Mex3b, a Coreceptor to Present Dsrna to TLR3

Mex3B: a coreceptor to present dsRNA to TLR3 Cell Research (2016) 26:391-392. doi:10.1038/cr.2016.29; published online 4 March 2016

Toll-Like Receptors (TLRs) play independent pathway associated with experiments in 293-TLR3 stable cells, critical roles in the early innate im- the stimulation of interferons. TLR3 uti- they showed that Mex3B was involved mune response to invading pathogens lizes TRIF to mediate its signaling [3]. in poly (I:C)-induced, TLR3-mediated by sensing microorganisms; a num- A number of accessory molecules signaling, but not SeV-induced signal- ber of accessory molecules have been have been shown to assist microbial ing. To further confirm their observa- shown to assist microbial recognition recognition by TLRs. For example, tion, they generated Mex3B-deficient by TLRs. In a recent paper in Cell LPS is extracted from the bacterial mice. Consistently, poly(I:C)-induced Research, Yang et al. demonstrate that membrane by the LPS-binding protein IRF3 and IκBα phosphorylation and Mex3B is associated with TLR3 in (LBP), after which it is transferred to IFN-β production were impaired in the endosomes and promotes dsRNA CD14. Subsequent transfer from CD14 Mex3B-/- MEFs, BMDMs or BMDCs, binding and proteolytic processing to an additional accessory molecule while SeV-, VSV- and EMCV-induced of TLR3, suggesting that Mex3B acts MD-2 then allows TLR4-mediated LPS signalings were not affected. In vivo, as a coreceptor of TLR3 in response recognition [4, 5]. HMGB1 can also they also observed less IFN-β and to dsRNA. mediate LPS transfer to CD14 to initi- TNFα production in the serum and less Toll-Like Receptors (TLRs), homo- ate a TLR4-mediated pro-inflammatory mortality in Mex3B-/- mice challenged logues of Drosophila Toll protein, are response [6]. For TLR3, the structure of with poly(I:C). These data suggest that evolutionarily conserved receptors dis- the extracellular domain of TLR3 was Mex3B is specifically required and covered to be important for host defense revealed by crystallography studies as important for TLR3-mediated but not against microbial infection [1]. TLRs a large horseshoe-shape [7]. It has been RLR-mediated signaling. recognize highly conserved structural shown that CD14, scavenger receptor The authors then investigated how motifs known as pathogen-associated class-A and clathrin-mediated endo- Mex3B facilitates TLR3 signaling. They molecular patterns (PAMPs), which cytic pathways participate in cellular constructed a series of truncation and are exclusively expressed by microbial uptake of extracellular dsRNA [8-10]. point mutants of Mex3B and TLR3 to pathogens, such as lipopolysaccharide However, how endocytosed dsRNA determine the binding and functional (LPS), peptidoglycan (PGN) and lipo- is presented to TLR3 in intracellular domains. Through co-IP experiments peptides from bacterial cell wall, as well compartments is still poorly understood. and IFN-β promoter reporter assay with as flagellin, bacterial DNA and double- In a recent paper in Cell Research these truncations, the authors found stranded RNA (dsRNA) from viruses. [11], the authors found that an RNA- that two RNA-binding KH domains TLRs also recognize danger-associated binding protein Mex3B is involved in in Mex3B and proteolytic cleavage of molecular patterns (DAMPs) that are this process. Mex3B is associated with TLR3 are indispensable for Mex3B to endogenous molecules released from TLR3 in the endosomes and promotes potentiate TLR3-mediated signaling. necrotic cells [1]. TLR3 is involved in the dsRNA binding and proteolytic pro- The authors also demonstrated that recognizing virus-derived dsRNA [2]. cessing of TLR3 through its own RNA- Mex3B and TLR3-mediated poly (I:C)- Stimulation of TLRs by the correspond- binding ability, and consequently helps induced signaling requires endosomal ing PAMPs or DAMPs initiates signal- TLR3 