Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Apoptosis Regulator BAX

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101518

Synonyms

Historical Background

Bax is a proapoptotic protein encoded by Bax gene belonging to B-cell lymphoma 2 (Bcl-2) protein family. Bcl-2 family is classified into two groups: antiapoptotic proteins (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1) and proapoptotic proteins according to their different functions. proapoptotic proteins are comprised of multidomain proteins (e.g., Bax and Bak) and BH3-only proteins (e.g., Bid, Bim, Puma, Bad, Noxa, Bik, Bmf, and Hrk) based on the presence of Bcl-2 homology domains (BH1–4 domains) (Liu et al. 2016a, b; Han et al. 2015). Interactions between Bcl-2 protein members regulate the mitochondrial signaling which is critical for normal cellular homeostasis and participates in the pathogenesis of different diseases including cancer, diabetes, obesity, and neurodegenerative disorders.

Bax was originally identified as a heterodimer with Bcl-2 in 1993 (Oltvai et al. 1993). Overexpressed Bax neutralizes the death repressor activity of Bcl-2 and accelerates apoptotic cell death. Bax acts as a regulator of apoptosis in various cell types given it is expressed in essentially all organs. Bax-deficient mice display selective expansion of cell population, selective hyperplasias, and resistance to certain apoptotic stimuli (Knudson et al. 1995). Three-dimensional structure of Bax was resolved by nuclear magnetic resonance (NMR) in 2000 (Suzuki et al. 2000). Bax was initially considered to be composed of transmembrane domain, BH1, BH2, and BH3 domain without BH4. The presence or absence of BH4 domain is taken as a distinguishing feature of the anti- and proapoptotic family members. Until 2008, the presence of BH4 motif of Bax was illustrated (Kvansakul et al. 2008). Crystal structures of C-terminally truncated BaxΔC21 with detergents and Bid BH3 peptides were determined in 2013, which defined critical Bax transitions toward apoptosis (Czabotar et al. 2013).

Bax is predominantly found as an inactive monomer in the cytosol of healthy cells or loosely attached to the mitochondrial, nuclear, or endoplastic reticulum membrane and is commonly believed to translocate to mitochondria following an apoptotic stimulus. Once activated, it will induce mitochondrial targeting and outer-membrane permeabilization, promote the formation of pores, and enable the release of cytochrome c and Smac/DIABLO from the intermembrane space into cytosol. There are mainly three different models of Bax activation-mediated mitochondrial outer membrane permeabilization (MOMP) including direct activation models, displacement models, and unified models (Liu et al. 2016a). Numerous anticancer agents including both chemotherapeutic and chemopreventive agents take effects via Bax activation indirectly (Zhang et al. 2000). Bax activators represent a new class of apoptotic sensitizers to combat pathologic cell survivals (e.g., cancers), while Bax inhibitors may serve as novel methods of removing unwanted or premature cell death (e.g., autoimmune disorders).

