NLRC3-like 1 inhibits NOD1-RIPK2 pathway via targeting RIPK2
Introduction
The nucleotide oligomerization domain (NOD)-like receptor (NLR) family proteins are intracellular pattern recognition receptors (PRRs) that have many functions including pattern recognition, antigen presentation, autophagy, immune modulation and embryonic development (Kufer and Sansonetti, 2011; Meunier and Broz, 2017). The most commonly studied subfamily of NLRs is NOD or NLRC subfamily, which includes NOD1 (NLRC1), NOD2 (NLRC2), NLRC3 (NOD3), NLRC4 (IPAF), NLRC5 (NOD27) and NLRX1 (Meunier and Broz, 2017). Among them, NOD1 and NOD2 are the best-characterized members as positive regulatory NLRs, and have been shown to form a multiprotein complex to modulate IFN and NF-κB signaling following bacterial or viral infection (Hasegawa et al., 2008; Coutermarsh-Ott et al., 2016).
In contrast to NOD1 and NOD2, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). NLRC3 consisted of an N-terminal caspase activation and recruitment domain (CARD), a central nucleotide-binding domain (NBD or NACHT) and a C-terminal leucine-rich repeat (LRR) domain (Conti et al., 2005; Schneider et al., 2012). In mammals, NLRC3 was originally identified as a suppressor of T cell activation (Conti et al., 2005). Subsequent studies revealed that NLRC3 inhibited TLR signaling through the interaction with TRAF6 to regulate NF-κB (Schneider et al., 2012), and reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses (Zhang et al., 2014). Among well-known inflammasome components, NLRC3 interacted with ASC, caspases 1 and 5, which negatively regulated NLRP3-mediated inflammatory responses (Gültekin et al., 2015; Eren et al., 2017). Furthermore, NLRC3 negatively regulated the PI3K–mTOR axis through the interaction of NLRC3 with PI3K subunits (Karki et al., 2016, 2017).
In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing (Hu et al., 2017; Wu et al., 2018). A few studies examined the function of piscine NLRC3 or NLRC3-like genes. In Japanese flounder (Paralichthys olivaceus), JfNLRC (containing NACHT and LRRs domains) and poNLRC3 (containing NACHT, FISNA, LRRs and PRY/SPRY domains) positively regulated the expression of proinflammatory cytokines (Unajak et al., 2011; Li et al., 2016a). Zebrafish NLRC3-like gene, which contains the canonical pyrin (PYD) and NACHT domains but lacks the common LRRs, can bind the inflammasome component ASC to regulate the inflammasome and is required for microglia development (Shiau et al., 2013). In goldfish, NLRC3L consisting of FISNA, NACHT, PRY, SPRY and LRRs domains interacted with ASC and cooperated with RIPK2 to regulate NF-κB activity (Xie and Belosevic, 2018). Since the functional diversities exist for NLRC3 or NLRC3-like genes, there is a need to reveal the functions and molecular mechanisms for the expanding analogues of piscine NLRC3-like genes.
In our previous studies, transcriptomic analysis and qRT-PCR results revealed that multiple NLRC3-like genes were regulated by NOD1 and RIPK2 deficiency (Hu et al., 2017; Wu et al., 2018). The present study focused on investigating the function of a zebrafish NLRC3-like gene consisting of FISNA, NACHT and LRRs domains (named as NLRC3-like 1) in bacterial infection. Our data suggest that zebrafish NLRC3-like 1 plays a negative role in bacterial infection and inhibits NOD1-RIPK2 antibacterial pathway via targeting RIPK2.
Section snippets
Maintenance of zebrafish
Wild-type AB/TU adult zebrafish were raised and maintained at 28 °C in the system water. Zebrafish embryos were obtained by artificial insemination and reared in the petri dishes at 28 °C.
