Research

We are broadly interested in mechanisms of signal transduction, namely how a cell communicates with its surroundings and within itself. In particular, we are fascinated by how a cell detects harmful or foreign insults and mounts an appropriate response to restore homeostasis.

Our research revolves around the common theme of cell signaling and host defense.

    Ubiquitin signaling in the NF-κB pathway

    IkB ubiquitination, IKK, and IkB ubiquitin ligase

    NF-κB is a transcription factor that regulates a plethora of genes in response to diverse stimuli. Aberrant regulation of NF-κB has been linked to many human diseases, such as cancer and autoimmune diseases. NF-κB is normally sequestered in the cytoplasm by proteins of the IkB family. Upon stimulation, distinct signaling cascades converge on the IkB kinase complex (IKK), which phosphorylates IkBs and targets these inhibitors for degradation by the ubiquitin-proteasome pathway. NF-κB then enters the nucleus to turn on downstream target genes.

    Ubiquitin-mediated activation of TAK1 and IKK

    Recently, we have focused on how IKK is regulated by different pathways. Interestingly, we found that ubiquitination activates IKK through a proteasome-independent mechanism. We found that TRAF6, an essential protein for NF-κB activation by interleukin-1b and Toll-like receptors (TLRs), is a ubiquitin E3 ligase that functions together with an E2 complex, Ubc13/Uev1A, to synthesize polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. These ubiquitin chains bind to the TAB2 and TAB3 subunits of the TAK1 kinase complex, resulting in TAK1 kinase activation. TAK1 then phosphorylates and activates IKK. Recent research in our lab and others have supported a critical role of ‘non-degradative’ ubiquitination in the activation of TAK1 and IKK in different pathways, including those triggered by TNF receptor, T cell receptor, RIG-I like receptor, NOD-like receptors, and DNA damage. 

    Representative Publications

    IkB ubiquitination, IKK, and IkB ubiquitin ligase

    • Chen, Z.J., Hagler, J. Palombella, V. J., Melandri, F., Scherer, D., Ballard, D., and Maniatis, T. (1995) Signal-induced site-specific phosphorylation targets IkBa to the ubiquitin-proteasome pathway. Genes & Dev. 9: 1586-1597
    • Chen, Z.J., Parent, L., and Maniatis, T. (1996) Site-specific phosphorylation of IkBa by a novel ubiquitination-dependent protein kinase activity. Cell 84, 853-862.
    • Spencer, E., Jiang, J., and Chen, Z.J. (1999) Signal-induced ubiquitination of IkBa by the F-box protein, Slimb/b-TrCP. Genes & Dev. 13, 284-294.

    Ubiquitin-mediated activation of TAK1 and IKK

    • Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z.J. (2000) Activation of the IkB kinase complex requires a dimeric ubiquitin-conjugating enzyme complex and the formation of a unique polyubiquitin chain. Cell 103, 351-361.
    • Wang, C., Deng, L., Hong, M. Akkaraju, G.R., Inoue, J-i., and Chen, Z.J. (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346-351.
    • Sun, L., Deng, L., Ea, C-K., Xia, Z-P., and Chen, Z.J. (2004) The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Molecular Cell 14, 289-301.
    • Kanayama, A., Seth, R.B., Ea, C-K, Hong, M., Shaito, A., Deng, L., and Chen, Z.J. (2004) TAB2 and TAB3 activate the NF-κB pathway through binding to polyubiquitin chains. Molecular Cell 15, 535-548.
    • Ea, C-K., Deng, L., Xia, Z-P., Pineda, G., and Chen, Z.J. (2006) Activation of  IKK by TNFa requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Molecular Cell 22, 245-257.
    • Xu, M., Skaug, B., Zeng, W., and Chen, Z.J. (2009) A ubiquitin replacement strategy reveals distinct mechanisms of IKK activation by TNFa and IL-1b. Molecular Cell 36, 315-325.
    • Xia, Z.P., Sun, L., Chen, X., Pineda, G., Jiang, X., Adhikari, A., Zeng, W., and Chen, Z.J. (2009). Direct activation of protein kinases by unanchored polyubiquitin chains. Nature. 461, 114-119.
    • Skaug, B., Chen, J., Du, F., He, J, Ma, A., and Chen, Z.J. (2011) Direct, non-catalytic mechanism of IKK inhibition by A20. Molecular Cell 44, 559-571.
    Innate immune sensing and signaling of cytosolic nucleic acids

    Discovery of MAVS and its role in immune defense

    NF-κB functions together with another transcription factor, IRF3, to induce type-I interferon production upon viral infection. After viral infection, viral RNA is detected by the RIG-I family of RNA helicases. In 2005, we identified the protein MAVS (also known as IPS-1, VISA, or CARDIF) as a key adaptor for RIG-I signaling. MAVS is localized on the mitochondrial outer membrane and this localization is indispensable for its function, as underscored by the finding that viral protease NS3/4A of hepatitis C virus efficiently cleaves MAVS off the mitochondrial membrane to suppress interferon induction. The essential role of MAVS in defense against RNA virus is further confirmed in MAVS knock-out mice. Recently, we found that the RIG-I pathway is also important for detecting commensal bacterial RNA and maintaining intestinal homeostasis.

