Rig I Like Receptors Proteins


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 Rig I Like Receptors Proteins Background

RIG-I like receptors (RLR) are cytoplasmic pathogen recognition receptors that detect pathogen-associated molecular patterns (PAMP) within viral RNA. There are three main members of the RLR family, RIG-I (retinoic acid inducible gene-I), MDA5 (melanoma differentiation associated gene 5) and LGP2 (Laboratory of genetics and physiology 2). The RLRs are widely expressed in multiple cell types and in many tissues to assist with pathogen recognition. The expression of the RLRs remains at low levels in the steady-state and is greatly increased upon IFN exposure or in response to virus infection. RIG-I is the prototypical RLR that is essential for recognition of many RNA viruses through the binding of viral RNA which initiates innate immune responses and antiviral programs that control RNA virus infections. RIG-I signaling results in the activation of transcription factors; IRF3/7, AP1 and NF-κB, production of type I interferons (type-I IFN), secretion of proinflammatory cytokines and drives the expression of innate immune genes. Immune responses induced by type-I IFNs are crucial in triggering innate antiviral responses through Janus kinases and signal transducer and activator of transcription protein (JAK-STAT) signaling pathway. Type-I IFNs, when secreted from the virus-infected cell, bind to IFN-α/β receptors (IFNAR) to induce the expression of hundreds of interferon-stimulated genes (ISG) in autocrine and paracrine fashion to establish an antiviral state that limits infection.

Fig. 1 RIG-I like receptor signaling and the interferon signaling cascade.

 

Structure of the RLRs

Rig-I mediated antiviral immunity starts with the sensing of non-self RNA by the RLRs. The RLRs RIG-I, MDA5 and LGP2 share a DExD/H box helicase domain that constitutes the proteins’ central core, a C-terminal domain (CTD) that is essential for PAMP recognition and in the case of RIG-I, for autoregulation and two tandem amino-terminal caspase activation and recruitment domains (CARDs) that are essential for signaling in the case of RIG-I and MDA5. The RLRs work through a central adapter mitochondrial antiviral signaling protein (MAVS) which is present on the mitochondrial-associated membranes (MAM). The MAM has emerged as a critical platform that facilitates innate immune signaling functions. The CARD’s of RIG-I and MDA5 are involved in interaction with the single CARD domain on MAVS adapter molecule to trigger signaling.

In an uninfected cell, the repressor domain (RD) on RIG-I, that is part of the CTD, binds the N-terminal CARDs and represses signaling. Upon sensing of non-self viral RNA by the RD followed by the helicase domain, ATP is hydrolyzed and RIG-I undergoes a conformational change and occupies an active conformation that releases the CARD domains for signaling to MAVS. Unlike RIG-I, MDA5 is less stringently regulated as ectopic over expression of MDA5 is sufficient to drive signaling even when the RNA ligand is absent. LGP2 lacks the N terminal CARD domains and is thought to have a role in regulating RIG-I and MDA5 signaling. The RLR activation, translocation and homotypic interactions between the RLRs CARDs and MAVS involve the dynamic recruitment of several co-factors that regulate the formation of a large signaling complex also called the “signalosome”.

 

Regulation of the RLR signaling pathway

Self versus non-self RNA recognition is a tightly regulated process that determines the establishment of innate immunity against virus infection. RIG-I is regulated by its expression level and its conformational state in uninfected cells. In the basal state, RIG-I is auto-repressed through the C-terminal repressor domain (RD), which binds and conceals the CARD domains. RIG-I is signaling “inactive” in the steady-state and requires the binding of non-self viral RNA to be activated. Viral RNA/PAMP binding to the RD and helicase domains triggers ATP hydrolysis followed by conformational change that releases the CARDs for signaling.

The RLR components are post-translationally modified and these modifications are dynamically controlled and altered based on the infection status. RIG-I transition from the “inactive” to “active” state requires recruitment of several co-factors as well as dynamic changes to the post-translational modifications (PTM) present on RIG-I. In the basal state, RIG-I is constitutively phosphorylated in its CARDs and C-terminal domain (CTD) and is rapidly dephosphorylated upon virus infection. The phosphorylation of Thr 770 and Ser 854/855 in the RIG-I CTD by casein kinase II and phosphorylation of Ser 8/Thr 170 in the CARD domains of RIG-I by protein kinase C-α/β maintains RIG-I in an autorepressed basal state. Dephosphorylation of the Ser 8/Thr 170 of RIG-I and Ser 88 of MDA5 by protein phosphatase 1 α/γ is essential for RLR activation.

RIG-I activation occurs in multiple steps and starts with viral PAMP binding, followed by dephosphorylation of RIG-I, which promotes binding of E3 ubiquitin ligase tripartite motif-containing protein 25 (TRIM25) to CARD1 of RIG-I, followed by K63- linked ubiquitination of Lys 172 of CARD 2 as well as riplet (a ring finger protein) dependent K63-linked ubiquitination of Lys 788 of CTD of RIG-I. The K63 polyubiquitination of RIG-I allows for its multimerization and activation. Activated RIG-I in combination with TRIM25 associates with 14-3-3ε, a mitochondrial targeting chaperone translocates from the cytoplasm and redistributes to the mitochondria associated membranes (MAM) to interact with MAVS. This interaction acts as a platform for recruitment of several signaling molecules and cofactors and forms the signalosome complex that leads to amplification of signal as well as transcription factor activation (IRF3/7 and NFκB).

The ubiquitination and deubiquitination status of RIG-I and the other signaling molecules downstream of RIG-I further regulates the pathway. RIG-I is regulated by the deubiquitinating (DUB) enzymes; tumor suppressor protein cylindromatosis (CYLD), USP3 and USP2. These DUBs negatively regulate RIG-I by removing K63-linked ubiquitin chains, thus dampening RIG-I activity. RIG-I is also regulated by K48- linked ubiquitination through the action of Ring-finger protein125 (RNF125). RNF125 induces K48-linked ubiquitination of RIG-I, MDA5 and MAVS to promote proteasomal degradation triggering a negative feedback loop that prevents excessive cytokine production. The ubiquitin-editing protein A20 and linear ubiquitin assembly complex (LUBAC) composed of two E3 ligases, HOIL-1L and HOIP, negatively regulate RIG-I signaling whereas USP4 and USP15 positively regulate the pathway by removing Lys48-linked ubiquitin chains.

The extensive post-translational modifications that control and regulate the activation of the RLR serve as a check point to prevent aberrant signaling and allow for rapid modulation of the steady-state in case of infection.