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1.
Cell ; 186(14): 3013-3032.e22, 2023 07 06.
Article in English | MEDLINE | ID: mdl-37352855

ABSTRACT

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.


Subject(s)
Cardiotoxicity , DNA, Mitochondrial , Animals , Mice , DNA, Mitochondrial/metabolism , Immunity, Innate , Interferons/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Phosphorylation
2.
Cell ; 185(17): 3214-3231.e23, 2022 08 18.
Article in English | MEDLINE | ID: mdl-35907404

ABSTRACT

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.


Subject(s)
Mitochondria , Necroptosis , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Animals , Humans , Inflammasomes , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Macrophages , Mice , Mitochondria/metabolism , Reactive Oxygen Species/metabolism
3.
Cell ; 175(2): 488-501.e22, 2018 10 04.
Article in English | MEDLINE | ID: mdl-30270045

ABSTRACT

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.


Subject(s)
Capsid Proteins/immunology , Nuclear Matrix-Associated Proteins/immunology , Nuclear Matrix-Associated Proteins/physiology , Octamer Transcription Factors/immunology , Octamer Transcription Factors/physiology , RNA-Binding Proteins/immunology , RNA-Binding Proteins/physiology , Capsid/metabolism , Capsid Proteins/metabolism , Capsid Proteins/physiology , Cell Nucleus/metabolism , DNA, Viral/genetics , DNA, Viral/immunology , DNA-Binding Proteins , Dendritic Cells/immunology , HIV Infections/immunology , HIV-1/genetics , HIV-1/immunology , HIV-2/genetics , HIV-2/immunology , Host-Pathogen Interactions , Humans , Immunity, Innate/immunology , Macrophages/immunology , Membrane Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/physiology , RNA-Binding Proteins/metabolism , Signal Transduction/immunology
4.
Nat Immunol ; 20(7): 879-889, 2019 07.
Article in English | MEDLINE | ID: mdl-31182807

ABSTRACT

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.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cysteine Endopeptidases/metabolism , Interleukin-15/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cysteine Endopeptidases/deficiency , Deubiquitinating Enzymes/metabolism , Disease Models, Animal , Energy Metabolism , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interleukin-15/genetics , Melanoma, Experimental , Mice , Mice, Transgenic , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Interleukin-15/metabolism , Self Tolerance/genetics , Self Tolerance/immunology , Signal Transduction , T-Cell Antigen Receptor Specificity , Ubiquitination
5.
Immunity ; 50(3): 591-599.e6, 2019 03 19.
Article in English | MEDLINE | ID: mdl-30893587

ABSTRACT

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.


Subject(s)
DNA, Viral/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interferon Type I/metabolism , Membrane Proteins/metabolism , Animals , Cell Line , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Nucleic Acids/metabolism , Protein Binding/immunology
6.
Immunity ; 50(1): 51-63.e5, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30635239

ABSTRACT

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.


Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , DNA Virus Infections/immunology , DNA Viruses/physiology , RNA Virus Infections/immunology , RNA Viruses/immunology , 2',5'-Oligoadenylate Synthetase/genetics , Animals , Cyclic AMP/metabolism , Humans , Interferon Type I/genetics , Interferon Type I/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nucleotidyltransferases/metabolism , RNA, Small Interfering/genetics , Signal Transduction , THP-1 Cells , Virus Replication
7.
PLoS Pathog ; 20(7): e1012398, 2024 Jul.
Article in English | MEDLINE | ID: mdl-39038050

ABSTRACT

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1ß. Caspase-1 is involved in the maturation of IL-1ß and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1ß and other IL-1ß-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1ß (sPro-IL-1ß) exists as an oligomeric precursor unlike monomeric human Pro-IL-1ß (hPro-IL-1ß). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1ß to produce mature IL-1ß, while it cleaves hPro-IL-1ß but does not produce mature IL-1ß in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1ß through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1ß serves as an 'exosite' with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1ß generates a monomeric and active form of IL-1ß, initiating the downstream signaling. Thus, these studies provide IL-1ß is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.