Structure of Bax

Bax is a 21kD protein of 192 amino acids comprised of 9 α-helices. It has a similar tertiary structure as other Bcl-2 family members (Fig. 1). Helices α5 (Hα5) and Hα6 form the core of the protein and are embedded within the other seven helices. There is a long and unstructured loop between Hα1 and Hα2, whose function remains to be elucidated. It was suggested that conversion of this loop from a closed to an opened conformation is necessary for Bax activation (Gavathiotis et al. 2010). Hα2, Hα3, Hα4, and Hα5 constitute the canonical BH3-binding site, also known as hydrophobic groove. Hα2 comprises the BH3 domain which is necessary for the heterodimerization with other Bcl-2 family members. The hydrophobic side chains (e.g., Leu59, Leu63, Ile66, and Leu 70) of the BH3 helix point inward toward Hα5/ Hα6 and are covered by Hα9. Once Bax is directly triggered, allosteric release of the Hα9 from its binding groove is required for regulated Bax translocation and functional insertion into the outer mitochondrial membrane. Also, Bax BH3 domain becomes exposed on ligand-induced direct Bax activation and itself propagates Bax activation (Gavathiotis et al. 2010). Recently, a new model for activation of MOM-anchored Bax was revealed with the support of cocrystal structures of BaxΔC21-Bid BH3 peptide (PDB code: 4BD2) and BaxΔC26-Bim BH3 peptide (PDB code: 4ZIE, Fig. 1). In the Bax:BH3 peptide complex, cavities appear at the interface between the core and latch domains, while no cavities are formed in the equivalent Bax without the bound peptide or Bcl-xL:BH3 peptide. The destabilizing cavities facilitate release of the core domain (Hα1–Hα5) from the latch domain (Hα6–Hα8) and dislodgement of Bax Hα2 (BH3 domain) when an activator BH3-only protein inserts its BH3 domain into the canonical groove of Bax. The exposed Bax BH3 domain, competing with activator BH3-only proteins, binds the groove of another Bax molecule to form a stable BH3-in-groove symmetric dimer which nucleates oligomerization of the Bax core domain and then provokes MOMP (Czabotar et al. 2013 and Robin et al. 2015).
Apoptosis Regulator BAX, Fig. 1

Sequence of Bax and different conformations of inactive Bax (PDB Code: 1F16) versus activated BaxΔC26 by Bim BH3 (PDB Code: 4ZIE)

Functions of Bax in Apoptotic Signaling Pathways

Bax and Bak are essential executive proteins responsible for MOMP in the intrinsic (mitochondrial) signaling pathway and requisite gateway to mitochondrial dysfunction (Wei et al. 2001). Upon various stresses, Bax is activated by BH3-only proteins directly or indirectly. The activated Bax is characterized as conformational changes from inactive to active, location changes from cytosol to mitochondria, and aggregation status changes from monomer to dimer or oligomers. Oligomerized Bax facilitates MOMP and promotes the formation of pores that can release apoptotic cytochrome c from the intermembrane space into cytosol (Kuwana et al. 2002). Cytochrome c, apoptotic protease activating factor 1 (Apaf-1), and procaspase 9 form a complex known as apoptosome which activates caspase 9 and other downstream executive caspases thereby ultimately leading to cell death (Liu et al. 2016a) (Fig. 2).
Apoptosis Regulator BAX, Fig. 2

Bax is a unique entry point for intrinsic (mitochondrial) signaling and participates in extrinsic apoptotic signaling pathway. Cyto c: cytochrome c

Bax also participates in the extrinsic signaling which is mediated by transmembrane death receptors. It reinforces the extrinsic signaling when caspase 8 cleaves Bid to generate the activated tBid. Moreover, it was reported that Bax or Bak is required for normal fusion of mitochondria into elongated tubules, indicating that Bcl-2 family proteins may regulate apoptosis via organelle morphogenesis machineries (Karbowski et al. 2006). In addition, Bax is involved in the endoplasmic reticulum (ER) signaling pathway. Mouse embryonic fibroblasts without Bax and Bak were found to be resistant to proapoptotic agents that induce unfolded protein response (UPR) and show a defect in steady-state ER calcium homeostasis under nonapoptotic conditions (Scorrano et al. 2003).

Bax interacts with many non-Bcl-2 proteins such as tumor suppressor p53 and inositol-requiring 1α (IRE1α) as well as Bcl-2 family members. The protein p53, a key tumor suppressor, is frequently inactivated in human cancers. In 1995, tumor suppressor p53 was found to be a direct transcriptional activator of the human bax gene (Miyashita and Reed 1995). p53 can activate Bax directly without other proteins to permeabilize mitochondria and engage the apoptotic program. It was proposed that when p53 accumulates in cytosol, it acts like the BH3-only subset of proapoptotic Bcl-2 members to activate Bax and trigger apoptosis (Chipuk et al. 2004).

