Aug 2026· Fish and Shellfish Immunology· pp.
111680
· 0 citations· 32 references
Medicine
TL;DR
SiTRAF3 is identified as a pivotal scaffold protein in the echinoid innate immune system, as it significantly drove the promoter activities of nuclear factor kappa-B, activator protein-1, signal transducer and activator of transcription 3, along with multiple inflammatory cytokines and interferon-related elements.
Abstract
Tumor necrosis factor receptor-associated factor 3 (TRAF3) serves as a versatile protein in innate immune signaling cascades, playing a pivotal role in modulating inflammatory responses, cellular homeostasis, and anti-infection immunity. Here, we cloned and identified a novel TRAF3 homolog (SiTRAF3) from the sea urchin Strongylocentrotus intermedius. Sequence analysis revealed that the open reading frame (ORF) of SiTRAF3 is 1914 bp in length, encoding a polypeptide of 637 amino acids. Structural prediction indicated the presence of a typical N-terminal RING finger domain, zinc finger motifs, and a highly conserved C-terminal MATH domain. Phylogenetic analysis demonstrated that SiTRAF3 clustered closely with the TRAF3-like protein from the sea urchin Strongylocentrotus purpuratus, underscoring the evolutionary conservation of TRAF3 among echinoderms. Quantitative reverse transcription PCR (qRT-PCR) assays revealed ubiquitous expression of SiTRAF3 transcripts across various tissues of S. intermedius, with the highest levels detected in the gill and coelomocytes. Following immune challenges with lipopolysaccharide (LPS), poly(I:C), and peptidoglycan, SiTRAF3 mRNA expression was significantly upregulated, suggesting an active, broad-spectrum response to diverse pathogenic stimuli. In addition, RNA interference of SiTRAF3 suppresses the expression of IL-17 family members and strongylocins following LPS stimulation. Co-immunoprecipitation (Co-IP) assays further verified a direct physical interaction between SiTRAF3 and the core adaptor protein SiMyD88. Functional analyses in HEK293T cells demonstrated that ectopic expression of SiTRAF3 notably elevated the phosphorylation levels of JNK, Erk1/2, and p38 MAPKs, and synergistically enhanced LPS-induced signaling activation. Furthermore, dual-luciferase reporter (DLR) assays revealed the broad-spectrum transcriptional activation capacity of SiTRAF3, as it significantly drove the promoter activities of nuclear factor kappa-B, activator protein-1, signal transducer and activator of transcription 3, along with multiple inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6) and interferon-related elements (interferon α/β/γ, interferon-stimulated response element). Taken together, these findings identify SiTRAF3 as a pivotal scaffold protein in the echinoid innate immune system. By assembling a signaling complex with SiMyD88, SiTRAF3 mediates the activation of MAPK cascades and multiple downstream immune transcriptional pathways. This study not only elucidates the functional significance of SiTRAF3 within the invertebrate immune regulatory network but also provides a theoretical foundation for the molecular breeding of disease-resistant sea urchin strains.
This study provides a new perspective for understanding the evolutionary conservation and species specificity of molluscan innate immune signaling pathways and offers important theoretical support for further comprehension of the molecular mechanism of scallop immune defense and promoting the green control of aquatic diseases in marine farming.
Xiaoxue Lin, Zihao Zhang, Xiaolong Chu et al.· Fish and Shellfish Immunolog...· 0 citations
Toll-like receptor 2 (TLR2) is a pivotal pattern-recognition receptor in innate immunity. It is well characterized in vertebrates but remains poorly understood in mollusks. In this study, we identified a TLR2 homolog (RpTLR2) in Ruditapes philippinarum encoding a 575-amino acid protein with a canonical Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis placed RpTLR2 within the molluscan TLR2 clade, confirming its evolutionary conservation. RpTLR2 was constitutively expressed in all examined tissues, with the highest transcript levels in the gill and mantle. After Vibrio parahaemolyticus challenge, RpTLR2 mRNA and protein levels were significantly upregulated. Functional assays showed that RpTLR2 overexpression intensified inflammatory responses and increased the expression of key inflammation-related genes, whereas RNA interference-mediated RpTLR2 knockdown markedly reduced V. parahaemolyticus-induced inflammation in gill cells. Overall, our findings indicate that RpTLR2 acts as a positive regulator of V. parahaemolyticus-induced inflammatory responses. These results improve understanding of TLR2-mediated innate immunity in mollusks and provide a molecular basis for studying host-pathogen interactions in bivalves.
