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1.  MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades 
eLife  2013;2:e00785.
RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses.
DOI: http://dx.doi.org/10.7554/eLife.00785.001
eLife digest
The innate immune system can detect and destroy viruses, bacteria and other pathogens that enter the human body. In particular, inside cells, viral RNA can bind to and activate a protein called RIG-I. This protein switches on another protein, called MAVS, which can activate other copies of itself. These MAVS molecules then aggregate together on the membrane of mitochondria and send a signal that leads to the production of small proteins, called cytokines, which stimulate an inflammatory response and ultimately neutralize the virus.
Although many of the proteins that are activated by MAVS in the innate immunity signaling pathway have been identified, precisely how MAVS transmits this signal is unknown. Now, Liu et al. explore how this protein can propagate signals in the innate immune response by monitoring activation of the transcription factors IRF3 and NF-κB, which transcribe cytokine genes.
Previous studies have suggested that a protein known as ubiquitin is needed to activate RIG-I, and that this protein collaborates with MAVS to signal through the innate immunity pathway. Liu et al. found that a group of proteins including TRAF2, TRAF5, TRAF6 and LUBAC relay the antiviral signal by binding to MAVS. These so-called ‘E3 ligases’ string ubiquitin together in chains called polyubiquitin, which is essential for activating signaling after, or downstream of, MAVS; however, the association of these E3 ligases with MAVS also requires that multiple copies of MAVS cluster together.
MAVS, the TRAF proteins and LUBAC collectively recruit other innate immunity pathway proteins to activate IRF3 and NF-κB, and thus transcription of the genes that control the innate immunity response. Together, these results show the intricate interplay of proteins needed to eliminate viruses from the body.
DOI: http://dx.doi.org/10.7554/eLife.00785.002
doi:10.7554/eLife.00785
PMCID: PMC3743401  PMID: 23951545
MAVS; innate immunity; virus; ubiquitin; signaling; mitochondria; Human; Mouse; Viruses
2.  Correction: Structural basis for the prion-like MAVS filaments in antiviral innate immunity 
eLife  null;4:e07546.
doi:10.7554/eLife.07546
PMCID: PMC4526786  PMID: 26314863
MAVS; innate immunity; prion-like filaments; cryoEM reconstruction; human; viruses
3.  Structural basis for the prion-like MAVS filaments in antiviral innate immunity 
eLife  2014;3:e01489.
Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments.
DOI: http://dx.doi.org/10.7554/eLife.01489.001
eLife digest
When infected by a virus, the body will generally launch an immune response to eliminate the infectious agent. Activation of the innate immune system–the first line of defense against infection—requires the host cells to recognize the presence of a pathogen and to sound the alarm once the invader is detected.
Viruses can contain DNA or RNA, and when a virus containing double stranded RNA enters a cell, or starts replicating within the cytoplasm, proteins called RIG-I-like receptors (RLRs) will detect these RNA molecules. This will trigger a signaling cascade that results in the production of type I interferons, the proteins that activate cells of the innate immune system.
Members of the RLR family of receptors, including RIG-I and MDA5, initiate the signaling cascade by interacting with the mitochondrial antiviral-signaling (MAVS) protein. Recent work revealed that upon activation by RIG-I or MDA5, MAVS proteins aggregate on the surface of mitochondria and form protein filaments. These filaments then activate inactive MAVS proteins, leading to the formation of more filaments. While a region of the MAVS protein called caspase activation and recruitment domain (CARD) is known to be involved in the formation of the filaments, the chemical interactions that govern the formation process have yet to be described.
Now, using cryo-electron microscopy, Xu et al. have shown that these filaments are comprised of three-stranded helixes. This came as something of a surprise because other similar filaments known as prions are made of tightly packed beta sheets. Xu et al. went on to visualize full-length MAVS filaments in virus-infected cells, and to verify that mutations that impair the assembly of MAVS filaments also prevent RNA viruses from triggering the production of interferon. These results have the potential to inform future studies of the innate immune response, as well as investigations into the assembly of proteins to form prion-like filaments.