Subject(s)
3C Viral Proteases , Inflammasomes , Interleukin-1beta , Picornaviridae , Viral Proteins , Animals , Interleukin-1beta/metabolism , 3C Viral Proteases/metabolism , Swine , Humans , Viral Proteins/metabolism , Inflammasomes/metabolism , Inflammation/metabolism , Picornaviridae Infections/metabolism , Picornaviridae Infections/virology , Cysteine Endopeptidases/metabolism , Species Specificity , Macrophages, Alveolar/virology , Macrophages, Alveolar/metabolism
8.
Nature ; 587(7835): 673-677, 2020 11.
Article in English | MEDLINE | ID: mdl-32911481

ABSTRACT

Nucleic acids derived from pathogens induce potent innate immune responses1-6. 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 axis7-11. cGAS was previously thought to not react with self DNA owing to its cytosolic localization2,12,13; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering14-18. 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 site19. 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.


Subject(s)
Nucleosomes/chemistry , Nucleosomes/metabolism , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry , Animals , Biocatalysis , Catalytic Domain , Cell Line , Cryoelectron Microscopy , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA/ultrastructure , Humans , Mice , Models, Molecular , Mutation , Nucleosomes/ultrastructure , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/ultrastructure , Protein Binding , Signal Transduction
9.
PLoS Pathog ; 19(9): e1011641, 2023 09.
Article in English | MEDLINE | ID: mdl-37708231

ABSTRACT

RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing attention. Recent studies showed that the cGAS-STING pathway plays an important role in restricting RNA viruses via mitochondria DNA (mtDNA) mediated activation. However, the mechanisms of cGAS mediated innate immune evasion by RNA viruses remain unknown. Here, we report that seneca valley virus (SVV) protease 3C disrupts mtDNA mediated innate immune sensing by cleaving porcine cGAS (pcGAS) in a species-specific manner. Mechanistically, a W/Q motif within the N-terminal domain of pcGAS is a unique cleavage site recognized by SVV 3C. Three conserved catalytic residues of SVV 3C cooperatively contribute to the cleavage of pcGAS, but not human cGAS (hcGAS) or mouse cGAS (mcGAS). Additionally, upon SVV infection and poly(dA:dT) transfection, pcGAS and SVV 3C colocalizes in the cells. Furthermore, SVV 3C disrupts pcGAS-mediated DNA binding, cGAMP synthesis and interferon induction by specifically cleaving pcGAS. This work uncovers a novel mechanism by which the viral protease cleaves the DNA sensor cGAS to evade innate immune response, suggesting a new antiviral approach against picornaviruses.


Subject(s)
Nucleotidyltransferases , Peptide Hydrolases , Picornaviridae , Animals , Humans , Mice , DNA, Mitochondrial , Endopeptidases , Mitochondria , Picornaviridae/physiology , Swine , Nucleotidyltransferases/metabolism
10.
PLoS Biol ; 20(3): e3001589, 2022 03.
Article in English | MEDLINE | ID: mdl-35324891

ABSTRACT

Clostridioides difficile secretes Toxin B (TcdB) as one of its major virulence factors, which binds to intestinal epithelial and subepithelial receptors, including frizzled proteins and chondroitin sulfate proteoglycan 4 (CSPG4). Here, we present cryo-EM structures of full-length TcdB in complex with the CSPG4 domain 1 fragment (D1401-560) at cytosolic pH and the cysteine-rich domain of frizzled-2 (CRD2) at both cytosolic and acidic pHs. CSPG4 specifically binds to the autoprocessing and delivery domains of TcdB via networks of salt bridges, hydrophobic and aromatic/proline interactions, which are disrupted upon acidification eventually leading to CSPG4 drastically dissociating from TcdB. In contrast, FZD2 moderately dissociates from TcdB under acidic pH, most likely due to its partial unfolding. These results reveal structural dynamics of TcdB during its preentry step upon endosomal acidification, which provide a basis for developing therapeutics against C. difficile infections.


Subject(s)
Bacterial Toxins , Clostridioides difficile , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Protein Domains , Virulence Factors/metabolism
11.
Nature ; 569(7758): 718-722, 2019 05.
Article in English | MEDLINE | ID: mdl-31118511

ABSTRACT

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. 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 IFNß 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.


