The Mechanism of DNA Sensing by AIM2-Like Receptors

AIM2-Like Receptors are a family of nuclear and cytosolic foreign-DNA sensors consisting of an N-terminal PYD and one or two C-terminal DNA-binding HIN200 domains. AIM2 is a cytosolic sensor which forms a supramolecular structure known as the inflammasome, while IFI16 resides in both the nucleus and cytoplasm, where it can associate with ASC and form its own inflammasome, as well as lead to activation of the interferon pathway by parallel means. Persistent questions in innate immunology remain of how such sensors respond to foreign DNA while remaining silent towards host DNA, as well as their role in activating downstream effectors. Using fluorescence anisotropy, FRET, EMSA, and various mutational studies, it is shown here that the PYD of IFI16 plays a positive, cooperative role in DNA binding, allowing it to oligomerize in a length-dependent manner. Disruption of the non-DNA-binding PYD in turn disrupts DNA binding, demonstrating that oligomerization and DNA binding are coupled events. By fluorescence anisotropy, FRET, EMSA, electron microscopy, and mutagenesis studies, a model which doesn’t invoke the previous autoinhibition is put forth to explain the behavior and activation of AIM2. Results also give clues into how AIM2 may then recruit the next member of the downstream pathway, ASC. Finally, using time-dependent FRET assays as well as competition anisotropy experiments, it is demonstrated that nucleosomes act as effective barriers to IFI16 DNA binding and oligomerization. The length-dependent rates of FRET signal also support a model in which IFI16 uses one-dimensional diffusion as an efficient means of oligomerization. Taken together, these results show that the AIM2-Like Receptor family share many common characteristics that give insight into their roles in host defense and autoimmune disorders

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period

21openInternationalInternational coauthor/editorWe subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.openMaixner, Frank; Sarhan, Mohamed S; Huang, Kun D; Tett, Adrian; Schoenafinger, Alexander; Zingale, Stefania; Blanco-Míguez, Aitor; Manghi, Paolo; Cemper-Kiesslich, Jan; Rosendahl, Wilfried; Kusebauch, Ulrike; Morrone, Seamus R; Hoopmann, Michael R; Rota-Stabelli, Omar; Rattei, Thomas; Moritz, Robert L; Oeggl, Klaus; Segata, Nicola; Zink, Albert; Reschreiter, Hans; Kowarik, KerstinMaixner, F.; Sarhan, M.S.; Huang, K.D.; Tett, A.; Schoenafinger, A.; Zingale, S.; Blanco-Míguez, A.; Manghi, P.; Cemper-Kiesslich, J.; Rosendahl, W.; Kusebauch, U.; Morrone, S.R.; Hoopmann, M.R.; Rota-Stabelli, O.; Rattei, T.; Moritz, R.L.; Oeggl, K.; Segata, N.; Zink, A.; Reschreiter, H.; Kowarik, K

Structural Basis of PP2A Inhibition by Small t Antigen

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST

Decoupling nucleosome recognition from DNA binding dramatically alters the properties of the Chd1 chromatin remodeler

PMC3561990Chromatin remodelers can either organize or disrupt nucleosomal arrays, yet the mechanisms specifying these opposing actions are not clear. Here, we show that the outcome of nucleosome sliding by Chd1 changes dramatically depending on how the chromatin remodeler is targeted to nucleosomes. Using a Chd1–streptavidin fusion remodeler, we found that targeting via biotinylated DNA resulted in directional sliding towards the recruitment site, whereas targeting via biotinylated histones produced a distribution of nucleosome positions. Remarkably, the fusion remodeler shifted nucleosomes with biotinylated histones up to 50bp off the ends of DNA and was capable of reducing negative supercoiling of plasmids containing biotinylated chromatin, similar to remodelling characteristics observed for SWI/ SNF-type remodelers. These data suggest that forming a stable attachment to nucleosomes via histones, and thus lacking sensitivity to extranucleosomal DNA, seems to be sufficient for allowing a chromatin remodeler to possess SWI/SNF-like disruptive properties.JH Libraries Open Access Fun

