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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.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
17.  Human Metapneumovirus M2-2 Protein Inhibits Innate Cellular Signaling by Targeting MAVS 
Journal of Virology  2012;86(23):13049-13061.
Human metapneumovirus (hMPV) is a leading cause of respiratory infections in pediatric populations globally, with no prophylactic or therapeutic measures. Recently, a recombinant hMPV lacking the M2-2 protein (rhMPV-ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making it a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein and host cells that regulates viral infection/propagation is largely unknown. By taking advantage of the available reverse genetics system and ectopic expression system for viral protein, we found that M2-2 not only promotes viral gene transcription and replication but subverts host innate immunity, therefore identifying M2-2 as a novel virulence factor, in addition to the previously described hMPV G protein. Since we have shown that the RIG-I/MAVS pathway plays an important role in hMPV-induced signaling in airway epithelial cells, we investigated whether M2-2 antagonizes the host cellular responses by targeting this pathway. Reporter gene assays and coimmunoprecipitation studies indicated that M2-2 targets MAVS, an inhibitory mechanism different from what we previously reported for hMPV G, which affects RIG-I- but not MAVS-dependent gene transcription. In addition, we found that the domains of M2-2 responsible for the regulation of viral gene transcription and antiviral signaling are different. Our findings collectively demonstrate that M2-2 contributes to hMPV immune evasion through the inhibition of MAVS-dependent cellular responses.
doi:10.1128/JVI.01248-12
PMCID: PMC3497653  PMID: 23015697
18.  Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection 
The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. Here, we demonstrate a non-redundant functional requirement for both nucleic acid-sensing Toll-like receptors (TLR) and RIG-I-like receptors (RLR) in the control of a mouse model of chronic viral infection. Whereas the RLR pathway was important for production of type I interferons and optimal CD8+ T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral antibody responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific antibody and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on antibody production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal antibody responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.
doi:10.4049/jimmunol.1103656
PMCID: PMC3331923  PMID: 22447976
19.  Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor 
Journal of Lipid Research  2013;54(5):1410-1420.
Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells.
doi:10.1194/jlr.M035774
PMCID: PMC3653405  PMID: 23419260
low density lipoprotein receptor; lysosomal protein degradation; proteasomal protein degradation; E3 ubiquitin ligase
20.  SnapShot: Pathways of Antiviral Innate Immunity 
Cell  2010;140(3):436-436.e2.
doi:10.1016/j.cell.2010.01.041
PMCID: PMC3586550  PMID: 20144765
21.  A20 Ubiquitin Ligase-Mediated Polyubiquitination of RIP1 Inhibits Caspase-8 Cleavage and TRAIL-Induced Apoptosis in Glioblastoma 
Cancer Discovery  2012;2(2):140-155.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a cancer therapeutic target. However, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL-targeted therapies. We show here that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through two signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 (DR5) and receptor-interacting protein 1 (RIP1), forms a plasma membrane bound preligand assembly complex (PLAC) under physiologic conditions. TRAIL treatment leads to the recruitment of caspase-8 to the PLAC for the assembly of a death-inducing signaling complex (DISC). In the DISC, the C-terminal Zinc finger (Znf) domain of A20 ubiquitin ligase mediates RIP1 ubiquitination through lysine (K)-63-linked polyubiquitin chains that bind the protease domain of caspase-8 and inhibits its dimerization, cleavage and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells.
doi:10.1158/2159-8290.CD-11-0172
PMCID: PMC3354650  PMID: 22585859
A20; apoptosis; caspase-8; TRAIL; ubiquitination
22.  STING Specifies IRF3 phosphorylation by TBK1 in the Cytosolic DNA Signaling Pathway 
Science signaling  2012;5(214):ra20.
Cytosolic double-stranded DNA (dsDNA) triggers type-I interferon production through the endoplasmic reticulum adaptor protein STING (also known as MITA, MPYS and ERIS), which activates the transcription factor IRF3. However, how STING activates IRF3 remains largely unknown. Here we show that STING stimulates IRF3 phosphorylation by the kinase TBK1 in an in vitro reconstitution system. Using this system, we identified a carboxyl terminal region of STING that is both necessary and sufficient to activate TBK1 and stimulate IRF3 phosphorylation. Interestingly, we found that STING interacts with both TBK1 and IRF3, and that mutations in STING that selectively disrupt its binding to IRF3 abrogate IRF3 phosphorylation without impairing TBK1 activation. These results suggest that STING functions as a scaffold to specify and promote IRF3 phosphorylation by TBK1. The scaffolding function of STING and other adaptors may explain why IRF3 is activated only in a subset of signaling pathways that activate TBK1.
doi:10.1126/scisignal.2002521
PMCID: PMC3549669  PMID: 22394562
23.  Intrinsic Antiviral Immunity 
Nature immunology  2012;13(3):214-222.
Intrinsic antiviral immunity refers to a form of innate immunity that directly restricts viral replication and assembly, thereby rendering a cell non-permissive to a specific class or species of viruses. Intrinsic immunity is conferred by restriction factors that are largely preexisting in certain cell types, although these factors can be further induced by virus infection. Intrinsic viral restriction factors recognize specific viral components, but unlike other pattern recognition receptors that inhibit viral infection indirectly by inducing interferons and other antiviral molecules, intrinsic antiviral factors block viral replication immediately and directly. This review focuses on recent advances in understanding the roles of intrinsic antiviral factors that restrict infection by human immunodeficiency virus (HIV) and influenza virus.
doi:10.1038/ni.2229
PMCID: PMC3549670  PMID: 22344284
24.  Ubiquitination in Signaling to and Activation of IKK 
Immunological reviews  2012;246(1):95-106.
A role of polyubiquitination in the activation of IκB kinase (IKK) through a proteasome-independent mechanism was first reported in 1996, but the physiological significance of this finding was not clear until 2000 when TRAF6 was found to be a ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination. Since then, several proteins known to regulate IKK have been linked to the ubiquitin pathway. These include the deubiquitination enzymes CYLD and A20 that inhibit IKK, and the ubiquitin binding proteins NEMO and TAB2 which are the regulatory subunits of IKK and TAK1 kinase complexes, respectively. Now accumulating evidence strongly supports a central role of K63 polyubiquitination in IKK activation by multiple immune and inflammatory pathways. Interestingly, recent research suggests that some alternative ubiquitin chains such as linear or K11 ubiquitin chains may also play a role in certain pathways such as the TNF pathway. Here I present a historical narrative of the discovery of the role of ubiquitin in IKK activation, review recent advances in understanding the role and mechanism of ubiquitin-mediated IKK activation, and raise some questions to be resolved in future research.
doi:10.1111/j.1600-065X.2012.01108.x
PMCID: PMC3549672  PMID: 22435549
25.  Ubiquitin-Induced Oligomerization of the RNA Sensors RIG-I and MDA5 Activates Antiviral Innate Immune Response 
Immunity  2012;36(6):959-973.
SUMMARY
RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.
doi:10.1016/j.immuni.2012.03.022
PMCID: PMC3412146  PMID: 22705106

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