboran I, Herold M, Bouillet P, Xu Q et al. (2013) CREB-binding protein (CBP) regulates beta-adrenoceptor (beta-AR)-mediated apoptosis. Cell Death Differ 20, 941–952. 68 Chang KC, Unsinger J, Davis CG, Schwulst SJ, Muenzer JT, Strasser A & Hotchkiss RS (2007) Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis. FASEB J 21, 708–719. 69 Yang E, Zha J, Jockel J, Boise LH, Thompson CB & Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80, 285–291. 70 Happo L, Strasser A & Cory S (2012) BH3-only proteins in apoptosis at a glance. J Cell Sci 125, 1081–1087. 71 Labi V, Woess C, Tuzlak S, Erlacher M, Bouillet P, Strasser A, Tzankov A, Villunger A (2014) Deregulated
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M. Doerflinger et al.
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74
75
76
cell death and lymphocyte homeostasis cause premature lethality in mice lacking BH3 only proteins Bim and Bmf. Blood 123, 2652–2662. Scatizzi JC, Bickel E, Hutcheson J, Haines GK & Perlman H (2006) Bim deficiency leads to exacerbation and prolongation of joint inflammation in experimental arthritis. Arthritis Rheum 54, 3182–3193. Scatizzi JC, Hutcheson J, Bickel E, Haines GK & Perlman H (2007) Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis. Arthritis Res Ther 9, R49. Diwan A, Krenz M, Syed FM, Wansapura J, Ren X, Koesters AG, Li H, Kirshenbaum LA, Hahn HS, Robbins J (2007) Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice. J Clin Investig 117, 2825–2833. Parrino J, Hotchkiss RS & Bray M (2007) Prevention of immune cell apoptosis as potential therapeutic strategy for severe infections. Emerg Infect Dis 13, 191–198. Ghosh AP, Klocke BJ, Ballestas ME & Roth KA (2012) CHOP potentially co-operates with FOXO3a in neuronal cells to regulate PUMA and BIM expression in response to ER stress. PLoS One 7, e39568.
1016
77 Wali JA, Rondas D, McKenzie MD, Zhao Y, Elkerbout L, Fynch S, Gurzov EN, Akira S, Mathieu C, Kay TM (2014) The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity. Cell Death Dis 5, e1124. 78 Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM & Strasser A (2003) p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036–1038. 79 Villunger A, Scott C, Bouillet P & Strasser A (2003) Essential role for the BH3-only protein Bim but redundant roles for Bax, Bcl-2, and Bcl-w in the control of granulocyte survival. Blood 101, 2393–2400.
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FEBS Journal 282 (2015) 1006–1016 ª 2015 FEBS
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