Effective immune defense against microbial infection depends upon efficient detection of pathogens by innate immune receptors. Among the proteins that ensure proper functioning of these immune receptors are ubiquitin (Ub) and E3 ligases that work through both proteasome-dependent and -independent mechanisms. In this grant, we explore the molecular mechanism of RIPLET, an E3 ligase that plays a proteasome-independent function in activating antiviral innate immune receptor, RIG-I. This grant builds upon our previous work on RIG- I and our recent findings on RIPLET. RIG-I is a conserved cytosolic innate immune receptor that recognizes RNAs from a broad range of viruses. RIG-I contains an N-terminal signaling domain (tandem CARD or 2CARD) and C-terminal RNA binding domain. Studies from our lab, and others, have identified at least three steps involved in the activation of RIG-I: (i) RNA binding, (ii) release of 2CARD auto-repression, and (iii) tetramerization of 2CARDs. The 2CARD tetramer then activates the downstream adaptor, MAVS, which in turn stimulates the antiviral signaling pathways. In particular, the third step of 2CARD tetramerization is stimulated by K63-linked polyubiquitin chains (K63-Ubn), which binds and stabilizes the 2CARD tetramer, as demonstrated by our crystal structures.! Despite the detailed understanding of the action of K63-Ubn on RIG-I, much remains debated about how and when K63-Ubn is placed on RIG-I. Accumulating evidence suggests that RIPLET, a poorly understood E3 ligase, plays an essential role in conjugating K63-Ubn required for 2CARD tetramerization. We found that RIPLET recognizes the RNA-binding domain of RIG-I, but only when it is pre-oligomerized on dsRNA in a filamentous form. We further revealed that RIPLET binds RIG-I filaments through two distinct binding modes: intra-filament binding and inter-filament bridging. The latter dominates for RIG-I filaments on longer dsRNAs, leading to RIG-I clustering and further amplification of RIG-I signaling in a dsRNA length-dependent manner. These findings showed the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination (Cadena et al, under revision, available in BioRxiv). These findings of ours now raise new and deeper questions about the RIG-I mechanism from the fresh perspective of RIPLET: precisely how RIG-I is ubiquitinated by RIPLET (Aim 1), how RIG-I is recognized by RIPLET (Aim 2), how the oligomeric state of RIG-I is altered by RIPLET (Aim 3), and whether RIPLET can be utilized to identify ligands for RIG-I (Aim 4). We here propose a combination of biochemistry, structural biology and cell biology to answer these questions, which we believe are the key to resolving the next layers of complexity in the RIG-I signaling pathway. The four aims will be pursued independently, but are highly synergistic. These four aims build upon our strong preliminary data, an established network of collaboration and biochemical and functional assays that our lab has developed over the last several years. !
Viral infection poses a major challenge to global health as demonstrated in the recent pandemics of H1N1 influenza virus, SARS and HIV. The goal of the current proposal is to understand the molecular mechanisms of viral recognition and signaling by RIG-I and RIPLET, which together constitute one of the principle innate immune pathways. These signaling pathways have been also implicated in pathogenesis of several autoimmune and inflammatory diseases, and were proposed to be a potential therapeutic target in anticancer immunotherapies.
|Ahmad, Sadeem; Mu, Xin; Yang, Fei et al. (2018) Breaching Self-Tolerance to Alu Duplex RNA Underlies MDA5-Mediated Inflammation. Cell 172:797-810.e13|
|Mu, Xin; Greenwald, Emily; Ahmad, Sadeem et al. (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res 46:5239-5249|
|Ruaud, Lyse; Rice, Gillian I; Cabrol, Christelle et al. (2018) Autosomal-dominant early-onset spastic paraparesis with brain calcification due to IFIH1 gain-of-function. Hum Mutat 39:1076-1080|
|de Carvalho, Luciana Martins; Ngoumou, Gonza; Park, Ji Woo et al. (2017) Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy. Arthritis Rheumatol 69:2081-2091|
|Cadena, Cristhian; Hur, Sun (2017) Antiviral Immunity and Circular RNA: No End in Sight. Mol Cell 67:163-164|
|Wu, Bin; Huoh, Yu-San; Hur, Sun (2016) Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors. Methods Mol Biol 1390:131-42|
|Sohn, Jungsan; Hur, Sun (2016) Filament assemblies in foreign nucleic acid sensors. Curr Opin Struct Biol 37:134-44|
|Mu, X; Ahmad, S; Hur, S (2016) Endogenous Retroelements and the Host Innate Immune Sensors. Adv Immunol 132:47-69|
|Ahmad, Sadeem; Hur, Sun (2015) Helicases in Antiviral Immunity: Dual Properties as Sensors and Effectors. Trends Biochem Sci 40:576-585|
|Yao, Hui; Dittmann, Meike; Peisley, Alys et al. (2015) ATP-dependent effector-like functions of RIG-I-like receptors. Mol Cell 58:541-548|
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