W D Fairlie

Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

Crystal structure of ABT-737 complexed with Bcl-xL: implications for selectivity of antagonists of the Bcl-2 family.

Crystal structure of ABT-737 complexed with Bcl-x L : implications for selectivity of antagonists of the Bcl-2 family

Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands

Apoptosis is an important part of the hosts defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the α1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.

Bcl-2, Bcl-x(L), and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells.

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

Conformational Changes in Bcl-2 Pro-survival Proteins Determine Their Capacity to Bind Ligands

Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-xL, and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-xL and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-xL, a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.

Structure-guided rational design of α/β-peptide foldamers with high affinity for BCL-2 family prosurvival proteins.

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐xL but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐xL was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐xL. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐xL validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.

Discovery and molecular characterization of a Bcl-2–regulated cell death pathway in schistosomes

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2–regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2–like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with “BH3 mimetic” drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

Functional genomics approaches in parasitic helminths

As research on parasitic helminths is moving into the post‐genomic era, an enormous effort is directed towards deciphering gene function and to achieve gene annotation. The sequences that are available in public databases undoubtedly hold information that can be utilized for new interventions and control but the exploitation of these resources has until recently remained difficult. Only now, with the emergence of methods to genetically manipulate and transform parasitic worms will it be possible to gain a comprehensive understanding of the molecular mechanisms involved in nutrition, metabolism, developmental switches/maturation and interaction with the host immune system. This review focuses on functional genomics approaches in parasitic helminths that are currently used, to highlight potential applications of these technologies in the areas of cell biology, systems biology and immunobiology of parasitic helminths.

Characterisation of a novel A1-specific monoclonal antibody.

Dear Editor, A1/BFL-1 is the least studied pro-survival BCL-2 family member. This can be largely attributed to the lack of proper tools to study A1/BFL-1 function. Owing to the genomic organisation of the A1 locus in mice (three expressed A1 genes and one pseudo-gene, interspersed by unrelated genes)1 a knockout is challenging. We generated shRNA transgenic mice in which all functional A1 isoforms were knocked down. In accordance with A1 mRNA expression studies, we found that A1 is critical for the development and survival of lymphocytes and granulocytes.2 As the A1/BFL-1 protein is regulated by ubiquitin-dependent proteasomal degradation, the A1 mRNA expression data may not truly reflect the A1/BFL-1 protein levels. Previous attempts to generate A1-specific antibodies have failed and commercially available antibodies do not reliably detect the endogenous protein. To generate A1-specific monoclonal antibodies, we immunised rats with a truncated/mutated A1 protein (delta-C20, P104K)3 together with two KLH-conjugated peptides corresponding to central and C-terminal residues of the A1 protein (aa71–84; aa129–154). Screening by ELISA and western blotting identified one monoclonal antibody that detected overexpressed A1-a, A1-b and A1-d, and to a lesser extent overexpressed human homologue BFL-1 (data not shown and Figure 1a). To test whether this antibody could reliably detect endogenous A1, we used the mouse WEHI-231 B lymphoma cells, known to express high levels of this protein.4 Western blotting revealed a single band of the molecular weight expected for A1 in untreated WEHI-231 cells (Figure 1b, first lane). Overexpressed A1 protein is highly unstable due to ubiquitin-dependent proteasomal degradation.5 To further verify the specificity of the A1 antibody, we tested the impact of protein synthesis inhibition or proteasome inhibition on the protein detected in WEHI-231 cells. As expected, the protein synthesis inhibitor cyclohexamide (CHX) decreased the intensity of the protein band, whereas the proteasome inhibitor (MG132) increased it substantially (Figure 1b). Furthermore, we were able to show that this antibody can be used to immunoprecipitate endogenous A1 protein from lysates of WEHI-231 cells (Figure 1c). Next we examined whether this antibody could also detect endogenous A1 in primary mouse cells. In accordance with previous reports on A1 mRNA expression,1 we could reliably detect A1 protein in haematopoietic tissues, such as the lymph nodes and spleen but not in the heart, kidney, liver or lungs (Figure 1d). Immunohistochemical staining using this antibody showed strong A1 protein staining within cell foci in the germinal centres of lymph nodes of non-immunised mice (Figure 1e). No staining with this antibody against A1 was observed in non-haematopoietic tissues, such as the pancreas or the heart (data not shown). To further validate the specificity of this A1 antibody in primary cells, mouse spleen cells were treated with crosslinking IgM antibodies, a stimulus known to upregulate A1 mRNA levels in B lymphocytes.6 Such BCR (B-cell receptor) stimulation increased the protein band detected by our A1 antibody and its density was further augmented when cells were additionally treated with the proteasome inhibitor MG132 during the last hour of the stimulation (Figure 1f). A1 mRNA levels are upregulated when bone marrow cells are treated with GM-CSF or when mast cells are stimulated with the calcium ionophore ionomycin.7, 8 These stimuli caused strong upregulation of the protein band detected by the A1 antibody and the density of this protein band was further increased by the addition of MG132 during the last hour of stimulation (Figures 1g and h). Finally, we validated the specificity of the antibody by using our A1 knockdown mice. In cells from these animals high GFP levels indicate high levels of A1 shRNA expression and thus low levels of endogenous A1 protein.2 We therefore FACS-sorted GFP-positive and GFP-negative spleen cells and treated them with concanavalin A (ConA), a stimulus known to upregulate A1 mRNA levels in T cells.9 As expected, our antibody detected a protein band of the molecular weight predicted for A1 in ConA-stimulated GFP-negative cells but not in the GFP-positive (i.e. A1 shRNA expressing) splenocytes (Figure 1i). This confirms the specificity of our A1 antibody. Figure 1 The newly developed A1 antibody reliably detects the endogenous levels of the pro-survival BCL-2 family member A1. (a) EYZ (control), A1-a, -b, -d and BFL-1 expression vectors were transiently transfected into 293T cells and protein lysates (total protein ... In conclusion, we present here for the first time a mouse A1-specific monoclonal antibody capable of detecting endogenous A1 protein in cell lines as well as in primary mouse cells. Unfortunately, this antibody does not recognise endogenous levels of human BFL-1 (data not shown). This antibody will be made available commercially.