A key step in cell death involves formation of Bak dimers via a BH3: groove interaction
G Dewson, TK Kratina, HW Sim, H Puthalakath, JM Adams, RM Kluck in collaboration with PM Colman (Structural Biology) Pub ref: 38
The pivotal step in a cell’s commitment to apoptosis is the puncture of the mitochondrial membrane by the pro-apoptotic Bcl-2 family members Bak or Bax. How they do so is poorly understood, but their assembly into large complexes (oligomers) in the mitochondrial membrane is strongly implicated.
Our studies on Bak have revealed how this process begins. Bak (and Bax) possess a helical segment, called the BH3 region, that can bind into a groove on the surface of other Bcl-2 family members. Upon apoptotic signalling, we found that Bak molecules expose this domain (α-helix 2). We showed that Bak then self associates by inserting the BH3 domain into the groove of another Bak molecule, (path B). The Bak doublet may then aggregate into the larger oligomers that puncture the mitochondria and provoke the cell’s self-destruction.
In cells that return active pro-survival family members, they can capture any activated Bak by its exposed BH3 domain (path A) and ensure cell survival. Thus cell fate is dicided when the Bak BH3 is exposed.
Our insights into how Bak, and presumably also Bax, changes conformation and oligomerizes to permeabilise mitochondria should assist in the development of drugs that can flick on the apoptosis “switch” to kill cancer cells more effectively and perhaps eventually also drugs that can switch off the unwarranted apoptosis in degenerative disorders.
Bak activation during apoptosis. Upon an apoptotic signal, Bak exposes its BH3 domain (α-helix 2). (A) In cells destined to survive, this domain may become re-buried within the groove of pro-survival member Bcl-xL or Mcl-1. (B) In cells destined to die because neither Bcl-xL nor Mcl-1 remains active, the Bak BH3 domain binds instead into the groove of another Bak molecule, perhaps as a symmetric dimer.
In several cell types the tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute
EM Michalak, JM Adams, A Strasser in collaboration with A Villunger (Biocenter, Innsbruck Medical University, Innsbruck, Austria) Pub ref: 103
Puma and Noxa are pro-apoptotic BH3-only proteins induced transcriptionally by the tumour suppressor p53. To examine their roles in p53-mediated apoptosis, we generated mice lacking both. In embryonic fibroblasts, their absence prevented induction of apoptosis by the DNA damaging agent etoposide. Moreover, following whole body γ-irradiation, their loss protected thymocytes better than loss of Puma alone, indeed as strongly as loss of p53 itself. These results indicate that p53-induced apoptosis proceeds principally via Puma, with Noxa having a minor role. The absence of tumours in the mice suggests that tumour suppression by p53 requires functions in addition to induction of apoptosis.
Pro-apoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic β cells
T Kaufmann, DCS Huang, A Strasser in collaboration with MD McKenzie, E Carrington, TWH Kay, HE Thomas (St. Vincent’s Institute of Medical Research, Melbourne) Pub ref: 98
Apoptosis of pancreatic β cells causes diabetes and failure of islet transplantation. To clarify the role of Bcl-2 family members, we studied the responses to diverse cytotoxic stimuli of islets from mice lacking the pro-apoptotic BH3-only protein Bid or the multi-BH-domain pro-apoptotic members Bax or Bak. Bid-deficient islets were protected from apoptosis induced by Fas ligand or TNFα plus cycloheximide and were partially resistant to pro-inflammatory cytokine-induced death. Loss of Bax or Bak provided partial protection. These results indicate that blocking Bid activity may help to protect islets in pathological states that destroy β cells, such as diabetes.
Targeted deletion of the BH3-only protein gene bmf impairs B cell homeostasis and accelerates γ-radiation-induced thymic lymphoma development
LA O’Reilly, A Strasser in collaboration with V Labi, M Erlacher, S Kiessling, C Manzl, A Frenzel, A Villunger (Biocenter, Innsbruck Medical University, Innsbruck, Austria) Pub ref: 83
The Bcl-2 protein family maintains tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Its pro-apoptotic BH3-only family members are the essential triggers of cell death. The eight or more BH3-only proteins in mammals are activated in both a stimulus- and cell type-specific manner. BH3-only proteins then activate, either directly or indirectly through binding pro-survival relatives, their pro-apoptotic relatives Bax and Bak, which impose cell death by damaging the mitochondria.
We have investigated the role in cell death signalling of the BH3-only protein Bmf by generating mice with its gene disrupted. Our studies revealed that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV-irradiation. Notably, however, Bmf loss protected lymphocytes against apoptosis induced by glucocorticoids or HDAC-inhibition. Moreover, the resistance of the B cells of bmf-/- mice to diverse apoptotic stimuli led to lymphadenopathy. Finally, Bmf-deficiency accelerated the development of γ-irradiation-induced thymic lymphomas.