Structural perspectives of antibody-dependent enhancement of infection of dengue virus

10.1016/j.coviro.2019.02.00

Optimization of Instrument Parameters for Efficient Phosphopeptide Identification and Localization by Data-dependent Analysis Using Orbitrap Tribrid Mass Spectrometers

The analysis of protein phosphorylation site identification by mass spectrometry-based methods continues to improve with increased efficiency at multiple points of the pipeline including affinity isolation, sample handling with automated protein digestion and phosphopeptide extraction routines, and more sensitive phosphopeptide detection capabilities. The role mass spectrometry parameters play in the quantity and quality of results is understudied, with some reports presenting improvements without sufficient details of how these parameters were derived, nor how they were fully optimized. Here, we systematically scrutinize and optimize parameters of two Orbitrap Tribrid mass spectrometers by varying instrument parameters at both the MS1 and MS2 levels using Titanium IMAC-enriched phosphopeptide samples to illustrate how the recovery of phosphopeptides, and the confidence of phosphosite localizations, vary accordingly. These optimized values are then used to compare different IMAC beads for phosphopeptide-enrichment, and optimal bead combinations used to further improve phosphopeptide detection. These results have implications for planning detailed phosphoprotein identification and quantitation experiments to provide confidence in detection, site localization and reproducibility

Single-molecule imaging at high fluorophore concentrations by local activation of dye

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluo- rescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimen- sional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore

Optimization of Instrument Parameters for Efficient Phosphopeptide Identification and Localization by Data-dependent Analysis Using Orbitrap Tribrid Mass Spectrometers

The analysis of protein phosphorylation site identification by mass spectrometry-based methods continues to improve with increased efficiency at multiple points of the pipeline including affinity isolation, sample handling with automated protein digestion and phosphopeptide extraction routines, and more sensitive phosphopeptide detection capabilities. The role mass spectrometry parameters play in the quantity and quality of results is understudied, with some reports presenting improvements without sufficient details of how these parameters were derived, nor how they were fully optimized. Here, we systematically scrutinize and optimize parameters of two Orbitrap Tribrid mass spectrometers by varying instrument parameters at both the MS1 and MS2 levels using Titanium IMAC-enriched phosphopeptide samples to illustrate how the recovery of phosphopeptides, and the confidence of phosphosite localizations, vary accordingly. These optimized values are then used to compare different IMAC beads for phosphopeptide-enrichment, and optimal bead combinations used to further improve phosphopeptide detection. These results have implications for planning detailed phosphoprotein identification and quantitation experiments to provide confidence in detection, site localization and reproducibility

Crystal structures of the extracellular domain of LRP6 and its complex with DKK1

Low-density-lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6) are Wnt co-receptors essential for Wnt/β-catenin signaling. Dickkopf 1 (DKK1) inhibits Wnt signaling by interacting with the extracellular domains of LRP5/6 and is a drug target for multiple diseases. Here we present the crystal structures of a human LRP6-E3E4-DKK1 complex and the first and second halves of human LRP6's four propeller-epidermal growth factor (EGF) pairs (LRP6-E1E2 and LRP6-E3E4). Combined with EM analysis, these data demonstrate that LRP6-E1E2 and LRP6-E3E4 form two rigid structural blocks, with a short intervening hinge that restrains their relative orientation. The C-terminal domain of DKK1 (DKK1c) interacts with the top surface of the LRP6-E3 YWTD propeller and given their structural similarity, probably also that of the LRP6-E1 propeller, through conserved hydrophobic patches buttressed by a network of salt bridges and hydrogen bonds. Our work provides key insights for understanding LRP5/6 structure and the interaction of LRP5/6 with DKK, as well as for drug discovery. © 2011 Nature America, Inc. All rights reserved