AREQUIPA – PERÚ
2. MARCO CONCEPTUAL 1 Síndrome de Burnout
RLRs are in an inactive state and only get activated upon recognition of foreign RNA. In this respect, the RD regions in RIG-I and LGP2 play an important role. RD in RIG-I keeps the molecule inactive until RNA binding triggers a conformation change (Wilkins and Gale, 2010). A region in the CTD of RIG-I overlapping with RD is responsible for the specific recognition of viral RNA (Yoneyama and Fujita, 2009)
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In uninfected cells, RIG-I is in an auto-repressed conformation, with the CTD exposed and scanning the cytoplasm in search of RNA ligands (Weber, 2015). The open conformation of the domains in the helicase region, with the second CARD (2CARD) bound to the Hel2i domain, sterically blocks CARDs interactions with polyubiquitin or CARDs from other binding partners, and thus prevents signalling to the signalling adaptor MAVS (Kowalinski et al., 2011).An adenosine-5'-triphosphate (ATP)-dependent conformational change occurs in RIG-I after RNA binding that releases the CARD domains for signalling (Wilkins and Gale, 2010). Lack of CARDs in LGP2 prevents further signalling upon activation of the molecule. In the past, it was assumed that LGP2 acted as an inhibitor of RIG-I/Mda5 activation, but recent studies have shown that LGP2
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plays a more extensive role in modulating RIG-I/Mda5 activation (Venkataraman et al., 2007). Mda5 CTD does not contain RD and this allows the molecule to signal constitutively when expressed in abundance, reflecting an important role for Mda5 in amplifying IFN production and the host response (Saito et al., 2007).
RIG-I activation in infected cells, when the CTD recognizes a specific RNA ligand, is shown in Fig. 1.4. 3 steps are required: RNA recognition, ATPase activity, and exposure of the CARDs for signalling (Anchisi et al., 2015). First, CTD in RIG-I recognizes and binds 5'ppp dsRNA independently of ATP binding (Patel et al., 2013), and the recognition is helped by zinc coordination in cysteine residues in RD (Cui et al., 2008). RIG-I surrounds dsRNA, adopting a more compact conformation (Jiang et al., 2011), and this permits ATP to bind RIG-I (Luo et al., 2011). Effective RIG-I binding of short RNA ligands is dependent on the presence of triphosphate, and the ATPase activity requires the presence of a blunt-end formation at the triphosphate end (Schlee et al., 2009). An ATP-dependent conformational change occurs, inducing a packed complex formation of the helicase domain: CTD with dsRNA (Kowalinski et al., 2011). This conformational change disrupts the CARD:Hel2i interaction, as the CARDs and dsRNA bind partially to the same helicase region (Anchisi et al., 2015). The CARDs, released from auto-repression, are available for interaction with MAVS (Kowalinski et al., 2011). Although RIG-I binding to RNA is independent of ATPase activity, ATP hydrolysis is essential for RIG-I signalling (Anchisi et al., 2015).
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Fig. 1.4. Schematic of RIG-I activation. (A) RIG-I leaves the auto-repressed conformation (B) upon capture of 5'ppp dsRNA by the CTD. (C) Cooperative ATP and dsRNA binding to the helicase drives a conformational switch that leads to a closed form with high affinity for dsRNA, where CARDs (C1 and C2) are released. ATP hydrolysis could allow cycling between (C) and (D) pending CARD ubiquitination. (D) K63-linked polyubiquitination of the CARDs allows downstream signalling via MAVS. (E) ATP hydrolysis leads to helicase re-opening and recycling of RIG-I as long as the CARDs no longer bind polyubiquitin. Dotted lines represent flexible linkers. Adapted from Kowalinski et al., 2011.
The helicase domain and CTD of Mda5 surrounds dsRNA in the same manner as RIG-I. Contrary to RIG-I, Mda5 CTD interacts with the dsRNA stem allowing both ends of dsRNA to be free from interaction (Wu et al., 2013). Mda5 CTD
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brings the dsRNA closer as compared to the RIG-I structure, allowing contact of CTD with Hel1 and forming a closed ring around the dsRNA. This promotes cooperative filament formation along the dsRNA that is initiated from internal sites in the dsRNA, as opposed to RIG-I where filament formation begins from one of the ends (Peisley et al., 2011). Filament formation along dsRNA is essential for the activation of RIG-I and Mda5, and is dependent on ATP hydrolysis (Patel et al., 2013). Mda5 specificity for long dsRNA is determined by filament assembly and disassembly dynamics (Reikine et al., 2014), and LGP2 could play a role in RNA recognition by Mda5 and filament formation (Bruns et al., 2014). Mda5 ATP-dependent filaments are longer and more stable than RIG-I filaments (Peisley et al., 2013).
RIG-I/Mda5 oligomerization is essential for signalling and is driven by CARD- CARD interactions (Patel et al., 2013; Saito et al., 2007). Large oligomers on RNA possibly induce more robust aggregation of MAVS, thereby leading to a strong activation of IFN (Patel et al., 2013). The minimal signalling unit for Mda5 contains no less than 11 Mda5 molecules, much larger than that required for RIG-I (Peisley et al., 2013). Increased levels of RIG-I during the IFN response may further promote its self-association and potentiate signalling to drive an IFN amplification loop (Saito et al., 2007).
Ubiquitination is a versatile post-translational modification involved in various cellular functions. RIG-I, but not Mda5, requires E3 ubiquitin ligase tripartite motif containing (TRIM) 25-dependent ubiquitination to interact with MAVS (Gack et al., 2007). K63-polyubiquitination of RIG-I by TRIM25 also promotes oligomerization (Gack et al., 2007), facilitating the assembly of RIG-I into a tetrameric 'lock-washer' stabilized by CARD-CARD interactions (Reikine et al., 2014). This provides stabilization of the oligomer, facilitating interaction with
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MAVS and thereby increasing IFN signalling capacity, as shown in Fig. 1.4.D. The requirement for ubiquitination by RIG-I seems not to be completely strict, as ubiquitin-independent signal activation of RIG-I has also been reported (Peisley et al., 2013).