Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

BCL-2 Family

  • Zuzana Saidak
  • Zakaria Ezzoukhry
  • Jean-Claude Maziere
  • Antoine Galmiche
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_578

Synonyms

Historical Background

The BCL-2 protein, the founding member of this family of proteins, was discovered in 1985. The gene BCL2 was identified as the main protagonist in the chromosomal translocation t(14;18) in a subset of B-cell lymphomas, placing it under the control of the promoter of the immunoglobulin heavy chain genes (Cotter 2009). In contrast to previously identified oncogenes that mainly promote cell proliferation, BCL-2 was the first oncogene shown to inhibit cell death (Vaux et al. 1988). Since then other members of the BCL-2 family have been discovered, and the family of BCL-2 proteins now consists of approximately 20 members. In mammalian organisms, BCL-2 proteins play an essential role in the control of programmed cell death, and in particular apoptosis. Recent investigations have helped to unveil some facets of their regulation and their molecular mode of action on membrane organelles and mitochondria.

Structure and Classification of BCL-2 Proteins

BCL-2 proteins are divided into three groups, based on functional as well as structural criteria (Chipuk et al. 2010): (1) functionally, depending on their effect on apoptosis – either pro- or anti-apoptotic; (2) structurally according to the presence of one or multiple homology domains – all members of the BCL-2 family share one or more regions of sequence homology, called the BCL-2 homology domains 1–4 (BH1 to BH4). The anti-apoptotic proteins of the BCL-2 family constitute the first group of proteins. They are multidomain proteins, usually containing BH1-BH4 domains. This group principally includes the proteins BCL-2, BCL-XL, MCL-1, and A1 (Fig. 1). The corresponding proteins functionally counteract the pro-apoptotic proteins of the BCL-2 family. The pro-apoptotic proteins are divided into two groups: (1) the multidomain pro-apoptotic proteins (BAX, BAK, BOK) of the BCL-2 family, and (2) the BH3-only proteins, containing this sole homology domain (such as BID, BIM, PUMA, BAD, NOXA, BMF, HRK, BIK) (Fig. 1). The BH3 domain is, therefore, the only conserved region of homology among the proteins of the BCL-2 family. While this domain is an essential region for the activity of the BCL-2 proteins, it is short and consists of approximately 15 amino acids organized in an amphipathic helix (Fig. 1). The BH3 domain is also present in proteins that are only loosely connected to the BCL-2 family, such as the proteins MULE and Beclin-1.
BCL-2 Family, Fig. 1

Structural domains and organization of the BCL-2 proteins. BCL-2 proteins can be classified according to their pro- or anti-apoptotic effects and the presence of one or multiple BCL-2-homology (BH) domains. The BH3 domain is the only domain of homology shared by all members of the family. It consists of 15 AA with a preference for the motif depicted in the sequence logo in the lower part of the figure (adapted from the server Prosite, http://expasy.org/cgi-bin/prosite/). While the membrane localization domains are indicated here for the multidomain BCL-2 proteins, several members of the BH3-only proteins also possess membrane targeting domains with an affinity for lipids

Remarkably, despite important differences in their amino-acid sequences, all multidomain proteins of the BCL-2 family possess a similar secondary structure consisting mostly of α-helices and a similar overall fold (Chipuk et al. 2010). This similarity extends to the proteins that have opposing functions, either pro- or anti-apoptotic. In BCL-XL, the spatial juxtaposition of α-helices from the BH1-BH3 regions defines a globular structure with a hydrophobic groove on the surface of the molecule. This hydrophobic groove enables BCL-XL to interact with the BH3 domain of pro-apoptotic proteins. In contrast to the multidomain proteins of the BCL-2 family, the BH3-only proteins are structurally diverse, with the exception of BID, which has an overall fold reminiscent of the multidomain proteins. Members of the BH3-only subset, such as BAD or BIM, tend to be intrinsically unfolded proteins and they probably acquire a stable fold only upon their interaction with other members of the BCL-2 family.

Mitochondrial Membrane Permeabilization by BCL-2 Proteins

In mammalian cells, mitochondrial outer membrane permeabilization (MOMP) is an early and crucial event during the induction of apoptosis (Tait and Green 2010). The MOMP leads to the release of pro-apoptotic factors, such as cytochrome c, into the cytosol. There, cytochrome c induces a cascade of biochemical events that lead to the activation of caspases, a family of proteases involved in the execution of the death sentence.

