Utility of epimerization domains for the redesign of nonribosomal peptide synthetases

Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block. Very often, d-amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound l-intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two 'peptidyl-' and two 'aminoacyl-'E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP-E bidomain apo-proteins (TycB(3)-, FenD(2)-, TycA- and GrsA-PCP-E) were primed with various peptidyl-CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl-S-Ppant epimerization products were chemically cleaved and analyzed for their l/d-ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl-S-Ppant-substrates as evaluated by k(obs) values and final l/d-product equilibria determined for each reaction. The two C-terminal amino acids of the substrate seem to be recognized by 'peptidyl-'E domains. Interestingly, the 'aminoacyl-'E domains GrsA- and TycA-E were also able to convert the elongated intermediates. All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation (C) domain of TycB(1) is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts.     

A novel approach to specific allergy treatment: the recombinant fusion protein of a bacterial cell surface (S-layer) protein and the major birch pollen allergen Bet v 1 (rSbsC-Bet v 1) combines reduced allergenicity with immunomodulating capacity

Counterregulating the disease-eliciting Th2-like immune response of allergen-specific Th lymphocytes by fostering an allergen-specific Th1-like response is a promising concept for future immunotherapy of type I allergy. The use of recombinant allergens combined with more functional adjuvants has been proposed. In this respect, we present a novel approach. The gene sequence encoding the major birch pollen allergen, Bet v 1, was fused with the gene encoding the bacterial cell surface (S-layer) protein of Geobacillus stearothermophilus, resulting in the recombinant protein, rSbsC-Bet v 1. rSbsC-Bet v 1 contained all relevant Bet v 1-specific B and T cell epitopes, but was significantly less efficient to release histamine than rBet v 1. In cells of birch pollen-allergic individuals, rSbsC-Bet v 1 induced IFN-gamma along with IL-10, but no Th2-like response, as observed after stimulation with Bet v 1. Intracellular cytokine staining revealed that rSbsC-Bet v 1 promoted IFN-gamma-producing Th cells. Moreover, rSbsC-Bet v 1 induced IFN-gamma synthesis in Bet v 1-specific Th2 cell clones, and importantly, increased IL-10 production in these cells. In conclusion, genetic fusion of an allergen to S-layer proteins combined reduced allergenicity with immunomodulatory capacity. The strategy described in this work may be generally applied to design vaccines for specific immunotherapy of type I allergy with improved efficacy and safety.     

Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations

Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. Here, we address the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. First, we find that Fgf signalling is required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. Second, combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with our model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations.     

Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. We report here that the bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together our data demonstrate a role of Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homologue Hairy.     

Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1)

Glycosylation determines essential biological functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially determined by O-glycosylation at its threonine with either GalNAcalpha1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.     

Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.