Subject(s)
Amino Acid Motifs , Conserved Sequence , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Crystallography, X-Ray , Enzyme Activation , HEK293 Cells , Humans , Interferon-beta/metabolism , Membrane Proteins/genetics , Models, Molecular , Mutation , Nucleotides, Cyclic/metabolism , Protein Binding , Signal Transduction
12.
Nature ; 557(7706): 516-521, 2018 05.
Article in English | MEDLINE | ID: mdl-29769717

ABSTRACT

Chromatin remodelling factors (CHRs) typically function to alter chromatin structure. CHRs also reside in ribonucleoprotein complexes, but little is known about their RNA-related functions. Here we show that CHR2 (also known as BRM), the ATPase subunit of the large switch/sucrose non-fermentable (SWI/SNF) complex, is a partner of the Microprocessor component Serrate (SE). CHR2 promotes the transcription of primary microRNA precursors (pri-miRNAs) while repressing miRNA accumulation in vivo. Direct interaction with SE is required for post-transcriptional inhibition of miRNA accumulation by CHR2 but not for its transcriptional activity. CHR2 can directly bind to and unwind pri-miRNAs and inhibit their processing, and this inhibition requires the remodelling and helicase activity of CHR2 in vitro and in vivo. Furthermore, the secondary structures of pri-miRNAs differed between wild-type Arabidopsis thaliana and chr2 mutants. We conclude that CHR2 accesses pri-miRNAs through SE and remodels their secondary structures, preventing downstream processing by DCL1 and HYL1. Our study uncovers pri-miRNAs as a substrate of CHR2, and an additional regulatory layer upstream of Microprocessor activity to control miRNA accumulation.


Subject(s)
Adenosine Triphosphatases/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , MicroRNAs/biosynthesis , RNA-Binding Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , MicroRNAs/genetics , MicroRNAs/metabolism , Protein Binding , RNA Folding , RNA Processing, Post-Transcriptional , Transcription, Genetic
13.
Nat Immunol ; 12(10): 1002-9, 2011 Aug 28.
Article in English | MEDLINE | ID: mdl-21874024

ABSTRACT

T cell activation is subject to tight regulation to avoid inappropriate responses to self antigens. Here we show that genetic deficiency in the ubiquitin ligase Peli1 caused hyperactivation of T cells and rendered T cells refractory to suppression by regulatory T cells and transforming growth factor-ß (TGF-ß). As a result, Peli1-deficient mice spontaneously developed autoimmunity characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency resulted in the nuclear accumulation of c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T cell activation. Peli1 negatively regulated c-Rel by mediating its Lys48 (K48) ubiquitination. Our results identify Peli1 as a critical factor in the maintenance of peripheral T cell tolerance and demonstrate a previously unknown mechanism of c-Rel regulation.


Subject(s)
Autoimmunity , Lymphocyte Activation , Nuclear Proteins/physiology , T-Lymphocytes/immunology , Animals , CD28 Antigens/physiology , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/metabolism , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes, Regulatory/physiology , Transforming Growth Factor beta/physiology , Ubiquitin-Protein Ligases , Ubiquitination
14.
Proc Natl Acad Sci U S A ; 117(12): 6550-6558, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32152123

ABSTRACT

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85ß subunit. Here we present the mechanism underlying the molecular recognition of the p85ß subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85ß of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85ß. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85ß-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85ß. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85ß compared to Ud NS1ED, although the former has a lower affinity to p85ß than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.


Subject(s)
Influenza A virus/physiology , Influenza, Human/virology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Host-Pathogen Interactions , Humans , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H3N2 Subtype/pathogenicity , Influenza A Virus, H3N2 Subtype/physiology , Influenza A virus/classification , Influenza A virus/pathogenicity , Influenza, Human/epidemiology , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Species Specificity , Structure-Activity Relationship , Virulence Factors/chemistry , Virulence Factors/metabolism
15.
Immunity ; 39(6): 1019-31, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24332030

ABSTRACT

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-ß gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-ß reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization.


Subject(s)
DNA/metabolism , Models, Molecular , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Animals , Binding Sites/genetics , Catalytic Domain , Humans , Mice , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/genetics , Protein Binding , Protein Structure, Quaternary
16.
J Immunol ; 205(7): 1886-1896, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32826280

ABSTRACT

The innate immune system is the first line of defense against bacterial and viral infections. The recognition of pathogen-associated molecular patterns by the RIG-I-like receptors, TLRs, and cGAS leads to the induction of IFN-I by activating the transcription factor IRF-3. Although the mechanism of IRF-3 activation has been extensively studied, the structural basis of IRF-3 activation upon phosphorylation is not fully understood. In this study, we determined the crystal structures of phosphorylated human and mouse IRF-3 bound to CREB-binding protein (CBP), which reveal that phosphorylated IRF-3 forms a dimer via pSer386 (pSer379 in mouse IRF-3) and a downstream pLxIS motif. Size-exclusion chromatography and cell-based studies show that mutations of key residues interacting with pSer386 severely impair IRF-3 activation and IFN-ß induction. By contrast, phosphorylation of Ser396 within the pLxIS motif of human IRF-3 only plays a moderate role in IRF-3 activation. The mouse IRF-3/CBP complex structure reveals that the mechanism of mouse IRF-3 activation is similar but distinct from human IRF-3. These structural and functional studies reveal the detailed mechanism of IRF-3 activation upon phosphorylation.


