Elsevier

Fish & Shellfish Immunology

Volume 88, May 2019, Pages 627-635
Fish & Shellfish Immunology

Full length article
Characterisation and function of TRIM23 in grass carp (Ctenopharyngodon idella)

https://doi.org/10.1016/j.fsi.2019.03.025Get rights and content

Highlights

  • The TRIM23 gene in grass carp (Ctenopharyngodon idella) was identified and analysed.

  • Expression and transcriptional regulation of TRIM23 gene were investigated.

  • TRIM23 involved in the regulation of the promoter activity of IRF3 and IRF7.

  • The TRIM23 protein could colocalise and interact with TRAF6 and MyD88 in CIK cells.

  • The TRIM23 protein could induce autophagy.

Abstract

Tripartite motif (TRIM) proteins are key components of the innate immune system, functioning as antiviral restriction factors or modulating signaling cascades that lead to proinflammatory cytokine induction. In the present study, the TRIM family gene TRIM23 from grass carp (Ctenopharyngodon idella) was cloned and characterised. TRIM23 was moderately expressed in the examined tissues, and the significantly altered expression was observed after grass carp reovirus (GCRV) and poly(I:C) infection. Dual-luciferase activity assay showed that TRIM23, especially its C-terminal domain ARF, depressed the promoter activity of IRF3 and IRF7. The subcellular localisation showed that TRIM23 protein was located in the cytoplasm and could be recruited by both TRAF6 and MyD88. Furthermore, TRIM23 was confirmed to interact with either TRAF6 or MyD88 by the bimolecular fluorescence complementation (BiFC) system in CIK cells. Additionally, autophagy was enhanced by over-expressed TRIM23 in 293T cells. Taken together, our results demonstrate that TRIM23 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the TRIM23 in teleosts.

Introduction

Innate immune responses are activated by the engagement of pattern recognition receptors (PRRs) recognizing the invaded pathogen [[1], [2], [3]]. Subsequently, various immune responses were initiated, including the production of cytokines and the initiation of pro-inflammatory and adaptive immune responses [4]. The tripartite motif-containing (TRIM) superfamily (also known as RBBC superfamily) contains about 100 members in humans and most of them have emerged as key components of the innate immune system, functioning as antiviral restriction factors or involving in a broad range of biological processes that are associated with innate immunity [[5], [6], [7], [8], [9]].

TRIM proteins belong to the larger family of RING E3 ligases and most of the numbers compose a Really Interesting New Gene (RING) zinc finger domain, one or two B-box domains and an associated coiled-coil domain in the amino-terminal region [5]. The RING domain is a specialised type of zinc finger that is located in the N-terminal portion of almost all TRIM proteins and confers E3 ligase activity by binding to an ubiquitin-loaded E2 enzyme and promoting the transfer of ubiquitin to a target protein [5,7,10,and11]. Recently, many TRIM family members have been confirmed to mediate ubiquitylation events depending on the RING domain. The RING-finger-type E3 ubiquitin ligase of TRIM5α could ubiquitinate itself in cooperation with the E2 ubiquitin-conjugating enzyme UbcH5B [12]. Activator-recruited cofactor 105-kDa component (ARC105)-mediated transcriptional activation induced by transforming growth factor β (TGFβ) signaling is regulated by TRIM11 through an ubiquitin-mediated degradation pathway [13]. The RING domain of TRIM21 facilitates and enhances the polyubiquitination chains of mitochondrial antiviral-signaling protein (MAVS) [14,15]. The catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity [16]. TRIM25 involves in not only for RIG-I ubiquitination but also for RIG-I-mediated interferon-β production and antiviral activity in response to RNA virus infection [17]. The B-box domains flanked RING domain have different consensus sequences between members of the TRIM superfamily [4]. Interestingly, some evidences have showed that B-box domains may contribute to innate resistance to HIV. Studies on HIV showed that B-box 2 of TRIM5α influences the recognition of the viral capsid by the carboxy-terminal PRYSPRY domain and the ability of TRIM15 to mediate HIV restriction [18,19]. B-Box C-terminal domain (BBC) is always a coiled-coil domain which mediates homomeric and heteromeric interactions among TRIM family members and other proteins, in particular self-association [4]. The C terminus of TRIM family members contain various domains among different species. The contribution of these domains to the regulation of innate immune responses has yet to be well established.

The number of TRIMs varies greatly in different species. More than 100 members encoded by the humans genome excluding pseudogenes, 208 trim genes in the zebrafish (Danio rerio) and 66 in pufferfish (Tetraodon nigroviridis) were identified by whole genome sequencing [9], [20], [21], [22], thus demonstrating their ancient separation from each species [23]. Recent studies showed that a large family numbers of TRIMs play a significant role in fish immunity [9]. TRIM14, 51, 67, 72, 82, 83 and 99 in grass carp were identified by bioinformatics analysis [23], however, little TRIMs functional information of regulation in innate immune was reported. In the study, the TRIM family gene TRIM23 in grass carp (Ctenopharyngodon idella) was cloned and characterised. Expression profiles in different tissues and in response to GCRV and poly(I:C) infection were examined. In C. idella kidney (CIK) cells, the regulation of promoter activity of IRF3 and IRF7 by TRIM23 was analysed. Additionally, the subcellular localisation of TRIM23 protein and the interaction with TRAF6 and MyD88 were visualized. Furthermore, the enhanced autophagy in 293T cells transfected with pCMV-HA-TRIM23 was explored. These findings will provide new insights for understanding the functions of TRIM23 gene in teleosts.

Section snippets

Experimental fish and cells

Six-month-old grass carps (weight, 40 ± 10 g; length, 15 ± 3 cm) used in the study were provided by the GuanQiao Experimental Station, Institute of Hydrobiology, Chinese Academic of Sciences, and acclimatized in aerated freshwater at 28 °C. The fish were fed twice a day with a commercial feed (Tong Wei, China). Grass carps were acclimatized for 1 week and used for the following experiments after no abnormal symptoms were observed. The CIK cells (China Center for Type Culture Collection, China)

Molecular characterisation and phylogenetic analysis of TRIM23

The full length cDNA of TRIM23 (Genbank accession number: MH892482) is 2390 bp long, with a 1785 bp ORF encoding a predicted polypeptide of 594 amino acids, 72 bp 5′ UTR, and 533 bp 3’ UTR (Fig. S1). Structure analysis revealed that TRIM23 consisted of two conserved BBOX (aa 142–188, aa 193–239) flanked by an N-terminal conserved Ring finger domain (RING, aa 51–95), and a B-Box C-terminal domain (BBC, aa 246–390) in the middle, followed by a C-terminal domain of ADP-ribosylation factor (ARF)

Discussion

TRIM family proteins are critical E3 ligase family in many cellular functions, including the regulation and coordination of innate immunity and antiviral responses [[30], [31], [32], [33], [34]]. In recent years, a great number of studies have been focused on their regulatory role in innate immunity and the pathogenesis of many diseases [35]. In the study, dramatic changes of TRIM23 expression levels after GCRV infection predicted that TRIM23 may be involved in grass carp hemorrhagic disease (

Acknowledgments

This work was funded by the National Natural Science Foundation of China [grant number 31572614], the Strategic Pilot Science and Technology Projects (A) Category, Chinese Anatomical Society [grant number XDA08030203], the State Key Laboratory of Freshwater Ecology and Biotechnology [grant number 2016FBZ02], the National Natural Science Foundation of China [grant number 31702332], and the Hubei Natural Science Foundation of China [grant number 2017CFB374].

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