DOI: http://dx.doi.org/10.7554/eLife.01489.002
doi:10.7554/eLife.01489
PMCID: PMC3932521  PMID: 24569476
MAVS; innate immunity; prion-like filaments; cryoEM reconstruction; three-stranded filaments; human; viruses
4.  Cyclic GMP-AMP Synthase (cGAS) Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis 
Cell host & microbe  2015;17(6):820-828.
Summary
Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.
doi:10.1016/j.chom.2015.05.005
PMCID: PMC4499468  PMID: 26048137
5.  Sequence specific detection of bacterial 23S ribosomal RNA by TLR13 
eLife  2012;1:e00102.
Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity.
DOI: http://dx.doi.org/10.7554/eLife.00102.001
eLife digest
A central feature of the immune system is the ability to detect bacteria, viruses and other pathogens so that they can be repelled or neutralized before they cause lasting damage to an organism. Cells employ a number of different receptors that can detect these pathogens or the molecules they produce. Many of these are called pattern recognition receptors because they recognize certain signatures of microorganisms such as nucleic acids or carbohydrates. An important class of pattern recognition receptor is the toll-like receptor: there are many different families of the receptors, each recognizing a unique feature of bacteria or viruses. (The word toll, which means ‘great’ in German, refers to a gene whose mutations lead to striking phenotypes in flies, and has nothing to do with road and bridge tolls.)
Toll-like receptors have two parts that perform two different functions: when one part binds the relevant microbial molecules, the other part sends a signal that results in the production of effector proteins. These proteins include interleukin-1β, which helps to fight infection by causing the inflammation of tissue. To date, 12 different types of toll-like receptors have been found in mice, including three—known as TLR11, TLR12 and TLR13—that are not present in humans. Very little is known about the functions of TLR12 and TLR13. Humans, on the other hand, possess 10 different TLRs, only one of which, TLR10, is not found in mice.
Li and Chen have now discovered that TLR13 is responsible for detecting a certain type of ribosomal RNA called 23S ribosomal RNA that are present in bacteria but not in eukaryotic cells. Moreover, they have shown that a short sequence of 13 residues within the 23S ribosomal RNA triggers this pathway and leads to the production of interleukin-1β. The sequence of 13 residues is located at an active site in the RNA that catalyzes the synthesis of peptide bonds, and changing just one of these residues stops the production of interleukin-1β. Other forms of ribosomal RNA are unable to trigger the production of interleukin-1β. These results show that TLR13 differs from all other pattern recognition receptors because it is able to recognize a specific RNA sequence. Li and Chen went on to generate mice lacking TLR13 and showed that immune cells isolated from these mice failed to respond to bacterial RNA. These mice can be used to investigate the role of TLR13 in immune responses to bacterial infections in vivo.
DOI: http://dx.doi.org/10.7554/eLife.00102.002
doi:10.7554/eLife.00102
PMCID: PMC3482692  PMID: 23110254
Innate Immunity; Toll-like receptor; bacteria; Ribosomal RNA; E. coli; Mouse
6.  STING-dependent Cytosolic DNA Sensing Promotes Radiation-induced Type I interferon-dependent Antitumor Immunity in Immunogenic Tumors 
Immunity  2014;41(5):843-852.
SUMMARY
Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but the several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-β induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-β treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests cGAMP treatment may provide a new strategy to improve radiotherapy.
doi:10.1016/j.immuni.2014.10.019
PMCID: PMC5155593  PMID: 25517616
7.  Getting to grips with hepatitis 
eLife  2012;1:e00301.
The receptor that allows hepatitis B and hepatitis D viruses to enter human liver cells has been identified as a protein that transports bile acids in the liver.
doi:10.7554/eLife.00301
PMCID: PMC3485613  PMID: 23150799
Sodium taurocholate cotransporting polypeptide; receptor; hepatitis B virus; hepatitis D virus; liver; virus infection; Viruses; Other
8.  Prion-like polymerization as a signaling mechanism 
Trends in immunology  2014;35(12):622-630.