The proteins of the BCL-2 family are key players in the MOMP (Kuwana et al. 2002). The pro-apoptotic multidomain proteins of the BCL-2 family, i.e., BAX and BAK, play an essential role in the MOMP through their ability to form membrane-inserted oligomers (Chipuk et al. 2010; Westphal et al. 2010). How BAX and BAK insert and ultimately permeabilize mitochondrial membranes is a complex question and represents the focus of intense research. According to a commonly accepted model, several steps are required for BAX/BAX oligomerization and MOMP (Fig. 2). The first step consists of the mitochondrial recruitment of these proteins. While BAK is constitutively present at the mitochondrial level, BAX is cytosolic in healthy cells. In its cytosolic form, the C-terminal extremity of BAX is sequestered in its BH3-binding pocket and BAX is therefore locked in a monomeric, inactive form. The first step in BAX activation consists of the release of BAX from this intramolecular lock, and this step is a requisite for the insertion of BAX into the MOM. The next step is common to BAX and BAK, and consists of the direct activation of these proteins. Some proteins of the BH3-only subset, in particular BID, BIM, or PUMA, can directly activate BAX and BAK through direct contacts (Gavathiotis et al. 2008; Gallenne et al. 2009). The multimerization of BAX and BAK also requires the release of these molecules from the inhibitory effect of anti-apoptotic proteins of the BCL-2 family, such as BCL-2, BCL-XL, or MCL-1. Anti-apoptotic proteins of the BCL-2 family negatively regulate the multimerization of BAX and BAK through two mechanisms: (1) the direct sequestration of BAX and/or BAK, and (2) indirectly, through the neutralization of the BH3-only proteins endowed with the ability to activate BAX/BAK, such as BID (Billen et al. 2008). Overall, the MOMP is a complex process that is intimately associated with the formation of complexes between proteins of the BCL-2 family. While the role of BCL-2 proteins in MOMP is well accepted, many questions about the precise mechanisms still remain, such as the exact nature of the pore formed by BAX and BAK and the contribution of accessory mitochondrial proteins to this process.
BCL-2 Family, Fig. 2

A model for the activation of BAX/BAK and the induction of MOMP. The activation of the pro-apoptotic multidomain proteins BAX and BAK is an essential step that leads to mitochondrial membrane permeabilization and apoptosis. While BAK is constitutively present at the mitochondrial level, BAX is normally cytosolic. The first step in the activation of BAX consists of a cytosolic to membrane translocation, possibly occurring as a consequence of the release of the carboxy-terminal tail of BAX from an inhibitory internal interaction with the hydrophobic groove of this molecule. The second step consists of the activation of BAX/BAK per se and probably results in the shaping of the BH3 domains of BAX or BAK. Reciprocal interactions and homodimer formation are rendered possible once this shaping has allowed reciprocal interactions between their BH3 domains and hydrophobic grooves. Further interactions implicating other parts of BAX/BAK lead to higher order complex- and pore-formation, resulting in MOMP and apoptosis

Regulation of the BCL-2 Network: Role of the BH3-Only Proteins

Cell survival is the result of a delicate balance between the activities of the pro- and anti-apoptotic proteins of the BCL-2 family. Apoptosis occurs when this balance is tipped over in favor of the pro-apoptotic signal. The BH3-only proteins play an upstream regulatory role in the BCL-2 network. While the proteins of this subset generally stimulate apoptosis, a complex picture of the mode of action of BH3-only proteins has recently emerged.

All BH3-only proteins are able to neutralize the anti-apoptotic proteins of the BCL-2 family, but the interaction of BH3-only proteins with anti-apoptotic BCL-2 proteins is characterized by its selectivity. There are large differences in the interaction spectra among BH3-only proteins (Chen et al. 2005; Certo et al. 2006). Some BH3-only proteins, such as BAD, neutralize selected anti-apoptotic proteins, such as BCL-2 and BCL-XL, while others, such as BIM and PUMA, bind all pro-survival proteins. Some BH3-only proteins, such as BIM, BID, and PUMA, do not only neutralize the anti-apoptotic proteins of the BCL-2 family, but can also activate the pro-apoptotic proteins BAX and BAK through labile interactions (Gavathiotis et al. 2008; Gallenne et al. 2009). These differences in terms of mode of action of the BH3-only proteins translate into differences in apoptotic potency, and proteins such as BAD behave more as sensitizers toward apoptosis rather than true inducers. The study of how the effector BCL-2 proteins are regulated in living cells has until now been a difficult task. New biochemical as well as functional approaches will certainly help to track the dynamic interactions between BCL-2 proteins, and to clarify the regulation of BCL-2 proteins during the life/death decision.

