Species‐specific detection of the antiviral small‐molecule compound CMA by STING

Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10‐carboxymethyl‐9‐acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell‐intrinsic type I IFN response, depending on the ER‐resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C‐terminal ligand‐binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate ( c‐diGMP)‐binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c‐diGMP. Altogether, these results provide novel insight into ligand‐sensing properties of STING and, furthermore, unravel unexpected species‐specific differences of this innate sensor.     

A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii: SUBSTRATE SELECTION ACHIEVED THROUGH MULTIMERIZATION*

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers'' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states.     

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Mechanistic insights into the superoxide–cytochrome c reaction by lysine surface scanning

This study summarizes results which have been obtained by a mutational study of human cytochrome c. The protein can be used as a recognition element in analytical assays and biosensors for superoxide radicals since ferricytochrome c reacts with superoxide to form ferrocytochrome c and oxygen. Here lysine mutagenesis of the distal surface (i.e., of exposed residues around the Met80 axial ligand) of human cytochrome c was pursued to evaluate the effect of the surface charges on the reaction rate with the superoxide anion radical and on the redox properties of the heme protein. The latter feature is particularly relevant when the protein is immobilized on a negatively charged self-assembled monolayer on an electrode to be used as a biosensor. The observed effects of the mutations are rationalized through structural investigations based on solution NMR spectroscopy and computational analysis of the surface electrostatics. The results suggest the presence of a specific path that guides superoxide toward an efficient reaction site. Localized positive charges at the rim of the entry channel are effective in increasing the reaction rate, whereas diffused positive charges or charges far from this area are not effective or are even detrimental, resulting in a misguided approach of the anion to the protein surface.     

Vegetation controls methane emissions in a coastal brackish fen

Climate change and associated sea level rise will likely affect coastal ecosystems and lead to more frequent inundations. Plants are an important control for methane (CH₄) emissions in peatlands because the metabolism of the living plant can either enhance or attenuate CH₄ emissions and plant litter supplies an easily available carbon source for methanogenesis. Here we compare the contribution of various dominant plant species to methane emissions in a degraded, rewetted coastal brackish fen at the southern Baltic Sea coast in Northeast Germany. We analyse one year of bi-weekly static closed chamber data gathered at measurement spots that were located in different mono-dominant vegetation stands (Bolboschoenus maritimus (L.) Palla, Schoenoplectus tabernaemontani (C.C.Gmel.) Palla, Carex acutiformis Ehrh.). Furthermore, data on water level, water temperature, conductivity (sulphate), and several peat characteristics were recorded. Generally, the annual methane emissions were low with an average across vegetation stands of 14 kg CH₄ ha^?1 a^?1, which we related to high decomposition of peat after drainage and to relatively low water levels in summer. Nevertheless, methane emissions varied between different vegetation types with significantly higher methane fluxes (31.8 ± 5.7 kg CH₄ ha^?1 a^?1) from Bolboschoenus maritimus stands compared to Carex acutiformis and Schoenoplectus tabernaemontani stands (4.3 ± 1.2 and 5.7 ± 2.4 kg CH₄ ha^?1 a^?1, respectively). None of the environmental variables that have been recorded can explain this difference. Thus, vegetation composition seems to be an important driver for methane emissions in coastal brackish fens and may therefore be crucial with regard to recreation measures.     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.