Beschreibung: Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa . We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX 1 -Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX 1 -Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.

Beschreibung: Genes involved in detoxification of foreign compounds exhibit complex spatiotemporal expression patterns in liver. Cytochrome P450 1A1 ( CYP1A1 ), for example, is restricted to the pericentral region of liver lobules in response to the interplay between aryl hydrocarbon receptor (AhR) and Wnt/β-catenin signaling pathways. However, the mechanisms by which the two pathways orchestrate gene expression are still poorly understood. With the help of 29 mutant constructs of the human CYP1A1 promoter and a mathematical model that combines Wnt/β-catenin and AhR signaling with the statistical mechanics of the promoter, we systematically quantified the regulatory influence of different transcription factor binding sites on gene induction within the promoter. The model unveils how different binding sites cooperate and how they establish the promoter logic; it quantitatively predicts two-dimensional stimulus-response curves. Furthermore, it shows that crosstalk between Wnt/β-catenin and AhR signaling is crucial to understand the complex zonated expression patterns found in liver lobules. This study exemplifies how statistical mechanical modeling together with combinatorial reporter assays has the capacity to disentangle the promoter logic that establishes physiological gene expression patterns.

Beschreibung: Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l –/– mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.

Beschreibung: miR-661 expression in SNAI1-induced epithelial to mesenchymal transition contributes to breast cancer cell invasion by targeting Nectin-1 and StarD10 messengers Oncogene 34, 3884 (July 2015). doi:10.1038/onc.2015.265 Authors: G Vetter, A Saumet, M Moes, L Vallar, A Le Béchec, C Laurini, M Sabbah, K Arar, C Theillet, C-H Lecellier & E Friederich

Beschreibung: In intestinal and pyloric epithelia leucine-rich repeat-containing G protein-coupled receptor 5 ( Lgr5 )-expressing cells represent long-lived adult stem cells which give rise to all epithelial cell types including endocrine cells. Ablation of the Apc gene in Lgr5 -expressing cells leads to intestinal and pyloric adenomas. To assess whether all epithelial tumours of the gastrointestinal tract are derived from LGR5-positive stem cells we crossed Lgr5-EGFP-IRES-creER T2 mice which express EGFP and Cre recombinase driven by the Lgr5 promoter with CEA424-SV40-TAg mice which develop pyloric neuroendocrine carcinomas of epithelial origin. In 19-day-old mice, single SV40 T antigen (TAg)-positive cells were identified preferentially at the the base of pyloric glands close to the stem cell compartment. However, contrary to previous publications describing subpopulations of LGR5-positive cells in gastrointestinal neoplasia we could not detect Lgr5- EGFP-positive tumour cells in malignant lesions. The lack of expression of the Wnt target gene Lgr5 is probably not caused by suppression of Wnt signalling by TAg since β-catenin-mediated Wnt signalling, as measured by the TOPflash assay, was not inhibited. To determine the cellular origin of CEA424-SV40-TAg tumours we performed tracing experiments using Lgr5-EGFP-IRES-creERT2:CEA424-SV40-TAg:ROSA26-tdRFP mice. Following tamoxifen induction it was possible to efficiently trace the progeny of Lgr5 -expressing cells in gastrointestinal tissue via red fluorescent protein (RFP) expression. No RFP-positive tumour cells were detected even when RFP gene activation occurred in 7-day-old mice well before the appearance of TAg-positive tumour cells. Hence we conclude that Lgr5 -expressing stem cells probably do not constitute the cells-of-origin in CEA424-SV40-TAg mice. Consequently, not all epithelial tumours in the pyloric region are initiated by transformation of LGR5-positive stem cells. Thus additional long-lived LGR5-negative stem cells or progenitor cells with low turnover rate might exist in the pyloric region which can give rise to tumours.

Beschreibung: Nature Structural & Molecular Biology 22, 695 (2015). doi:10.1038/nsmb.3065 Authors: Melanie Vetter, Ralf Stehle, Claire Basquin & Esben Lorentzen

Beschreibung: Providing enantiomerically pure products is of key importance in the fine chemicals, food, and pharmaceutical industries. A continuous preferential crystallization process is presented that allows the separation of conglomerate forming enantiomers in a stable, robust, and flexible way. This is achieved by coupling two continuous crystallizers by exchanging their clear liquid phases. Each crystallizer is connected to a suspension mill responsible for in situ seed generation through particle breakage. The dynamic and steady-state behavior of this process is extensively analyzed for racemic feed streams through process simulations, and parameter regions, which yield pure enantiomers in both crystallizers, are identified. For enriched feed streams, it is further shown when this novel flow sheet is capable of outperforming an ideal batch process in terms of solvent consumption per unit mass of desired enantiopure product produced. © 2015 American Institute of Chemical Engineers AIChE J , 61: 2810–2823, 2015

Beschreibung: Background: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid β-oxidation and a target disease of newborn screening in many countries.Case presentationWe report on two siblings with mild MCAD deficiency associated with a novel splice site mutation in the ACADM gene. The younger sibling was detected by newborn screening, while the older sister was missed, but diagnosed later on by genetic family testing. Both children were found to be compound heterozygous for the common c.985A 〉 G (p.K329E) mutation and a novel splice site mutation, c.600-18G 〉 A, in the ACADM gene. To determine the biological consequence of the c.600-18G 〉 A mutation putative missplicing was investigated at RNA level in granulocytes and monocytes of one of the patients. The splice site mutation was shown to lead to partial missplicing of the ACADM pre-mRNA. Of three detected transcripts two result in truncated, non-functional MCAD proteins as reflected by the reduced octanoyl-CoA oxidation rate in both patients. In one patient a decrease of the octanoyl-CoA oxidation rate was found during a febrile infection indicating that missplicing may be temperature-sensitive. Conclusions: Our data indicate that the c.600-18G 〉 A variant activates a cryptic splice site, which competes with the natural splice site. Due to only partial missplicing sufficient functional MCAD protein remains to result in mild MCADD that may be missed by newborn screening.

Beschreibung: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (Na V ) channels, we screened spider venoms for inhibitors of human Na V 1.7 (hNa V 1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na V 1.7 inhibitor, μ -TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens . Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa V 1.7 〉 hNa V 1.6 〉 hNa V 1.2 〉 hNa V 1.1 〉 hNa V 1.3 channels in fluorescent assays. Na V 1.7 inhibition was diminished (IC 50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC 50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNa V 1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μ M. Unlike most spider toxins that modulate Na V channels, Tp1a inhibited hNa V 1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa V 1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa V 1.7 inhibitors for treatment of chronic pain.

Beschreibung: Biochemistry DOI: 10.1021/acs.biochem.7b00079