Nucleic acids derived from pathogens induce potent innate immune responses
. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis of the cyclic dinucleotide ...cyclic GMP-AMP, which mediates the induction of type I interferons through the STING-TBK1-IRF3 signalling axis
. cGAS was previously thought to not react with self DNA owing to its cytosolic localization
; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering
. Here we show that cGAS binds to nucleosomes with nanomolar affinity and that nucleosome binding potently inhibits its catalytic activity. To elucidate the molecular basis of cGAS inactivation by nuclear tethering, we determined the structure of mouse cGAS bound to human nucleosome by cryo-electron microscopy. The structure shows that cGAS binds to a negatively charged acidic patch formed by histones H2A and H2B via its second DNA-binding site
. High-affinity nucleosome binding blocks double-stranded DNA binding and maintains cGAS in an inactive conformation. Mutations of cGAS that disrupt nucleosome binding alter cGAS-mediated signalling in cells.
Baicalin is one of the most abundant flavonoids found in the dried roots of
Georgi (SBG) belonging to the genus
. While baicalin is demonstrated to have anti-inflammatory, antiviral, antitumor, ...antibacterial, anticonvulsant, antioxidant, hepatoprotective, and neuroprotective effects, its low hydrophilicity and lipophilicity limit the bioavailability and pharmacological functions. Therefore, an in-depth study of baicalin's bioavailability and pharmacokinetics contributes to laying the theoretical foundation for applied research in disease treatment. In this view, the physicochemical properties and anti-inflammatory activity of baicalin are summarized in terms of bioavailability, drug interaction, and inflammatory conditions.
Nucleic acids from bacteria or viruses induce potent immune responses in infected cells
. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and ...initiates protective immune responses
. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor
. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)
, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis
. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase
. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK1
. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons
. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNβ after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.
CD8
T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 ...also mediates homeostatic priming of CD8
T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8
T cells to IL-15, rendering naive CD8
T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8
T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8
T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.
The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 ...(NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.
Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and ...macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.
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•HIV-2, not HIV-1, activates innate immunity in macrophages and dendritic cells•NONO protein binds to the HIV-2 capsid protein with more affinity than HIV-1•NONO is an innate immune sensor of the HIV capsid in the nucleus•NONO associates with the sensor cGAS in the nucleus and enables sensing of HIV DNA
The cellular factor NONO activates cGAS-mediated innate immune defenses against HIV-2 infection via viral capsid binding.
Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial ...double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.
Abstract
Elucidating how individual mutations affect the protein energy landscape is crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because ...of epistasis—the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of influenza A viruses (IAVs). We integrate structural, kinetic, thermodynamic, and conformational dynamics analyses of four NS1s of influenza strains that emerged between 1918 and 2004. Although functionally near-neutral, strain-specific NS1 mutations exhibit long-range epistatic interactions with residues at the p85β-binding interface. We reveal that strain-specific mutations reshaped the NS1 energy landscape during evolution. Using NMR spin dynamics, we find that the strain-specific mutations altered the conformational dynamics of the hidden network of tightly packed residues, underlying the evolution of long-range epistasis. This work shows how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.
Artemisia is important medicinal plants in China and are widely used in medicine, agriculture, and food. Pharmacologically active components of the plants remain to be investigated.
This study sought ...to identify and compare the chemical constituents of three species of Artemisia in Tibet using a widely-targeted metabolomics approach and their antibacterial and antioxidant capacities were determined.
A total of 1109 metabolites within 10 categories were detected from the three species of Artemisia, including lipids, amino acids, nucleotides, flavonoids, terpenes, coumarins, organic acids, and phenolic acids. 732 different metabolites have been identified between Artemisia sieversiana and Artemisia annua, 751 different metabolites were identified between Artemisia wellbyi and A. sieversiana, and 768 differential metabolites were differentially detected from A. wellbyi and A. annua. Differentially identified compounds included flavonoids, phenolic acids, artemisinins and coumarin. A. annua contained the highest relative content of artemisinin among three Artemisia. The antimicrobial experiments showed that the three Artemisia species had strong antibiotic activities against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa. The biochemical analysis showed that the three species of Artemisia have strong antioxidant capacity.
This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The COVID‐19 pathogen, SARS‐CoV‐2, requires its main protease (SC2MPro) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication ...and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID‐19 treatment option. Guided by previous medicinal chemistry studies about SARS‐CoV‐1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain β‐(S‐2‐oxopyrrolidin‐3‐yl)‐alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active‐site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS‐CoV‐2‐induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS‐CoV‐2‐induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID‐19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra‐high antiviral potency.
Small but strong: A series of SARS‐CoV‐2 MPro covalent inhibitors exhibit excellent activity. Protein crystallography analysis and a live virus‐based microneutralization assay found two of the most potent anti‐SARS‐CoV‐2 small molecules so far. Due to the urgent matter of the COVID‐19 pandemic, these two inhibitors could be quickly advanced to preclinical and clinical tests for COVID‐19.
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