    Mechanism of signal transduction in the RIG-I–MAVS pathway

    Our recent work has focused on the mechanism of signal transduction in the RIG-I pathway. Through the development of a cell-free system that recapitulates the RIG-I pathway from detection of viral RNA to activation of IRF3, we found that ubiquitination also plays a key role in RIG-I activation. Specifically, after binding to RNA, RIG-I undergoes a conformational change that exposes its N-terminal CARD domains, which then bind to unanchored K63 polyubiquitin chains. This binding promotes the formation of RIG-I tetramer, which interacts with MAVS and promotes MAVS aggregation. Strikingly, MAVS aggregates catalyze the polymerization of other MAVS on the mitochondria through a prion-like mechanism. The structure of MAVS aggregates is solved by electron microscopy and the prion-like mechanism turns out to be a universal mechanism in cell signaling. These prion-like fibers of MAVS recruit multiple ubiquitin E3 ligases, including TRAF2 TRAF5 and TRAF6, to potently activate the cytosolic kinases IKK and TBK1, leading to the activation of NF-κB and IRF3, respectively. Interestingly, MAVS is phosphorylated by IKK and TBK1, which provides a binding site for IRF3. This signal-induced phosphorylation adds another layer of precise regulation for IRF3 activation.

    Interferon induction by cytosolic DNA

    We are also interested in how cytosolic DNA induces type-I interferons, which are important for immune defense against DNA viruses and intracellular bacteria. Furthermore, the inappropriate presence of cytosolic self-DNA could trigger autoimmune diseases. We have found that AT-rich DNAs are transcribed by RNA polymerase III into RNAs bearing 5’-triphosphates, which then induce interferons through the RIG-I pathway. However, most DNA induces interferons in a sequence-independent manner that depends on the endoplasmic reticulum protein STING (also known as MITA). We have recently shown that after stimulation, STING recruits both TBK1 and IRF3, thereby specifying IRF3 phosphorylation by TBK1.

    Discovery of cGAS-cGAMP pathway and its role in immune defense

    We identified cGAS, which directly binds to cytosolic DNA, forms a dimer, and synthesizes cGAMP from GTP and ATP. cGAMP with mixed phosphodiester linkages binds to STING with high-affinity and induces STING conformational change and interferon expression. We generated cGAS knockout mice and cell lines. Using these reagents we found that cGAS-cGAMP pathway is important for the immune response against DNA viruses, retroviruses, and bacteria. In addition, we found that cGAS is responsible for the severe inflammation found in DNAse deficient mice, suggesting cGAS can also be activated by self DNA and may cause inflammation in some lupus patients.

    Novel functions and regulations of TLRs

    Our recent work on commensal bacteria led us to discover the role of TLR13 in detecting the bacterial 23S ribosomal RNA (rRNA). Remarkably, we found that TLR13 recognizes a specific sequence of about 13 nucleotides near the active site of the 23S rRNA, which catalyzes peptide bond synthesis. Thus, unlike other innate immune sensors that detect a ‘pattern’ of microbial components, TLR13 detects bacterial RNA with exquisite sequence specificity. IRF5 is a transcription factor that is essential for immune signaling of multiple TLRs, we recently discovered that IKKb phosphorylated IRF5 and induces IRF5 dimerization and nuclear translocation.

    Representative publications

    Discovery of MAVS and its role in immune defense

    • Seth, R.B., Sun, L., Ea, C., and Chen, Z.J. (2005) Identification and characterization of MAVS: a mitochondrial antiviral signaling protein that activates NF-κB and IRF3. Cell 122, 669-682.
    • Li, X-D., Sun, L., Seth, R.B., Pineda, G., and Chen, Z.J. (2005) The Hepatitis C Virus Protease NS3/4A Cleaves MAVS off the Mitochondria to Evade Innate Immunity. Proc. Natl. Acad. Sci. U S A102, 17717-17722.
    • Sun, Q., Sun, L., Liu, H-H., Chen, X., Seth, R.B., Forman, J. and Chen, Z.J. (2006) The specific and essential role of MAVS in antiviral innate immune responses. Immunity 24, 633-642.
    • Bhoj, V.G., Sun, Q., Bhoj, E., Somers, C., Chen, X., Torres, J-P., Mejias, A., Gomez., A., Jafri, H., Ramilo, O., Chen, Z.J. (2008). MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus. Proc. Natl. Acad. Sci. U S A 105, 14046-14051.
    • Li, X.D., Chiu, Y.H., Ismail, A.S., Behrendt, C.L., Wight-Carter, M., Hooper, L.V., and Chen, Z.J. (2011). Mitochondrial antiviral signaling protein (MAVS) monitors commensal bacteria and induces an immune response that prevents experimental colitis. Proc Natl Acad Sci U S A108, 17390-17395.

