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.

BCL-XL is an actionable target for treatment of malignant pleural mesothelioma.

Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM.

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.

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 host's 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 alpha1 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.

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13.

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

Relaxin and prostaglandin E(2) regulate interleukin 11 during human endometrial stromal cell decidualization.

Decidualization of endometrial stromal cells and IL-11 signaling are essential for embryo implantation in the mouse. We investigated the effects of relaxin (RLX) and prostaglandin E(2) (PGE(2)) on IL-11 secretion by human endometrial stromal cells (HESC) and during cAMP or medroxyprogesterone acetate (P)-induced decidualization. cAMP-decidualized HESC secreted high levels of IL-11. RLX, cAMP, or PGE(2) increased IL-11 mRNA and IL-11 secretion, with maximal response to RLX and cAMP. Addition of the cAMP/protein kinase A inhibitor Rp-adenosine-3,5-cyclic-monophosphorothioate to either RLX- or PGE(2)-treated cells decreased IL-11 secretion. Indomethacin treatment decreased IL-11 secretion, which was largely restored by cotreatment with PGE(2) or RLX. Cotreatment of HESC with RLX, PGE(2), or cAMP and estrogen plus P down-regulated IL-11 mRNA and IL-11 secretion at 24 h, before secretion of prolactin (decidualization marker). Addition of W147AIL-11 (IL-11 signaling inhibitor) reduced prolactin secretion stimulated by RLX or PGE(2) and estrogen plus P. This is the first demonstration that cAMP-decidualized HESC secrete IL-11 and that IL-11 mRNA and IL-11 secretion are regulated by RLX and PGE(2), partly via a cAMP/protein kinase A-dependent pathway. Blocking IL-11 signaling reduced RLX+P- or PGE(2)+P-induced decidualization, suggesting that RLX and PGE(2) act via IL-11. This is important in understanding implantation and regulation of fertility.

MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation.

As part of a study to identify novel genes associated with macrophage activation, we have cloned a new member of the transforming growth factor beta (TGF-beta) superfamily designated macrophage inhibitory cytokine 1 (MIC-1). MIC-1 is synthesized as a 62-kDa intracellular protein, which, after cleavage by a furin like protease, is secreted as a 25-kDa disulfide-linked dimeric protein. Sequence analysis indicates that it does not cluster within any existing TGF-beta families, suggesting it may be the first member of a new grouping within the TGF-beta superfamily. Tissue Northern blots show that MIC-1 transcripts are only found abundantly in placenta, although smaller amounts are seen in a limited number of other adult and fetal tissues. MIC-1 is not expressed in resting macrophages but is induced by a number of different activation agents, including phorbol myristate acetate, interleukin 1, tumor necrosis factor alpha, and macrophage colony-stimulating factor but not by lipopolysaccharide or interferon-gamma. We have hypothesized that it may be an autocrine inhibitor of macrophage activation but its major biological role is still uncertain.

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.

The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women.

Macrophage inhibitory cytokine-1 (MIC-1) is a recently described divergent member of the transforming growth factor-ss superfamily. MIC-1 transcription up-regulation is associated with macrophage activation, and this observation led to its cloning. Northern blots indicate that MIC-1 is also present in human placenta. A sensitive sandwich enzyme-linked immunosorbent assay for the quantification of MIC-1 was developed and used to examine the role of this cytokine in pregnancy. High levels of MIC-1 are present in the sera of pregnant women. The level rises substantially with progress of gestation. MIC-1 can also be detected, in large amounts, in amniotic fluid and placental extracts. In addition, the BeWo placental trophoblastic cell line was found to constitutively express the MIC-1 transcript and secrete large amounts of MIC-1. These findings suggest that the placental trophoblast is a major source of the MIC-1 present in maternal serum and amniotic fluid. We suggest that MIC-1 may promote fetal survival by suppressing the production of maternally derived proinflammatory cytokines within the uterus.

Expression of a TGF-beta superfamily protein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine. This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism. The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems. Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system. Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase. However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted.

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Antibody-based approach to high-volume genotyping for MIC-1 polymorphism.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.

Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays.

Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein-protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein-protein interactions within the cell.

The BH3 mimetic compound, ABT-737, synergizes with a range of cytotoxic chemotherapy agents in chronic lymphocytic leukemia.

As chronic lymphocytic leukemia (CLL) is characterized by overexpression of pro-survival BCL2, compounds that mimic its physiological antagonists, the BH3-only proteins, may have a role in treatment of this disease. ABT-737 is a BH3 mimetic compound that selectively targets BCL2 and BCLX(L). In the present work, we report that ABT-737 is highly effective (LC(50)<50 nM) as a single agent against most (21/30) primary CLL samples, but that a sizable minority is relatively insensitive. In vitro sensitivity to ABT-737 could not be simply predicted by the patients' clinical features, including response to prior therapy or known prognostic markers (CD38 expression, 17p deletion), or the relative expression of BCL2 family proteins (BCL2, MCL1, BAX, BIM). Strikingly, co-incubation with cytotoxic agents (dexamethasone, etoposide, fludarabine, doxorubicin) sensitized most CLL samples to ABT-737, but this could not be predicted by responses to either ABT-737 or the cytotoxic agent alone. Of 17 samples least sensitive to ABT-737, 13 were sensitized by co-treatment with at least one cytotoxic agent. These data indicate that combination of ABT-737 with a second anti-leukemic agent would improve response rates and suggest a potential role for combination therapies that include BH3 mimetics for the treatment of this disease.

The disulphide bond structure of thyroid-stimulating hormone beta-subunit.

Previously only one of the six disulphide bonds within the beta-subunit of bovine thyrotropin (bTSH beta) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSH beta, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88-Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16-Cys67 and Cys19-Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatography, of a partly reduced bTSH beta derivative in which only the half-cystine residues Cys31, Cys85, Cys88 and Cys95 were labelled enabled the assignment of a previously uncharacterized disulphide bond to Cys31-Cys85. The remaining two assignments, Cys2-Cys52 and Cys27-Cys83, were made by comparison with the recently published human chorionic gonadotropin crystal structure. The flexibility of the double-labelling approach used in these studies demonstrates that only very small quantities are required for proteins containing an extensive number of half-cystine residues such as TSH beta, owing to the combination of the high resolution of the reversed-phase HPLC peptide mapping procedures and the sensitivity of the fluorimetric detection method.

Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites.