W. Douglas Fairlie

Structural insights into the degradation of Mcl-1 induced by BH3 domains

Apoptosis is held in check by prosurvival proteins of the Bcl-2 family. The distantly related BH3-only proteins bind to and antagonize them, thereby promoting apoptosis. Whereas binding of the BH3-only protein Noxa to prosurvival Mcl-1 induces Mcl-1 degradation by the proteasome, binding of another BH3-only ligand, Bim, elevates Mcl-1 protein levels. We compared the three-dimensional structures of the complexes formed between BH3 peptides of both Bim and Noxa, and we show that a discrete C-terminal sequence of the Noxa BH3 is necessary to instigate Mcl-1 degradation.

Bax Crystal Structures Reveal How Bh3 Domains Activate Bax and Nucleate its Oligomerization to Induce Apoptosis.

In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each others surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family and its receptor, Fas, are critical for shutdown of chronic immune responses1-3 and prevention of autoimmunity4,5. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding10,11. We generated gene-targeted mice that selectively lack either secreted FasL (ΔsFasL) or membrane-bound FasL (ΔmFasL) to resolve which of these forms is required for cell killing and to explore their hypothetical non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, (on a C57BL/6 background) FasLΔm/Δm mice succumbed to SLE-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasLΔm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1.

Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-β receptor II, extracellular signal–regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.

Concentration in plasma of macrophage inhibitory cytokine-1 and risk of cardiovascular events in women: a nested case-control study.

BACKGROUND Macrophage inhibitory cytokine-1 (MIC-1) is part of the TGF-beta superfamily. Raised concentrations of MIC-1 in serum arise in several disease states, can be detected in normal individuals, and can partly be genetically determined. We aimed to investigate whether MIC-1 has a role in atherothrombosis. METHODS We did a prospective, nested, case-control study in 27628 initially healthy women. Of these women, we established baseline concentrations of MIC-1 in 257 who subsequently had myocardial infarction, stroke, or died from a cardiovascular event (cases) and in 257 matched for age and smoking status, who did not report cardiovascular disease during 4-year follow-up (controls). We also assessed polymorphisms in the MIC-1 gene (MIC-1 H and MIC-1 D) in all 514 women. FINDINGS MIC-1 concentrations were higher at baseline in women who subsequently had cardiovascular events than in those who did not (618 vs 538 pg/mL, p=0.0002). Concentrations above the 90th percentile (>856 pg/mL) were associated with a 2.7-fold increase in risk (95% CI 1.6-4.9, p=0.001). This effect was independent of traditional cardiovascular risk factors and at least additive to that of C-reactive protein. There was no significant association between MIC-1 polymorphism and vascular events. INTERPRETATION MIC-1 could be a novel target for cardiovascular disease prevention.

Structure of the BH3 Domains from the p53-Inducible BH3-Only Proteins Noxa and Puma in Complex with Mcl-1

Pro-survival proteins in the B-cell lymphoma-2 (Bcl-2) family have a defined specificity profile for their cell death-inducing BH3-only antagonists. Solution structures of myeloid cell leukaemia-1 (Mcl-1) in complex with the BH3 domains from Noxa and Puma, two proteins regulated by the tumour suppressor p53, show that they bind as amphipathic alpha-helices in the same hydrophobic groove of Mcl-1, using conserved residues for binding. Thermodynamic parameters for the interaction of Noxa, Puma and the related BH3 domains of Bmf, Bim, Bid and Bak with Mcl-1 were determined by calorimetry. These unstructured BH3 domains bind Mcl-1 with affinities that span 3 orders of magnitude, and binding is an enthalpically driven and entropy-enthalpy-compensated process. Alanine scanning analysis of Noxa demonstrated that only a subset of residues is required for interaction with Mcl-1, and these residues are localised to a short highly conserved sequence motif that defines the BH3 domain. Chemical shift mapping of Mcl-1:BH3 complexes showed that Mcl-1 engages all BH3 ligands in a similar way and that, in addition to changes in the immediate vicinity of the binding site, small molecule-wide structural adjustments accommodate ligand binding. Our studies show that unstructured peptides, such as the BH3 domains, behave like their structured counterparts and can bind tightly and selectively in an enthalpically driven process.

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition*

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24–Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2–like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)–only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.

Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax

A central issue in the control of apoptosis is whether its essential mediators Bax and Bak must be restrained by Bcl-2-like prosurvival relatives to prevent their damaging mitochondria and unleashing apoptosis. The issue is particularly vexed for Bax, which is largely a cytosolic monomer in unstressed cells. To determine whether Bax regulation requires its binding by prosurvival relatives, we replaced a conserved aspartate in its BH3 interaction domain with arginine. Bax D68R functioned and behaved like wild-type Bax in localization and activation but had greatly impaired binding to the prosurvival family members. Nevertheless, Bcl-xL remained able to block apoptosis induced by Bax D68R. Whereas cells with sufficient Bcl-xL tolerated expression of Bax D68R, it provoked apoptosis when Bcl-xL was absent, downregulated, or inactivated. Moreover, Bax D68R rendered membrane bound by a C-terminal anchor mutation overwhelmed endogenous Bcl-xL and killed cells. These unexpected results suggest that engagement of Bax by its prosurvival relatives is a major barrier to its full activation. We propose that the Bcl-2-like proteins must capture the small proportion of Bax molecules with an exposed BH3 domain, probably on the mitochondrial membrane, to prevent Bax-imposed cell death, but that Bcl-xL also controls Bax by other mechanisms.

High-Resolution Structural Characterization of a Helical α/β-Peptide Foldamer Bound to the Anti-Apoptotic Protein Bcl-xL†

Get into the groove: The first high-resolution structure of a foldamer bound to a protein target is described (see picture; foldamer in sticks). The foldamer consists of alpha- and beta-amino acid residues and is bound to the anti-apoptotic protein Bcl-x(L). The overall binding mode and key interactions observed in the foldamer/Bcl-x(L) complex mimic those seen in complexes of Bcl-x(L) with natural alpha-peptide ligands. Additional contacts in the foldamer/Bcl-x(L) complex involving beta-amino acid residues appear to contribute to binding affinity.