activate downstream signals. acidification. By immunofluorescence ing cascades leading to the activation Initially, from a cDNA array contain- confocal microscopy, they found that of transcription factors, such as nuclear ing 352 expression clones of ubiquitin- Mex3B and TLR3 colocalized with factor-κB (NF-κB) and interferon related proteins, the authors identified the endosome marker EEA1, though regulatory factors (IRFs). Signaling of an RNA-binding protein Mex3B as a overexpressing both Mex3B and TLR3 all TLRs except TLR3 consists of two positive regulator of TLR3-mediated in 293T cells may not be a perfect re- distinct pathways: a MyD88-dependent signaling using IFN-β promoter reporter flection of an endogenous situation with pathway that leads to the production of assay in 293-TLR3 stable cell line. By physiologic protein levels. Proteinase inflammatory cytokines, and a MyD88- in vitro overexpression and knockdown K protection assay also suggests that npg 392 Mex3B and TLR3 are in the lumen of of TLR3 to poly (I:C). These results Xuqiu Lei1, Richard A Flavell1, 2 the endosomes. suggest that the RNA-binding ability of The author further analyzed whether Mex3B is required for the facilitation of 1Department of Immunobiology, Yale University Mex3B physically interacts with TLR3 ligand binding of TLR3 and initiation of School of Medicine, New Haven, CT 06520, USA; 2Howard Hughes Medical Institute, Yale Univer- and the mechanism whereby Mex3B downstream signals. sity, New Haven, CT 06520, USA participates in TLR3 signaling. By Taken together, in this interesting Correspondence: Richard A Flavell endogenous co-IP experiments in cells study, the authors identified Mex3B E-mail: [email protected] treated with poly (I:C), the authors as a coreceptor of TLR3 in mediating found that Mex3B is associated with virus dsRNA-induced innate immune References TLR3, and bound to TRIF in a poly (I:C) response. The proposed model is: by 1 Medzhitov R. Nat Rev Immunol 2001; signaling-dependent manner. Further, to sensing viral infection, TLR3 is trans- 1:135-145. test whether Mex3B affects the cleav- ported from ER to endosomes where it 2 Alexopoulou L, Holt AC, Medzhitov R, et age of TLR3, the authors overexpressed is cleaved and the stable form of cleaved al. Nature 2001; 413:732-738. 3 Akira S, Takeda K. Nat Rev Immunol 2004; or knocked down Mex3B in 293 cells TLR3 represents the sensor to the 4:499-511. transfected with TLR3, and found that dsRNA from virus. The RNA-binding 4 Jiang Z, Georgel P, Du X, et al. Nat Immu- Mex3B promotes TLR3 processing. protein Mex3B promotes ligand binding nol 2005; 6:565-570. Lastly, the authors tested whether the and proteolytic cleavage of TLR3 in 5 Shimazu R, Akashi S, Ogata H, et al. J Exp Med 1999; 189:1777-1782. RNA-binding protein Mex3B modulat- endosomes, resulting in full activation 6 Youn JH, Oh YJ, Kim ES, et al. J Immunol ed the dsRNA-binding ability of TLR3. of TLR3 and subsequently TLR3-medi- 2008; 180:5067-5074. By performing biotin-labeled poly (I:C) ated interferon and cytokine induction. 7 Choe J, Kelker MS, Wilson IA. Science pull-down experiments, they found that This study, together with other reports 2005; 309:581-585. 8 Lee HK, Dunzendorfer S, Soldau K, et al. full-length Mex3B bound to poly (I:C) that described the coreceptors of TLRs, Immunity 2006; 24:153-163. directly and facilitated the binding of highlighted the requirement of a number 9 Itoh K, Watanabe A, Funami K, et al. J Im- TLR3 to poly (I:C), while dominant- of distinct co-receptors and accessory munol 2008; 181:5522-5529. negative form of Mex3B, which lacks molecules for individual ligands. 10 Limmon GV, Arredouani M, McCann KL, et al. FASEB J 2008; 22:159-167. RNA-binding activity, could not bind 11 Yang Y, Wang SY, Huang ZF, et al. Cell Res 1 1 to poly (I:C) and inhibited the binding Shu Zhu , Geng Wang , 2016; 26:288-303.

Cell Research | Vol 26 No 4 | April 2016