Research in my laboratory is directed towards understanding two signaling pathways: ubiquitin-mediated activation of protein kinases and antiviral innate immunity. Ubiquitin is best known for targeting protein degradation by the proteasome. Recently my lab has uncovered a novel function of ubiquitin in activating protein kinases in the NF-κB signaling cascade through a proteasome-independent mechanism. Our current effort is focused on elucidating the biochemical mechanism underlying the regulatory function of ubiquitin. In viral signaling, we are particularly interested in dissecting the signaling pathway by which a host cell mounts an immune response to RNA virus infection. We are also interested in how some viruses evade the host immune system.
Ubiquitin Signaling in the NF-κB Pathway
NF-κB is a master transcription factor that regulates the expression of a large array of genes involved in inflammation, immunity and cell survival. The activity of NF-κB is tightly regulated through its subcellular localization. In unstimulated cells, NF-κB is sequestered in the cytoplasm through its association with the inhibitor IκB. Stimulation of cells with various agents leads to the activation of a large kinase complex, termed IKK, which phosphorylates IκB and targets this inhibitor for degradation by the ubiquitin-proteasome pathway. NF-κB is then liberated to carry out its nuclear functions.
In the course of studying how IKK is activated by TRAF6, a key protein required for signaling by proinflammatory cytokines and microbial products, we found that TRAF6 is a ubiquitin E3 ligase that functions together with a specific ubiquitin E2 complex to catalyze the synthesis of a unique polyubiquitin chain linked through Lys-63 (K63) of ubiquitin. This form of polyubiquitination activates a protein kinase complex consisting of TAK1 and its regulatory proteins TABs (TAB1-3). The activation of the TAK1 complex requires the binding of K63-linked polyubiquitin chains to a specialized ubiquitin-binding domain of TAB2 and TAB3. After TAK1 is activated, it phosphorylates IKKβ at two serine residues in the activation loop, leading to IKK activation.
The importance of TRAF6-catalyzed ubiquitination in the activation of TAK1 and IKK raises the question of how TRAF6 ubiquitin E3 activity is regulated. We gained insights into this question from studying the T cell receptor (TCR) signaling pathway. Stimulation of TCR leads to the activation of a protein kinase cascade that subsequently recruits a signaling complex consisting of CARMA1, BCL10 and MALT1 to the receptor. We found that MALT1 contains binding sites for TRAF6. The binding of MALT1 to TRAF6 promotes its oligomerization and activates its ubiquitin ligase activity. TRAF6 then activates TAK1 and IKK through a mechanism similar to that in the innate immunity pathways. This T cell signaling pathway, from BCL10 to IκB phosphorylation, can be reconstituted with purified proteins in vitro. In addition, we have recently generated conditional deletion of TAK1 in the T cell lineage of mice, and demonstrated that TAK1 is essential for thymocyte development and activation.
Recent studies in our lab have also elucidated the mechanism of IKK activation by TNFα. Stimulation of cells with TNFα leads to the trimerization of the TNF receptor (TNF-R1) and subsequent recruitment of signaling proteins including the ubiquitin ligase TRAF2 and protein kinase RIP1. We found that RIP1 is polyubiquitinated, likely by TRAF2, at a specific lysine (K377) in response to TNFα stimulation. A point mutation at K377 of RIP1 abrogated its polyubiquitination as well as its ability to activate IKK. Polyubiquitination of RIP1 mediates the recruitment of TAK1 and IKK complexes to the TNF receptor. The recruitment of TAK1 is through the interaction between K63 polyubiquitin chains and the ubiquitin-binding domain of TAB2 or TAB3, whereas the recruitment of IKK is through the binding between K63 polyubiquitin chains and a newly identified ubiquitin-binding domain of NEMO, an essential regulatory subunit of IKK. Thus, K63 polyubiquitin chains serve as a scaffold to bring together TAK1 and IKK complexes, allowing TAK1 to phosphorylate and activate IKK. These studies reveal the biochemical basis underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 mediates the activation of IKK in the TNFα pathway.