Thus, our results demonstrate that Bmf plays a critical role in apoptosis signalling in several cell types and can function as a tumor suppressor.
Accelerated development of γ-irradiation-induced thymic lymphomas in mice lacking the bmf gene. The acceleration from Bmf-deficiency was similar to that in mice lacking one allele of the p53 tumour suppressor.
BH3-only proteins Bim and Puma are essential for the death of antigen-specific B and T cells during shutdown of acute immune responses
SF Fischer, P Bouillet, A Strasser in collaboration with DM Tarlinton, GT Belz (Immunology Division) Pub ref: 50, 51
During acute T cell immune responses, such as to viral infection, antigen-specific B and T cells proliferate and differentiate into effector cells, but after pathogen clearance most are deleted by apoptosis to preclude collateral damage to healthy tissues. However, using gene-targeted mice immunised with a B cell antigen or infected with herpes simplex virus, we found that mice lacking either the pro-apoptotic BH3-only protein Bim or Puma had persistent antigen-stimulated B and T effector lymphocytes. These findings show that Bim and Puma are essential for eliminating antigen-specific B and T cells during shutdown of immune responses.
A crystal structure for Bcl-xL bound to ABT-737
EF Lee, PE Czabotar, BJ Smith, PM Colman, WD Fairlie in collaboration with K Deshayes, K Zobel (Genentech, San Francisco, CA USA)
ABT-737 is the precursor of a new cancer therapeutic, ABT-263, which Abbott Laboratories have recently taken into clinical trials. A detailed understanding of the interaction between ABT-737 and its targets, members of the Bcl-2 protein family, will help to explain its selectivity for certain family members and guide future drug discovery. We obtained an atomic resolution image of the complex with Bcl-xL, which showed the extent to which the compound is mimicking the natural cell death-inducing ligand, a helical peptide known as a BH3 domain. The structure also highlighted novel features of the family member Mcl-1, to which ABT-737 does not bind.
A cut-away drawing of the surface of Bcl-xL (grey) binding to the BH3 peptide of Bim (green) or to ABT-737 (yellow). The BH3 peptide projects four hydrophobic amino acids into pockets in the Bcl-xL surface. ABT-737 similarly engages two of these four pockets.
A novel BH3 ligand selectively neutralises Mcl-1 pro-survival activity without triggering its degradation
EF Lee, PE Czabotar, PM Colman, WD Fairlie in collaboration with MF van Delft, E Michalak, MJ Boyle, SN Willis, H Puthalakath, P Bouillet, DCS Huang, (Molecular Genetics of Cancer Division) Pub ref: 85
Mcl-1 is an important survival factor for many cancers. However, unlike other pro-survival proteins, Mclâ€‘1 stability is acutely regulated. We have discovered and characterised a novel BH3-like ligand derived from Bim, BimS2A, highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mclâ€‘1 degradation yet, like Noxa, BimS2A promotes cell killing only when Bclâ€‘xL is inactivated. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonised by BH3-like ligands engaging its binding groove. Our data have important implications for discovering compounds that kill cells whose survival depends on Mcl-1.
The structure of the Bcl-2 family protein A1
M Hinds, PE Czabotar in collaboration with C Smits, CL Day (University of Otago, New Zealand) Pub ref: 129
We have solved the structures of the pro-survival Bcl-2 protein A1 in complex with the binding domains of the pro-death BH3-ligands: Puma, Bid, Bmf and Bak. A1 provides a unique molecular surface for BH3 binding and significant conformational plasticity was observed in its interactions, but contacts between conserved residues on the BH3-ligand and A1 defined optimal binding. A1’s ability to interact with multiple BH3-ligands is critical to its ability to regulate cell death and understanding the atomic features of these interactions may provide a molecular basis for designing antagonists to specifically target A1 and initiate cell death in tumour cells.
The structure of vaccinia virus F1L
M Kvansakul, H Yang, WD Fairlie, PE Czabotar, PM Colman in collaboration with SF Fischer, DCS Huang (Molecular Genetics of Cancer Division), MA Perugini (University of Melbourne) Pub ref: 82
The F1L protein of vaccinia (and smallpox) virus is believed to perform a similar function to the Bcl-2 protein family, i.e. to promote cell survival. This may be important in keeping the virus-infected cell alive to sustain viral replication. The crystal structure of F1L is strongly reminiscent of the Bcl-2 structure despite having no amino acid sequence similarity to any previously known Bcl-2 family member. One fascinating feature is that the molecule exists as a novel dimer, both in crystals and in solution, and has enabled us to characterise an engineered form of Bcl-xL that similarly dimerises.