An important aspect of the regulation of BH3-only proteins is that they are kept under control by specific stimuli. Apoptosis-modulating stimuli operate on each BH3-only protein, via an array of regulations ranging from transcriptional to posttranslational (Fig. 3). For example, PUMA is induced transcriptionally following severe DNA damage, essentially through the activation of the transcription factor  p53 (Yu and Zhang 2008). On the other hand, the BH3-only protein BAD is phosphorylated and thereby inactivated by pro-survival kinases, such as the kinase cascades RAF-MEK-ERK or PKB-mTOR. The survival of cells requires their constant exposure to trophic factors that activate these cascades, and BAD becomes activated by dephosphorylation in response to growth factor deprivation (Danial 2008). BID is another member of the BH3-only subset whose activity is under regulation through the engagement of a family of cell surface receptors known as death receptors. BID is activated by a proteolytic cleavage generating the truncated, active form of the molecule called tBID. Cellular studies have helped to establish the basics on the regulation of each BH3-only protein and the sentinel function of BH3-only proteins, but the regulation of BCL-2 proteins remains a complex topic, involving several protagonists with different tissue-specific expression patterns and partially redundant functions.
BCL-2 Family, Fig. 3

Posttranslational regulation of proteins of the BCL-2 family. The BCL-2 proteins are regulated through the direct modulation of their activation status, their subcellular localization, protein stability, or their functional sequestration. Posttranslational modifications, such as phosphorylations, proteolytic cleavage, ubiquitylation, lipidation, interaction with chaperones or with specific molecules are frequently encountered. For example, the protein BAD is regulated by phosphorylation and association with proteins of the 14-3-3 family (panel a). BID becomes active upon engagement of death receptors: A proteolytic cleavage by Caspase-8 creates a truncated version of this protein (tBID) and unmasks a site for N-myristoylation of this protein (panel b). The protein MCL-1 is regulated by ubiquitylation, a posttranslational modification that controls its turnover through proteasomal degradation (panel c). Finally, protein interactions can also regulate the activity of BCL-2 proteins. The protein p53 is able to functionally neutralize the anti-apoptotic proteins of the BCL-2 family, such as BCL-XL or MCL-1, despite the absence of a BH3 domain; by doing so, the cytosolic accumulation of p53 might release pro-apoptotic proteins of the BCL-2 family, such as BIM, from preexisting inhibitory interactions (panel d)

Various Physiological Functions

In addition to the regulation of programmed cell death, proteins of the BCL-2 family regulate several physiological processes. These processes are diverse, and range from the control of mitochondrial morphogenesis and Ca2+ fluxes in the endoplasmic reticulum to various aspects of cell metabolism. Cell proliferation and the integrity of the genome are also regulated by BCL-2 proteins, through interactions established with regulatory proteins of the cell cycle and DNA repair machinery. A detailed overview of these mechanisms is clearly beyond the scope of this chapter, but the regulation of autophagy and inflammatory cytokine production by BCL-2 proteins provide two well-known examples. Autophagy is a process whereby cellular macromolecules or organelles become isolated inside cellular membrane and fuse with lysosomes to promote their elimination and recycling of their components. BCL-2 and BCL-XL have been shown to interact with Beclin-1, an essential regulator of autophagy. The interaction between Beclin-1 and BCL-2 is possible because Beclin-1 possesses a BH3 motif (Maiuri et al. 2007). BCL-2 and BCL-XL also play a role in the regulation of the metabolism of inflammatory cytokines, such as Interleukin-1, through molecular interactions established with the inflammasome, an intracellular protein complex involved in the regulation of Caspase-1, the enzyme responsible for the maturation processing of this cytokine (Bruey et al. 2007). The proteins of the BCL-2 family therefore exert pleiotropic effects that extend far beyond the regulation of programmed cell death.