Viral Signaling
Viral diseases remain a major global health threat. Currently there are few effective antiviral drugs, so the best weapon in our possession to fight viruses is our immune systems. Upon viral infection, the host innate immune system produces interferons and cytokines to suppress viral replication and assembly. The production of interferons and cytokines is regulated by transcription factors including NF-κB and IRF (interferon regulatory factor), but how these transcription factors are activated by viruses is not well understood. We have recently embarked on a research program aiming to dissect the signaling pathways elicited by RNA viruses; members of this family include hepatitis C virus (HCV), Influenza virus and West Nile virus. Most of these viruses replicate in the cytosol and the viral RNA binds to the RNA helicases RIG-I and MDA-5, which then trigger a signaling cascade to activate NF-κB and IRF.
We have recently discovered the adaptor that links RIG-I/MDA-5 to the kinases (IKK and TBK1) that activate NF-κB and IRF. This adaptor, which we named MAVS (Mitochondrial Anti-Viral Signaling), contains a CARD-like effector domain at the N-terminus and a mitochondrial membrane targeting domain (TM) at the C-terminus. The TM domain localizes MAVS to the mitochondrial outer membrane and this localization is essential for the signaling functions of MAVS. This is the first mitochondrial protein known to play a key role in antiviral response, and it suggests a new role of mitochondria in immunity.
The importance of the mitochondrial localization of MAVS is underscored by our recent finding that hepatitis C virus (HCV) employs a serine protease called NS3/4A to cleave MAVS at a specific residue (Cys-508) adjacent to the C-terminal TM domain. This cleavage dislodges MAVS from the mitochondrial membrane, cripples its ability to induce interferons and other antiviral molecules, and allows HCV to evade the host immune system to establish chronic infection. This is medically significant, as HCV infects more than 170 million people worldwide, and about 80% of the infected individuals develop chronic infection. Inhibitors of the HCV protease are promising therapeutics because they should not only block viral replication and assembly, but also prevent the cleavage of MAVS, thereby restoring the host antiviral immune defense.
We have recently investigated the in vivo function of MAVS by generating a mouse strain lacking this gene. With only one exception (plasmacytoid dendritic cells), most cells depend on MAVS for the induction of interferons and cytokines in response to RNA virus infection. The MAVS knockout mice are viable and fertile, but they are highly susceptible to viral killing, confirming the importance of MAVS in antiviral defense.
Currently we are taking a multidisciplinary approach to investigate the mechanism and function of MAVS and other signaling molecules of the innate immunity pathways. We are also studying the potential role of ubiquitination in regulating the antiviral pathways. Collectively, these studies should provide a deeper understanding of ubiquitin as a versatile signaling molecule and enable us to harness the power of the immune system to fight viral and autoimmune diseases.
RESEARCH INTERESTS
Ubiquitin-Proteasome Pathway
NF-kappaB Signaling
Innate Immunity
T Cell Receptor Signaling
MAVS: mitochondrial antiviral signaling protein
RECENT PUBLICATIONS
Chiu, Y.H., Macmillan, J.B., and Chen, Z.J, "RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway" Cell, 138:576-591, August 2009
Xia, Z.P., Sun, L., Chen, X., Pineda, G., Jiang, X., Adhikari, A., Zeng, W., and Chen, Z.J, "Direct activation of protein kinases by unanchored polyubiquitin chains" Nature, 461:114-119, August 2009
Bhoj, V.G., and Chen, Z. J, "Ubiquitylation in innate and adaptive immunity" Nature, 458:430-437, March 2009
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, "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, September 2008
Chiu, Y-H., Sun, Q., and Chen, Z.J, "E1-L2 activates both ubiquitin and FAT-10" Molecular Cell, 27:1014-1023, August 2007
SIGNIFICANT PUBLICATIONS
Seth, R.B., Sun, L., Ea, C., and Chen, Z.J., "Identification and characterization of MAVS: a mitochondrial antiviral signaling protein that activates NF-κB and IRF3." Cell, 122:669-682, 2005
Sun, Q., Sun, L., Liu, H-H., Chen, X., Seth, R.B., Forman, J. and Chen, Z.J, "The specific and essential role of MAVS in antiviral innate immune responses" Immunity, 24:633-642, May 2006
Ea, C-K., Deng, L., Xia, Z-P., Pineda, G., and Chen, Z.J, "Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO" Molecular Cell, 22:245-257, April 2006
Wang, C., Deng, L., Hong, M. Akkaraju, G.R., Inoue, J-i., and Chen, Z, "TAK1 is a ubiquitin-dependent kinase of MKK and IKK" Nature, 412:346-351, July 2001
Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z, "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, October 2000
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