The innate immune system uses pattern recognition receptors such as RIG-I and NLRP3 to sense pathogen invasion and other danger signals. Activation of these receptors induces robust signal transduction cascades that trigger the production of cytokines important for host protection. MAVS and ASC are essential adaptor proteins downstream of RIG-I and NLRP3, respectively, and both contain N-terminal domains belonging to the death domain superfamily. Recent studies suggest that both MAVS and ASC form functional prion-like fibers through their respective death domains to propagate downstream signaling. Here, we review these findings, and in this context discuss the emerging concept of prion-like polymerization in signal transduction. We further examine the potential benefits of this signaling strategy, including signal amplification, host evolutionary advantage, and molecular memory.
doi:10.1016/j.it.2014.10.003
PMCID: PMC4429004  PMID: 25457352
prion-like polymerization; signaling; pattern recognition receptors; signal transduction
9.  Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA 
Cell  2014;159(7):1563-1577.
The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify of a mechanism by which mitochondria and downstream pro-apoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response, and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a pro-inflammatory type of cell death.
doi:10.1016/j.cell.2014.11.037
PMCID: PMC4272443  PMID: 25525875
10.  MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses 
Science (New York, N.Y.)  2014;346(6216):1486-1492.
Multivalent molecules with repetitive structures including bacterial capsular polysaccharides and viral capsids elicit antibody responses through B cell receptor (BCR) crosslinking in the absence of T cell help. We report that immunization with these T cell-independent type 2 (TI-2) antigens causes upregulation of endogenous retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a MAVS-dependent RNA sensing pathway or reverse transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a second, sustained wave of signaling that promotes specific IgM production. Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings suggest that ERV and two innate sensing pathways that detect them are integral components of the TI-2 B cell signaling apparatus.
doi:10.1126/science.346.6216.1486
PMCID: PMC4391621  PMID: 25525240
11.  Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation 
Cell  2014;156(6):1207-1222.
SUMMARY
Pathogens and cellular danger signals activate sensors such as RIG-I and NLRP3 to produce robust immune and inflammatory responses through respective adaptor proteins MAVS and ASC, which harbor essential N-terminal CARD and PYRIN domains, respectively. Here, we show that CARD and PYRIN function as bona fide prions in yeast and their prion forms are inducible by their respective upstream activators. Likewise, a yeast prion domain can functionally replace CARD and PYRIN in mammalian cell signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, a conserved fungal NOD-like receptor and prion pair can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion-like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.
doi:10.1016/j.cell.2014.01.063
PMCID: PMC4034535  PMID: 24630723
12.  Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2 
PLoS ONE  2015;10(3):e0119727.
Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13−/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.
doi:10.1371/journal.pone.0119727
PMCID: PMC4355416  PMID: 25756897
13.  Pivotal Roles of cGAS-cGAMP Signaling in Antiviral Defense and Immune Adjuvant Effects 
Science (New York, N.Y.)  2013;341(6152):10.1126/science.1244040.
Invasion of microbial DNA into the cytoplasm of animal cells triggers a cascade of host immune reactions that help clear the infection; however, self DNA in the cytoplasm can cause autoimmune diseases. Biochemical approaches led to the identification of cyclic GMP-AMP (cGAMP) synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here we show that cells from cGAS-deficient (cGas−/−) mice, including fibroblasts, macrophages and dendritic cells, failed to produce type-I interferons and other cytokines in response to DNA transfection or DNA virus infection. cGas−/− mice were more susceptible to lethal infection with herpes simplex virus-1 (HSV1) than wild type mice. We also show that cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice.
doi:10.1126/science.1244040
PMCID: PMC3863637  PMID: 23989956
14.  IKKε-mediated tumorigenesis requires K63-linked polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex 
Cell reports  2013;3(3):724-733.
SUMMARY
IκB kinase ε (IKKε, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ~30% of breast cancers, that promotes malignant transformation through NF-κB activation. Here we show that IKKε is modified and regulated by K63-linked polyubiquitination at Lysine 30 and Lysine 401. TNFα and IL-1β stimulation induces IKKε K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKKε kinase activity, IKKε-mediated NF-κB activation and IKKε-induced malignant transformation. Disruption of K63-linked ubiquitination of IKKε does not affect its overall structure but impairs the recruitment of canonical NF-κB proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKKε. Together, these observations demonstrate that K63-linked polyubiquitination regulates IKKε activity in both inflammatory and oncogenic contexts and suggests an alterative approach to target this breast cancer oncogene.
doi:10.1016/j.celrep.2013.01.031
PMCID: PMC4135466  PMID: 23453969
15.  The Cytosolic DNA Sensor cGAS Forms An Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop 
Cell reports  2014;6(3):421-430.