Subject(s)
CREB-Binding Protein/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon-beta/metabolism , Animals , Humans , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Mice , Mutagenesis, Site-Directed , Pathogen-Associated Molecular Pattern Molecules/immunology , Phosphorylation , Protein Binding , Protein Conformation , Protein Domains/genetics , Sf9 Cells , Species Specificity , Spodoptera , Structure-Activity Relationship
17.
J Immunol ; 205(1): 153-167, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32404352

ABSTRACT

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.


Subject(s)
Interferon Type I/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mycobacterium tuberculosis/immunology , Signal Transduction/immunology , Tripartite Motif Proteins/metabolism , Tuberculosis/immunology , Animals , Disease Models, Animal , Gene Expression Regulation/immunology , Gene Knockout Techniques , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/isolation & purification , Macrophages/immunology , Macrophages/metabolism , Membrane Proteins/metabolism , Mice , Nitric Oxide Synthase Type II/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/isolation & purification , Nucleotidyltransferases/metabolism , Phosphorylation/immunology , Primary Cell Culture , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/isolation & purification , Protein Serine-Threonine Kinases/metabolism , RAW 264.7 Cells , Receptor, Interferon alpha-beta/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , STAT3 Transcription Factor/metabolism , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/isolation & purification , Tuberculosis/microbiology
18.
Cell Mol Biol (Noisy-le-grand) ; 68(3): 158-170, 2022 Mar 31.
Article in English | MEDLINE | ID: mdl-35988176

ABSTRACT

Tumors are the biggest opponents in the history of human diseases, and they cannot be eliminated so far. The only way to treat tumors is to detect them early so that the survival rate can be improved by early treatment. For tumor detection, CT scan is the most commonly used, and PET/CT is an enhanced version of CT technology. Although PET/CT can produce relatively clear images of the human body, due to the complex structure of the human body, there are many ghosts and shadows, and the images cannot be accurately judged. Therefore, this paper aims to prepare high-definition nanoparticle contrast agents, hoping to make PET/CT images clearer and easier to distinguish. In this paper, the advantages of gold nanoparticles are fully analyzed for the preparation of contrast agents, and a gold nano-contrast agent coated with bovine serum albumin (BSA) is proposed. Gold nanoparticles (GNRs) were prepared by the traditional induction method and their properties were analyzed. Finally, taking mice as the experimental object, a comparative experiment was carried out, and the toxicological and optical properties were analyzed. The experimental results show that the adsorption performance of the BSA-coated gold nanoparticles prepared in this paper is more than 90% at different temperatures. And through the comparison experiment, the contrast agent prepared in this paper has an increased signal-to-noise(StN) ratio change rate of more than 50%, which can be well applied to PET/CT imaging.


Subject(s)
Metal Nanoparticles , Nanoparticles , Neoplasms , Animals , Contrast Media , Gold/chemistry , Humans , Mice , Nanoparticles/chemistry , Positron Emission Tomography Computed Tomography , Serum Albumin, Bovine/chemistry
19.
Proc Natl Acad Sci U S A ; 113(24): E3403-12, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27302953

ABSTRACT

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-ß) 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.


Subject(s)
Interferon Regulatory Factor-3/chemistry , Rotavirus/chemistry , Viral Nonstructural Proteins/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Amino Acid Motifs , CREB-Binding Protein/chemistry , CREB-Binding Protein/genetics , CREB-Binding Protein/immunology , Humans , Immune Evasion , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/immunology , Protein Domains , Rotavirus/genetics , Rotavirus/immunology , Rotavirus Infections/genetics , Rotavirus Infections/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology
20.
Biophys J ; 114(7): 1590-1603, 2018 04 10.
Article in English | MEDLINE | ID: mdl-29642029

ABSTRACT

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca2+-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca2+ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.


Subject(s)
Protein Kinase C-alpha/chemistry , Protein Kinase C-alpha/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Calcium/metabolism , Hydrophobic and Hydrophilic Interactions , Mutation , Protein Domains , Protein Kinase C-alpha/genetics , Rats , Static Electricity
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