Reconstitution of the RIG-I Pathway Reveals a Signaling Role of Unanchored Polyubiquitin Chains in Innate Immunity

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
Cell (Impact Factor: 32.24). 04/2010; 141(2):315-30. DOI: 10.1016/j.cell.2010.03.029
Source: PubMed


RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.

    • "It is well established that the E3 ubiquitin ligase TRIM25 activates RIG-I by generation of K63-linked polyubiquitin chains covalently attached to K172 of RIG-I (Gack et al, 2007). Meanwhile, separated K63-linked polyubiquitin chains can bind to RIG-I noncovalently for synergistic activation (Zeng et al, 2010). Furthermore, T170 was found constitutively phosphorylated under physiological conditions, preventing RIG-I from binding to TRIM25 and MAVS (Gack et al, 2010). "
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    ABSTRACT: RIG-I is a well-studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4-Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG-I. The p97 complex is able to directly bind both non-ubiquitinated RIG-I and the E3 ligase RNF125, promoting K48-linked ubiquitination of RIG-I at residue K181. Viral infection significantly strengthens the interaction between RIG-I and the p97 complex by a conformational change of RIG-I that exposes the CARDs and through K63-linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG-I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.
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    • "Previously, it was shown that TRIM25-mediated K63 linked polyubiquitination of RIG-I Lys172 is critical for its activation, linked polyubiquitination of RIG-I Lys172 is critical for its activation , although another study showed that non-covalently bound K63-linked polyubiquitin chain was sufficient to activate RIG-I signaling (Gack et al., 2007; Zeng et al., 2010). Whichever the case, the critical role of TRIM25 and K63-linked polyubiquitination has been proven by these studies. "
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    ABSTRACT: Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.
    Full-text · Article · Aug 2015 · Moleculer Cells
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    • "the unanchored form may serve as a scaffold for recruiting proteins in the signaling pathways of innate immunity and protein aggregate removal (Zeng et al., 2010; Hao et al., 2013). How does Lys63-linked polyubiquitin perform different functions? "
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    ABSTRACT: A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.
    Full-text · Article · Jun 2015 · eLife Sciences
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