Small Molecular Direct Bax Activators

Direct activation of Bax is considered as a novel and specific approach for cancer therapy and developing efforts on several direct Bax activators were recently reviewed (Liu et al. 2016a). Peptidomimetics developed from Bim and Bid were extensively explored as effective tools. They revealed novel binding site on Bax, defined Bax transitions toward apoptosis, and provided evidence for the therapeutic modulation of Bax. Several nonpeptide small molecular direct Bax activators (15, Fig. 3) have also been identified to induce cell death in a Bax-dependent fashion through direct binding to Bax in vitro and in vivo. BAM7 (1), obtained by an in silico screen using Glide 4.0, is a selective Bax binder with an IC50 value of 3.3 μM. It impairs the viability of Bak/ MEFs, but has no effects on Bax/ or DKO MEFs (Gavathiotis et al. 2012). BTC-8 (2), a derivative of BAM7, induces MOMP with an EC50 value of 700 nM which is more potent than BAM7 in cultured HuH7 cells. It is toxic for cancer cells and immortalized cells and has little effects on healthy cells (Stornaiuolo et al. 2015). ZINC 14750348 (3) fits well into the cavity in the Bax hydrophobic groove, according to the results of the virtual screening experiments. It triggers cell apoptosis in a Bax-dependent manner. Also, it inhibits lung tumor growth on the female C57BL/6 mice model implanted LLC cells at the dose of 40 mg/kg/day. In addition, it functions synergistically with carboplatin or ABT-737 (Zhao et al. 2014). SMBA1 (4), discovered by high throughput screening, suppresses nicotine-induced Bax phosphorylation in A549 cells and binds to Bax selectively with an IC50 value of 43.3 nM. It suppresses tumor growth in xenograft mice model bearing A549 cells while has no effects on Bax-deficient lung cancer xenograft derived from A549 expressing Bax siRNA at the effective dose of 40 mg/kg (Xin et al. 2014). Obatoclax (5), a BH3 mimetic, was also found to function as a direct activator of Bax. BH3-only protein mimetic was believed to bind the prosurvival Bcl-2 protein and acted as a sensitizer. This is the first time that BH3 mimetic was proposed as a direct activating stimulus for Bax activation (Smoot et al. 2010).
Apoptosis Regulator BAX, Fig. 3

Direct Bax activators 15

Summary

Bax as a critical proapoptotic member of Bcl-2 family proteins suppresses tumorigenesis and stimulates apoptosis. Interactions between Bcl-2 members control the fates of normal and cancer cells. Bax is not only the unique point for the intrinsic apoptotic pathway, but also participates in the extrinsic signaling. While a number of anticancer drugs in clinic induce Bax activation to facilitate apoptosis of cancer cells indirectly, it is promising to directly activate Bax with small molecules for cancer therapy. Currently, several classes of direct Bax activator have been identified to effectively induce Bax-mediate apoptosis in vitro and in vivo. It is believed that identification of more novel chemical entities directly targeting Bax is feasible facilitated by the assistance of modern drug discovery technologies and multidisciplinary approaches. The Bax activators alone or as combination therapies with other chemotherapeutic drugs are of great potential to benefit cancer patients in the years to come.