Jiaxi Liang, Tian Yu, Haodong Feng et al.· Fish and Shellfish Immunolog...· 0 citations
Toll-like receptor (TLR) family proteins act as pattern recognition receptors with pivotal roles in immune defense; however, the function of TLRs in marine mollusks requires further investigation. In this study, we identified a novel TLR gene (CfTLR-Like-1 [CfTLR-L1]) in Chlamys farreri and analyzed its immune functions. CfTLR-L1 has a coding sequence of 1,785 bp and encodes 594 amino acids. It includes typical protein domains; the amino acid sequences of the protein domains are conserved. Phylogenetic analyses demonstrated a close evolutionary relationship among proteins in the mollusk TLR subfamily. CfTLR-L1 is predominantly located on the cell membrane. CfTLR-L1 mRNA is expressed in all tissues, particularly in the hepatopancreas and mantle. Pathogenic stressors, including lipopolysaccharide, peptidoglycan, polyinosinic-polycytidylic acid, Vibrio anguillarum, Vibrio alginolyticus, Vibrio splendidus, and acute viral necrosis virus, significantly increase the levels of CfTLR-L1 mRNA transcripts. The Toll/interleukin 1 receptor (TIR) domain of CfTLR-L1 interacts with the MyD88 family proteins of scallops (including CfMyD88-1, CfMyD88-3, and CfMyD88-4), implying that multiple unique TLR signal transduction mechanisms exist in marine mollusks. CfTLR-L1-TIR interacts with CfMyD88-1-P3, which contains only the TIR structural domain. Overexpression of CfTLR-L1-TIR in HEK293T cells activated reporter genes, including IFNβ, IFNγ, and STAT3. Similarly, overexpression of CfTLR-L1 significantly affected mitogen-activated protein kinase phosphorylation, specifically of p38 and c-Jun N-terminal kinase. Our findings expand the knowledge of the role of TLR in mollusk immunity and may provide evidence for selecting disease-resistant scallops for cultivation.
Jilv Ma, Fengchen Liu, Zihao Zhang et al.· Developmental and Comparativ...· 0 citations
Findings suggest that BmRNF181 acts as a negative regulator of the silkworm immune response and dramatically increased the expression of antimicrobial peptide genes.
Jiang Liu, Ying Peng, Ming Yu et al.· Journal of Invertebrate Path...· 0 citations
Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a vital factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system. The structure and function of IRAK1BP1 have been well investigated in mammals, yet the piscine IRAK1BP1 is poorly understood. In the present study, the first teleost fish IRAK1BP1 from blunt snout bream, maIRAK1BP1, was identified and characterized, which was composed of a 750 bp open reading frame encoding a protein of 249 amino acids. Phylogenetic analysis indicated that maIRAK1BP1, is a novel member of IRAK1BP1 family and showed the highest similarity to that of grass carp (96.28%). Amino acid sequence analysis revealed that maIRAK1BP1 shared a conserved SIMPL domain. Spatial expression pattern analysis revealed that maIRAK1BP1 was constitutively expressed in various normal tissues including heart, spleen, head kidneys, gill, kidney, muscle, intestine and liver. The maIRAK1BP1 expression in immune-relevant tissues (liver, head kidneys and kidney) and caudal fin cells (MAF) were significantly up-regulated after LPS and bacteria injection. The result from subcellular localization analysis indicated that maIRAK1BP1 protein was localized in both the cytoplasm and nucleus of MAF cells, but predominantly existed in cytoplasm. The activity of NF-κB promoter could be inhibited by overexpression of maIRAK1BP1 in HEK-293T cells. Overexpression of maIRAK1BP1 in MAF cells inhibited dramatically the transcriptional expression level of lipopolysaccharide-induced proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). In contrast, an increased expression of these cytokines was observed when maIRAK1BP1 was knocked down via siRNA. Taken together, these results indicated that maIRAK1BP1 played an important role in mediating host innate immune response to pathogen invasion, and disclosed the involvement of maIRAK1BP1 in NF-κB-mediated production of proinflammatory cytokines, which provided foundational insights into the conserved role of IRAK1BP1 in teleost immune regulation.
Yuandong Sun, Chunfang Li, Hongyang Sun et al.· Fish and Shellfish Immunolog...· 0 citations
Toll-like receptors (TLRs) are important pattern recognition receptors involved in crustacean innate immunity. In this study, the full-length cDNA of
HtTLR2
was cloned from the mudflat crab
Helice tientsinensis
using rapid amplification of cDNA ends (RACE). The
HtTLR2
cDNA was 3854 bp in length and contained a 3036-bp open reading frame (ORF) encoding a 1011-amino-acid protein. Bioinformatics analysis showed that the HtTLR2 protein possessed conserved structural features typical of TLR family members. Phylogenetic analysis indicated that HtTLR2 was closely related to Toll2 from
Eriocheir sinensis
. Tissue distribution analysis revealed high
HtTLR2
expression in the gills and hepatopancreas and its expression was significantly upregulated in both tissues following infection with
Vibrio parahaemolyticus
(Fujino et al., 1951) Sakazaki et al., 1963. This study provides new insights into the
TLR
gene family of
H. tientsinensis
and lays a foundation for further investigation of the immune mechanisms of this species.
Lulu Chen, Youkun Fang, Yang Ge et al.· Crustaceana· 0 citations