The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type-I interferons and other cytokines. Here we report the crystal structures of human cGAS in its apo form, representing its auto-inhibited conformation, as well as cGAMP-bound and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide new insights into the mechanism of DNA sensing by cGAS.
doi:10.1016/j.celrep.2014.01.003
PMCID: PMC3969844  PMID: 24462292
16.  Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses 
Science (New York, N.Y.)  2013;341(6148):10.1126/science.1240933.
Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus (MLV) and Simian immunodeficiency virus (SIV). These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.
doi:10.1126/science.1240933
PMCID: PMC3860819  PMID: 23929945
17.  Regulation of NF-κB by Ubiquitination 
Current opinion in immunology  2013;25(1):4-12.
The nuclear factor κ enhancer binding protein (NF-κB) family of transcription factors regulates the expression of a large array of genes involved in diverse cellular processes including inflammation, immunity and cell survival. Activation of NF-κB requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Studies in the past few years have provided new insights into the mechanisms underlying regulation of NF-κB by ubiquitination, including the involvement of multiple linkages of ubiquitin, the essential role of ubiquitin binding, and the function of unanchored polyubiquitin chains. In this review, we will focus on recent advances in understanding the role of ubiquitination in NF-κB regulation in various pathways.
doi:10.1016/j.coi.2012.12.005
PMCID: PMC3594545  PMID: 23312890
18.  Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING 
Molecular cell  2013;51(2):10.1016/j.molcel.2013.05.022.
The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.
doi:10.1016/j.molcel.2013.05.022
PMCID: PMC3808999  PMID: 23747010
19.  The role of ubiquitylation in immune defence and pathogen evasion 
Nature reviews. Immunology  2011;12(1):10.1038/nri3111.
Ubiquitylation is a widely used post-translational protein modification that regulates many biological processes, including immune responses. The role of ubiquitin in immune regulation was originally uncovered through studies of antigen presentation and the nuclear factor-κB family of transcription factors, which orchestrate host defence against microorganisms. Recent studies have revealed crucial roles of ubiquitylation in many aspects of the immune system, including innate and adaptive immunity and antimicrobial autophagy. In addition, mounting evidence indicates that microbial pathogens exploit the ubiquitin pathway to evade the host immune system. Here, we review recent advances on the role of ubiquitylation in host defence and pathogen evasion.
doi:10.1038/nri3111
PMCID: PMC3864900  PMID: 22158412
20.  Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway 
Science (New York, N.Y.)  2012;339(6121):10.1126/science.1232458.
The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.
doi:10.1126/science.1232458
PMCID: PMC3863629  PMID: 23258413
21.  Cyclic-GMP-AMP Is An Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA 
Science (New York, N.Y.)  2012;339(6121):10.1126/science.1229963.
Cytosolic DNA induces type-I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA.
doi:10.1126/science.1229963
PMCID: PMC3855410  PMID: 23258412
22.  RNA Helicase Signaling Is Critical for Type I Interferon Production and Protection against Rift Valley Fever Virus during Mucosal Challenge 
Journal of Virology  2013;87(9):4846-4860.
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
doi:10.1128/JVI.01997-12
PMCID: PMC3624317  PMID: 23408632
23.  Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination 
Cell  2013;152(5):1051-1064.
SUMMARY
Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
doi:10.1016/j.cell.2013.01.051
PMCID: PMC3640276  PMID: 23452853
24.  Competing E3 Ubiquitin Ligases Determine Circadian Period by Regulated Degradation of CRY in Nucleus and Cytoplasm 
Cell  2013;152(5):1091-1105.
SUMMARY
Period determination in the mammalian circadian clock involves the turnover rate of the repressors, CRY and PER. Here we show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate (G149E) missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss-of-function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm, but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.
doi:10.1016/j.cell.2013.01.055
PMCID: PMC3694781  PMID: 23452855
25.  Cyclic di-GMP Sensing via the Innate Immune Signaling Protein STING 
Molecular cell  2012;46(6):735-745.
SUMMARY
Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.
doi:10.1016/j.molcel.2012.05.029
PMCID: PMC3697849  PMID: 22705373

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