    Mechanism of signal transduction in the RIG-I–MAVS pathway

    • Zeng, W., Xu, M., Liu, S., Sun, L., Chen, Z.J. (2009) Key role of Ubc5 and K63 polyubiquitination in viral activation of IRF3. Molecular Cell 36, 302-314.
    • Zeng, W., Sun, L., Jiang, X., Chen, X., Hou, F., Adhikari, A., Xu, M., and Chen, Z.J. (2010) Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315-330.
    • Hou, F., Sun, L., Zheng, H., Skaug, B., Jiang, Q.X., and Chen, Z.J. (2011). MAVS Forms Functional Prion-like Aggregates to Activate and Propagate Antiviral Innate Immune Response. Cell 146, 448-461.
    • Jiang, X., Kinch, L., Brautigam, C.A., Chen, X., Du, F., Grishin, N., Chen, Z.J. (2012) Ubiquitin-induced oligomerization of RIG-I and MDA5 activates antiviral innate immune response. Immunity 36, 959-973.
    • Liu, S., Chen, J., Cai, X., Wu, J., Chen, X., Wu, Y-T., Sun, L., and Chen, Z.J. (2013) MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades. eLife 2:e00785
    • Xu, H., He, X., Zheng, H., Huang, L.J., Hou, F., Yu, Z., de la Cruz, M.J., Borkowski, B., Zhang, X., Chen, Z.J., and Jiang, Q.X. (2014) Structural basis for the prion-like MAVS filaments in antiviral innate immunity. Elife. 3:e01489.
    • Cai, X., Chen, J., Xu, H., Liu, S., Jiang, Q.X., Halfmann, R., and Chen, Z.J. (2014) Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation. Cell. 156(6):1207-22.
    • Liu, S., Cai, X., Wu, J., Cong, Q., Chen, X., Li, T., Du, F., Ren, J., Wu, Y., Grishin, N., and Chen, Z.J. (2015) Phosphorylation of innate immune adaptor proteins MAVS, STING and TRIF induces IRF3 activation. Science 347, aaa2630.

    Interferon induction by cytosolic DNA

    • Chiu, Y.H., Macmillan, J.B., and Chen, Z.J. (2009). RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576-591.
    • Tanaka, Y., and Chen, Z.J. (2012). STING Specifies IRF3 Phosphorylation by TBK1 in the Cytosolic DNA Signaling Pathway. Science Signaling 5, ra20.

    Discovery of cGAS-cGAMP pathway and its role in immune defense

    • Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., Chen Z.J. (2012) Cyclic GMP-AMP Is an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA. Science 339, 826-830.
    • Sun, L., Wu, J., Du, F., Chen, X., Chen, Z.J. (2012) Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway. Science 339, 786-791.
    • Zhang, X., Shi, H., Wu, J., Zhang, X., Sun, L., Chen, C., and Chen, Z.J. (2013) Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell 51:226-235.
    • Gao, D., Wu, J., Wu, Y-T., Du, F., Aroh, C., Yan, N., Sun, L., and Chen, Z.J. (2013) Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses. Science 341:903-906.
    • Li, X.D., Wu, J., Gao, D., Wang, H., Sun, L., and Chen, Z.J. (2013) Pivotal Roles of cGAS-cGAMP Signaling in Antiviral Defense and Immune Adjuvant Effects. Science 341(6152):1390-4.
    • Zhang, X., Wu, J., Du, F., Xu, H., Sun, L., Chen, Z., Brautigam, C.A., Zhang, X., and Chen, Z.J. (2014) The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Rep. 6(3):421-30. 
    • Collins, A.C., Cai, H., Li, T., Franco, L.H., Li, X-D., Nair, V.R., Scharn, C.R., Stamm, C.E., Levine, B., Chen, Z.J*., Shiloh, M.U*. (2015) Cyclic GMP-AMP synthase is an innate immune sensor of Mycobacterium tuberculosis DNA. Cell Host Microbe 17, 820-828. *co-corresponding author
    • Gao, D., Li, T., Li, X-D., Chen, X., Li, Q-Z., Wight-Carter, M., and Chen, Z.J. (2015) Activation of Cyclic GMP-AMP Synthase by Self DNA Causes Autoimmune Diseases. Proc Natl Acad Sci U S A. 112, E5699-705.

    Novel functions of TLRs

    • Li, X. and Chen, Z.J. (2012). Sequence specific detection of bacterial 23S ribosomal RNA by TLR13. eLife 1, e00102
    • Ren, J., Chen, X., and Chen, Z.J. (2014) IKKbeta Is An IRF5 Kinase that Instigates Inflammation. Proc Natl Acad Sci USA 111, 17438–17443.

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