References

  1. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science. 2004;303:1010–4.CrossRefPubMedGoogle Scholar
  2. Czabotar PE, Westphal D, Dewson G, Ma S, Hockings C, Fairlie WD. Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell. 2013;152:519–31.CrossRefPubMedGoogle Scholar
  3. Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD. BH3-triggered structural reorganization drives the activation of proapoptotic BAX. Mol Cell. 2010;40:481–92.PubMedCentralCrossRefPubMedGoogle Scholar
  4. Gavathiotis E, Reyna DE, Bellairs JA, Leshchiner ES, Walensky LD. Direct and selective small-molecule activation of proapoptotic BAX. Nat Chem Biol. 2012;8:639–45.PubMedCentralCrossRefPubMedGoogle Scholar
  5. Han B, Park D, Li R, Xie M, Owonikoko TK, Zhang G, Sica GL, Ding C, Zhou J, Magis AT, Chen ZG, Shin DM, Ramalingam SS, Khuri FR, Curran WJ, Deng X. Small-molecule Bcl2 BH4 antagonist for lung cancer therapy. Cancer Cell. 2015;27:852–63.PubMedCentralCrossRefPubMedGoogle Scholar
  6. Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ. Role of Bax and Bak in mitochondrial morphogenesis. Nature. 2006;443:658–62.CrossRefPubMedGoogle Scholar
  7. Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 1995;270:96–9.CrossRefPubMedGoogle Scholar
  8. Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell. 2002;111:331–42.CrossRefPubMedGoogle Scholar
  9. Kvansakul M, Yang H, Fairlie WD, Czabotar PE, Fischer SF, Perugini MA. Vaccinia virus anti-apoptotic F1 L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands. Cell Death Differ. 2008;15:1564–71.CrossRefPubMedGoogle Scholar
  10. Liu Z, Ding Y, Ye N, Wild C, Chen H, Zhou J. Direct activation of Bax protein for cancer therapy. Med Res Rev. 2016a;36:313–41.CrossRefPubMedGoogle Scholar
  11. Liu Z, Wild C, Ding Y, Ye N, Chen H, Wold EA. BH4 domain of Bcl-2 as a novel target for cancer therapy. Drug Discov Today. 2016b;21:989–96.CrossRefPubMedGoogle Scholar
  12. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80:293–9.CrossRefPubMedGoogle Scholar
  13. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–19.CrossRefPubMedGoogle Scholar
  14. Robin AY; Krishna Kumar K; Westphal D; Wardak AZ; Thompson GV; Dewson G; Colman PM; Czabotar PE. Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction. Cell Death Dis. 2015;6:e1809.PubMedCentralCrossRefPubMedGoogle Scholar
  15. Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science. 2003;300:135–9.CrossRefPubMedGoogle Scholar
  16. Smoot RL, Blechacz BR, Werneburg NW, Bronk SF, Sinicrope FA, Sirica AE. A Bax-mediated mechanism for obatoclax-induced apoptosis of cholangiocarcinoma cells. Cancer Res. 2010;70:1960–9.PubMedCentralCrossRefPubMedGoogle Scholar
  17. Stornaiuolo M, La Regina G, Passacantilli S, Grassia G, Coluccia A, La Pietra V. Structure-based lead optimization and biological evaluation of BAX direct activators as novel potential anticancer agents. J Med Chem. 2015;58:2135–48.CrossRefPubMedGoogle Scholar
  18. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–54.CrossRefPubMedGoogle Scholar
  19. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001;292:727–30.PubMedCentralCrossRefPubMedGoogle Scholar
  20. Xin M, Li R, Xie M, Park D, Owonikoko TK, Sica GL, Corsino PE, Zhou J, Ding C, White MA, Magis AT, Ramalingam SS, Curran WJ, Khuri FR, Deng X. Small-molecule Bax agonists for cancer therapy. Nat Commun. 2014;5:4935.PubMedCentralCrossRefPubMedGoogle Scholar
  21. Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B. Role of BAX in the apoptotic response to anticancer agents. Science. 2000;290:989–92.CrossRefPubMedGoogle Scholar
  22. Zhao G, Zhu Y, Eno CO, Liu Y, Deleeuw L, Burlison JA. Activation of the proapoptotic Bcl-2 protein Bax by a small molecule induces tumor cell apoptosis. Mol Cell Biol. 2014;34:1198–207.PubMedCentralCrossRefPubMedGoogle Scholar

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Chemical Biology Program, Department of Pharmacology and ToxicologyUniversity of Texas Medical BranchGalvestonUSA