Plasmid construction and sequence analysis
Based on zebrafish sequence derived by automated computational analysis using gene prediction method (GenBank accession No: XM_003200085), the forward and reverse primers NLRC3-like 1F/NLRC3-like 1R (Table 1) were used for cloning the open reading frame (ORF) and inserted into p3XFLAG-CMV™-14 Expression Vector
Features of zebrafish NLRC3-like 1
In our previous study, transcriptomic analysis revealed that multiple NLRC3-like (namely NOD3-like) genes were found in zebrafish database (Hu et al., 2017). Among them, a EST sequence (gene26713) was consistent with the Danio rerio si:ch211-181d7.3 sequence (GenBank accession number XM_003200085), which was cloned by us. Phylogenetic analysis confirmed that this gene was the analogue of piscine NLRC3 (Fig. 1A), and named as NLRC3-like 1. The coding region of zebrafish NLRC3-like 1 consists of
Discussion
Both NLRC3 and NOD1 belong to NLRC subfamily according to their phylogenetic relationships or regulatory NLR subfamily based on their best-characterized function (Schroder and Tschopp, 2010; Coutermarsh-Ott et al., 2016). Here we identified and characterized a novel NLRC3 gene (termed as NLRC3-like 1) from the zebrafish, and investigated the expression and function of NLRC3-like 1 in response to E. piscicida infection in vivo and in vitro. The correlation between zebrafish NLRC3-like 1 and NOD1
Acknowledgements
This work was supported by National Natural Science Foundation of China (31672687 and 31873046), and Fund for State Key Laboratory of Freshwater Ecology and Biotechnology (2019FBZ04).
References (52)
- et al.
Insights into the diversity of NOD-like receptors: identification and expression analysis of NLRC3, NLRC5 and NLRX1 in rainbow trout
Mol. Immunol.
(2017) - et al.
Two novel proteins activate superoxide generation by the NADPH oxidase NOX1
J. Biol. Chem.
(2003) - et al.
The discrepancy function of NLRC5 isoforms in antiviral and antibacterial immune responses
Dev. Comp. Immunol.
(2018) - et al.
The negative regulation of piscine CD44c in viral and bacterial infection
Dev. Comp. Immunol.
(2019) - et al.
Structure and expression pattern of teleost caspase recruitment domain (CARD) containing proteins that are potentially involved in NF-kappaB signalling
Dev. Comp. Immunol.
(2010) - et al.
Nox3 regulation by NOXO1, p47phox, and p67phox
J. Biol. Chem.
(2004) - et al.
CATERPILLER 16.2 (CLR16.2), a novel NBD/LRR family member that negatively regulates T cell function
J. Biol. Chem.
(2005) - et al.
NLRC3 protein inhibits inflammation by disrupting NALP3 inflammasome assembly via competition with the adaptor protein ASC for pro-caspase-1 binding
J. Biol. Chem.
(2017) - et al.
Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus)
Fish Shellfish Immunol.
(2018) - et al.
Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge
Fish Shellfish Immunol.
(2017)
An induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways
J. Biol. Chem.
Noxa1 as a moderate activator of Nox2-based NADPH oxidase
Arch. Biochem. Biophys.
A genome-wide survey of expansive NLR-C subfamily in miiuy croaker and characterization of the NLR-B30.2 genes
Dev. Comp. Immunol.
Characterization of NLR-A subfamily members in miiuy croaker and comparative genomics revealed NLRX1 underwent duplication and lose in actinopterygii
Fish Shellfish Immunol.
Expression profiles of NODs in channel catfish (Ictalurus punctatus) after infection with Edwardsiella tarda, Aeromonas hydrophila, Streptococcus iniae and channel catfish hemorrhage reovirus
Fish Shellfish Immunol.
Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages
Fish Shellfish Immunol.
Evolutionary convergence and divergence in NLR function and structure
Trends Immunol.
Identification of Nod like receptor C3 (NLRC3) in Asian seabass, Lates calcarifer: characterisation, ontogeny and expression analysis after experimental infection and ligand stimulation
Fish Shellfish Immunol.
Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors
Dev. Comp. Immunol.
The inflammasomes
Cell
An anti-inflammatory NOD-like receptor is required for microglia development
Cell Rep.
Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes
Fish Shellfish Immunol.
Molecular characterization, expression and functional analysis of a nuclear oligomerization domain proteins subfamily C (NLRC) in Japanese flounder (Paralichthys olivaceus)
Fish Shellfish Immunol.
Neutrophil plays critical role during Edwardsiella piscicida immersion infection in zebrafish larvae
Fish Shellfish Immunol.
Characterization and functional assessment of the NLRC3-like molecule of the goldfish (Carassius auratus L.)
Dev. Comp. Immunol.
NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING
Immunity
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