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NF-kB pathway

Innate immune sensing and signaling of cytosolic nucleic acids

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Ubiquitin signaling in the NF-kB pathway.

NF-kB is a transcription factor that regulates a plethora of genes in response to diverse stimuli. Aberrant regulation of NF-kB has been linked to many human diseases, such as cancer and autoimmune diseases. NF-kB 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-kB then enters the nucleus to turn on downstream target genes. (Representative publications 1-3)

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-kB 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 4-11)

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Innate immune sensing and signaling of cytosolic nucleic acids

NF-kB 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 indispensible 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. (Representative publications 12-16)

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 binds 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. These prion-like fibers of MAVS are highly potent in activating the cytosolic kinases including IKK and TBK1, leading to the activation of NF-kB and IRF3, respectively. (Representative publications 17-20)

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, 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. How cytosolic DNA leads to STING activation is currently unknown and a subject of active investigation. (Representative publications 21-22)

We maintain a considerable degree of flexibility in venturing into other areas of interest in cell signaling and host defense. For example, 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. (Publication 23).

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IkB ubiquitination, IKK and IkB ubiquitin ligase

  1. 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

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  3. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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-kB pathway through binding to polyubiquitin chains. Molecular Cell 15, 535-548.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

Discovery of MAVS and its role in immune defense

  1. 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-kB and IRF3. Cell 122, 669-682.

  2. 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 A 102, 17717-17722.

  3. 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.

  4. 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.

  5. 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 A 108, 17390-17395.

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

  1. 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.

  2. 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.

  3. 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.

  4. 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 inate immune response. Immunity 36, 959-973.

Interferon induciton by cytosolic DNA

  1. 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.

  2. Tanaka, Y., and Chen, Z.J. (2012). STING Specifies IRF3 Phosphorylation by TBK1 in the Cytosolic DNA Signaling Pathway. Science Signaling 5, ra20.

Novel functions of TLRs

  1. Li, X. and Chen, Z.J. (2012). Sequence specific detection of bacterial 23S ribosomal

RNA by TLR13. eLife (in press)

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

  1. Sun, L., Wu, J., Du, F., Chen, X., Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type i interferon pathway. Science 339:786-91 (2012), PMC3863629.

  2. Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339:826-30 (2012), PMC3855410.

  3. Li, X.D., Wu, J., Gao, D., Wang, H., Sun, L., Chen, Z.J. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341:1390-94 (2013), PMC3863637.

  4. 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 (2013)

  5. Zhang, X., Wu, J., Du, F., Xu, H., Sun, L., Chen, Z., Brautigam, C.A., Zhang, X., Chen, Z.J. The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop. Cell Rep 6, 421-430 (2014)

  6. Liu, S., Cai, X., Wu, J., Cong, Q., Chen, X., Li, T., Du, F., Ren, J., Wu, Y.T., Grishin, N.V., Chen, Z.J. (2015). Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 347(6227):aaa2630 (2015), PMID: 25636800

  7. 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)

Discovery of the roles of cGAS in anti-tumor immunity, cellular senescence, autoimmunity, and the regulation of cGAS through liquid-liquid phase separation

  1. 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. doi: 10.1073/pnas.1516465112.

  2. Wang, H., Hu, SQ., Shi, H., Chen, C., Sun, L. and Chen, Z.J. (2017) cGAS is essential for the antitumor effect of immune checkpoint blockade. Proc Natl Acad Sci U S A. 114, 1637-1642

  3. Yang, H., Wang, H., Ren, J., Chen, Q. & Chen, Z. J. cGAS is essential for cellular senescence. Proc Natl Acad Sci U S A 114, E4612-E4620, doi:10.1073/pnas.1705499114 (2017), PMC5468617

  4. Luo, M., Wang, H., Wang, Z., Cai, H., Lu, Z., Li, Y., Du, M., Huang, G., Wang, C., Chen, X., Porembka, MR, Lea, J, Frankel, A.E, Fu, Y.X, *Chen, Z.J, *Gao, J. (2017). A STING-activating nanovaccine for cancer immunotherapy. Nat Nanotechnol. 12, 648-654. (* co-corresponding author)

  5. Du, M., and Chen, Z.J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361:704-9 (2018), PMID: 29976794.

  6. Zhang, C., Shang, G., Gui, X., *Zhang, X., *Bai, X.C., and *Chen, Z.J. Structural basis of STING binding with and phosphorylation by TBK1. Nature 567:394-8 (2019), (* co-corresponding author), PMC6862768.

  7. Shang, G., Zhang, C., *Chen, Z.J., *Bai, X.C., and *Zhang, X. (2019). Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP. Nature 567, 389-393. (* co-corresponding author).

  8. Gui, X., Yang, H., Li, T., Tan, X., Shi, P., Li, M., Du, F., and Chen, Z.J. (2019). Autophagy induction via STING trafficking is a primordial function of the cGAS pathway. Nature 567, 262-266.

  9. Ablasser, A., and Chen, Z.J. (2019). cGAS in action: Expanding roles in immunity and inflammation. Science 363. 1055 (eaat8657).

  10. Li, T., Huang, T., Du, M., Chen, X., Du, F., Ren, J., and Chen, Z.J. Phosphorylation and chromatin tethering prevent cGAS activation during mitosis. Science, eabc5386 (2021). PMID: 33542149.

  11. Yum, S., Li, M., Fang, Y., and Chen, Z.J. (2021). TBK1 recruitment to STING activates both IRF3 and NF-kappaB that mediate immune defense against tumors and viral infections. Proc Natl Acad Sci U S A 118.

  12. Hu, S., Fang, Y., Chen, X., Cheng, T., Zhao, M., Du, M., Li, T., Li, M., Zeng, Z., Wei, Y., et al. (2021). cGAS restricts colon cancer development by protecting intestinal barrier integrity. Proc Natl Acad Sci U S A 118.

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