The preparation of topologically nontrivial molecules is often assisted by covalent, supramolecular or coordinative templates that provide spatial pre-organization for all components. Herein, we ...report a trefoil knot that can be self-assembled efficiently in water without involving additional templates. The direct condensation of three equivalents of a tetraformyl precursor and six equivalents of a chiral diamine produces successfully a 3 + 6 trefoil knot whose intrinsic handedness is dictated by the stereochemical configuration of the diamine linkers. Contrary to the conventional wisdom that imine condensation is not amenable to use in water, the multivalent cooperativity between all the imine bonds within the framework makes this trefoil knot robust in the aqueous environment. Furthermore, the presence of water is proven to be essential for the trefoil knot formation. A topologically trivial macrocycle composed of two tetraformyl and four diamino building blocks is obtained when a similar reaction is performed in organic media, indicating that hydrophobic effect is a major driving force behind the scene.
The σ
2
receptor has attracted intense interest in cancer imaging
1
, psychiatric disease
2
, neuropathic pain
3
–
5
, and other areas of biology
6
,
7
. We determined the crystal structure of this ...receptor in complex with the clinical candidate roluperidone
2
and the tool compound PB28
8
. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. 127 new chemotypes with affinities superior to 1 μM were identified, 31 of which had affinities superior to 50 nM. Hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, achieving affinities ranging from 3 to 48 nM with up to 250-fold selectivity versus the σ
1
receptor. Crystal structures of two new ligands bound to the σ
2
receptor confirmed the docked poses. To investigate the contribution of the σ
2
receptor in pain, two potent σ
2
-selective ligands and one potent σ
1
/σ
2
non-selective ligand were tested for efficacy in a mouse neuropathic pain model. All three ligands demonstrated time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model
9
, supporting a role for the σ
2
receptor in nociception. This study illustrates the opportunities for rapid discovery of
in vivo
probes to study under-explored areas of biology using structure-based screens of diverse, ultra-large libraries.
The Hyper Suprime-Cam software pipeline Bosch, James; Armstrong, Robert; Bickerton, Steven ...
Publications of the Astronomical Society of Japan,
01/2018, Letnik:
70, Številka:
SP1
Journal Article
Recenzirano
Odprti dostop
Abstract
In this paper, we describe the optical imaging data processing pipeline developed for the Subaru Telescope’s Hyper Suprime-Cam (HSC) instrument. The HSC Pipeline builds on the prototype ...pipeline being developed by the Large Synoptic Survey Telescope’s Data Management system, adding customizations for HSC, large-scale processing capabilities, and novel algorithms that have since been reincorporated into the LSST codebase. While designed primarily to reduce HSC Subaru Strategic Program (SSP) data, it is also the recommended pipeline for reducing general-observer HSC data. The HSC pipeline includes high-level processing steps that generate coadded images and science-ready catalogs as well as low-level detrending and image characterizations.
The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding ...to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.
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•Sterols induce conformational change in Smoothened to activate Hedgehog signaling•The active Smoothened conformation resembles that of active G-protein-coupled receptors•Inactive Smoothened is stabilized by a “lock” that is broken in a cancer mutant•A longitudinal tunnel in Smoothened might provide a path for activating cholesterol
Crystal structures of sterol-bound Smoothened molecules reveal active conformations reminiscent of other classes of G-protein-coupled receptors.
Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular ...understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.
Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on ...dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.
Emerging evidence suggests that the T helper 17 (T sub(H)17) subset of alpha beta T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the alpha v beta ...8 integrin on dendritic cells did not generate T sub(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T sub(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T sub(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor Kappa light-chain enhancer of activated B cells (NF- Kappa B)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin alpha v beta 8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T sub(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.
Remodelling of the extracellular matrix (ECM) and cell surface by matrix metalloproteinases (MMPs) is an important function of monocytes and macrophages. Recent work has emphasised the diverse roles ...of classically and alternatively activated macrophages but the consequent regulation of MMPs and their inhibitors has not been studied comprehensively. Classical activation of macrophages derived in vitro from un-fractionated CD16(+/-) or negatively-selected CD16(-) macrophages up-regulated MMP-1, -3, -7, -10, -12, -14 and -25 and decreased TIMP-3 steady-state mRNA levels. Bacterial lipopolysaccharide, IL-1 and TNFα were more effective than interferonγ except for the effects on MMP-25, and TIMP-3. By contrast, alternative activation decreased MMP-2, -8 and -19 but increased MMP -11, -12, -25 and TIMP-3 steady-state mRNA levels. Up-regulation of MMPs during classical activation depended on mitogen activated protein kinases, phosphoinositide-3-kinase and inhibitor of κB kinase-2. Effects of interferonγ depended on janus kinase-2. Where investigated, similar effects were seen on protein concentrations and collagenase activity. Moreover, activity of MMP-1 and -10 co-localised with markers of classical activation in human atherosclerotic plaques in vivo. In conclusion, classical macrophage activation selectively up-regulates several MMPs in vitro and in vivo and down-regulates TIMP-3, whereas alternative activation up-regulates a distinct group of MMPs and TIMP-3. The signalling pathways defined here suggest targets for selective modulation of MMP activity.
Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use ...efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.
Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting ...evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-Glcnac), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-Glcnac functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-Glcnac sites. Thus, to advance insight into the role of O-Glcnac in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-Glcnac sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-Glcnac sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-Glcnac groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-Glcnac glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-Glcnac glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-Glcnac in T cell activation.
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