BCL-2 Proteins and Cancer

Reduced sensitivity to apoptosis is one of the hallmarks of cancer cells. Deregulation of BCL-2 protein expression is frequently observed and it was shown to contribute to this disease (Yip and Reed 2008; Frenzel et al. 2009). Overexpression of the anti-apoptotic proteins of the BCL-2 family was the mechanism first reported to account for apoptosis resistance in cancer cells. While it is now well accepted that most cancer cells present a reduced sensitivity to apoptosis due to modulation of the BCL-2 regulatory system, the mechanisms that lead to the altered regulation of BCL-2 proteins are complex. Alterations in the genome of cancer cells, epigenetic mechanisms, and posttranslational modifications often concur to shape the BCL-2 proteome in cancer cells (Yip and Reed 2008; Frenzel et al. 2009).

The regulation of the proteins of the BCL-2 family has attracted considerable attention as a possible approach for cancer treatment. Indeed, inducing tumor regression through the death of cancer cells is the main goal of cancer treatment, and most chemotherapeutic agents are apoptosis inducers in cancer cells (Fulda and Debatin 2006). In a growing number of situations, apoptosis of cancer cells induced by medical treatments was found to depend on the modulation of BCL-2 proteins: treatment-induced apoptosis could either be blocked by the overexpression of anti-apoptotic proteins, such as BCL-XL or MCL-1, or by the reduction of the expression of pro-apoptotic proteins of the BCL-2 family. For example, colorectal cancer cells with a BAX knockout were found to be insensitive to the commonly used chemotherapeutic agent 5-fluorouracil (Zhang et al. 2000). More recently, specific BH3-only proteins were found to account for cell death induced by specific targeted therapies. For example, the BH3-only protein BAD mediates the apoptotic response of liver cancer cells exposed to the kinase inhibitor sorafenib, currently the only medical treatment for this tumor (Galmiche et al. 2010).

The realization that BCL-2 proteins play a pivotal role in the response of cancers to medical treatments led to intense efforts aiming to identify compounds that would directly target these proteins and could be used as a new line of targeted therapies in oncology. In recent years great advancements have been made along this line, principally with the search for BH3-mimetic compounds that bind the hydrophobic groove formed by BH1-BH3 of the anti-apoptotic BCL-2 proteins, thus favoring apoptosis. To date, the compound with the best characterized BH3-mimetic activity is ABT-737 that was developed by the Abbott laboratories (Oltersdorf et al. 2005). ABT-737 binds with high affinity to the anti-apoptotic proteins BCL-2, BCL-XL, and BCL-W, but not to MCL-1, thus demonstrating a BAD-like reactivity. ABT-737 exerts a strong anticancer activity on Small Cell Lung Carcinoma cells, which frequently overexpress BCL-2 (Oltersdorf et al. 2005). An orally active derivative, ABT-263, has been developed. ABT-263 has shown promising effects in animal models with xenografted tumors, leading to sustained regression and demonstrating the safety of the inhibition of BCL-2 proteins in the entire organism. Studies aiming to test BCL-2 inhibitors in animal models that more closely mimic human tumors are now eagerly awaited. In parallel, the identification of compounds with reactivities that differ from those of ABT-737 as well as the understanding of cancer cell addiction to anti-apoptotic proteins of the BCL-2 family are the focus of future research. BCL-2 proteins have acquired the status of potential targets in oncology, and advances in this field are expected in the coming decade.

Summary

BCL-2 proteins are pivotal regulators of apoptosis. Over the past decade, intense research efforts have helped to better understand how these proteins mutually interact and regulate the mitochondrial membrane permeabilization, a critical step in apoptosis execution. In addition to their role as important effectors, BCL-2 proteins have also emerged as key integrators for the cell signaling pathways regulating programmed cell death. Extensive work still remains to fully understand the functionality of the intricate network of BCL-2 proteins, but recent advances have demonstrated the therapeutic potential of targeting BCL-2 proteins in cancer therapy. The introduction of drugs with a new mode of action, called BH3 mimetics, holds great promise in cancer research. It is also expected to facilitate the exploration of the physiological functions and the regulation of these important signaling molecules.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Zuzana Saidak
    • 1
  • Zakaria Ezzoukhry
    • 1
  • Jean-Claude Maziere
    • 1
  • Antoine Galmiche
    • 1
  1. 1.Laboratoire de Biochimie, Inserm ERI12 – EA4292Université de Picardie Jules Verne (UPJV)AmiensFrance