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Caveolin-1 (Cav-1), a major structural component of caveolae, is involved in various biological functions, such as endocytosis, cholesterol trafficking, transcytosis, signal transduction, and immunity. To date, three caveolin members have been identified in mammals: Cav-1, Cav-2, and Cav-3. In this study, we identified the Cav-1 sequence from Amphiprion clarkii (AcCav-1). The protein is 181 amino acids long, with a molecular weight of 20.73 kDa and a predicted isoelectric point of 5.48. The phylogenetic tree disclosed that AcCav-1 is closely related to teleost fish orthologs and clusters together with vertebrates. It shares the highest identity (99.4%) and similarity (100%) with Amphiprion ocellaris. Subcellular localization assays showed that Cav-1 expressed in the endoplasmic reticulum and cytoplasm. Further, AcCav-1 was ubiquitously expressed in all examined tissues, but most highly in the skin and the spleen. The up and downregulation of AcCav-1 was observed throughout the testing period after in-vivo immunostimulation with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (poly (I:C), and Vibrio harveyi (V. harveyi). The antiviral assay showed that AcCav-1 overexpression suppresses the replication of the viral hemorrhagic septicemia virus (VHSV) in Fathead minnow cells by activating antiviral genes. Further, LPS induced NO production and H 2 O 2 -mediated oxidative stress assays showed that AcCav-1 is involved in the regulation of oxidative stress. Collectively, these findings suggest the indispensable role of Cav-1 in the immune system of A.clarkii., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Interleukin 17C (IL17C) is a cytokine that regulates innate immunity by recruiting antimicrobial peptides and pro-inflammatory cytokines. In this study, we characterized properties of IL-17C from Amphiprion clarkii also known as yellowtail clownfish (AcIL-17C). The AcIL-17C gene is 489 base pairs long and encodes a 163 amino acid long protein. AcIL-17C includes a signal peptide for localization in the extracellular space and comprises the IL-17 domain. The transcription analysis revealed that AcIL-17C mRNA was ubiquitously expressed in 12 tested tissues. Blood cells treated with polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharides (LPS), and Vibrio harveyi, AcIL-17C mRNA expression was upregulated at 6 h (following poly (I:C) and LPS treatments) and at 24 h post-injection (following all treatments). The downstream gene analysis of the epithelial fathead minnow (FHM) cells showed upregulated expression of genes, such as FHM_NK-Lysin, FHM_Hepcidin-1, FHM_Defensin-β, encoding antimicrobial peptides, as well as of FHM_IL-1β, FHM_TNF-A, FHM_IL-11, and FHM_STAT3 genes encoding inflammation-related proteins and IL-17C receptor genes FHM_IL-17RA, and FHM_IL-17RE at 12 and 24 h after treatment with AcIL-17C. The bacterial colony counting assay showed lower colony counts of Escherichia coli grown on FHM cells transfected with AcIL-17C carrying vector compared to those grown on control FHM cells. Further, AcIL-17C had a concentration-dependent positive effect on the survival of FHM cells infected with E. coli compared to the percentage of survived control cells. There has been a lack of studies characterizing the functions of teleost IL-17C. Therefore, these findings provide important information about the teleost host defense mechanisms and insights on the IL-17C-mediated antibacterial immunity., (Copyright © 2021. Published by Elsevier Inc.)

Peroxiredoxins (Prxs) are cysteine-dependent antioxidant proteins that play a leading part in oxidative stress response. Peroxiredoxin 4 (Prx4) is located in the endoplasmic reticulum, where it is primarily involved in regulating the concentration of H 2 O 2 , generated during protein folding. Prx4 is also located in the extracellular space, where it activates the JAK/STAT-mediated stress response. Here, we focus on the identification and characterization of the sequence and function of Prx4 from the big-belly seahorse (Hippocampus abdominalis) (HaPrx4). The size of the HaPrx4 coding sequence was 777 bp, which encoded a 258 amino acid protein of 28.8 kDa molecular weight. The amino acid sequence comprises a signal peptide, two active sites with peroxidatic cysteine and resolving cysteine, catalytic triad, and peroxiredoxin superfamily domain. According to the tissue distribution results, ovaries exhibited the highest HaPrx4 expression level within fourteen examined tissues. The HaPrx4 transcriptional responses to four immune stimulants (lipopolysaccharides, polyinosinic: polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae) were evaluated in the blood and liver tissues. Additionally, the functions of recombinant HaPrx4 protein were evaluated by metal ion-catalyzed oxidation assay, peroxidase activity assay, insulin reduction assay, cell viability assay, and Hoechst staining. The assay results confirmed that the functions of HaPrx4 involved DNA protection, hydrogen peroxide (H 2 O 2 ) elimination, oxidoreductase activity, enhancing cell survival, and cell protection. The results of the current study propose that HaPrx4 is effectively involved in H 2 O 2 scavenging activity during stress conditions and in innate immune responses of the big-belly seahorse., (Copyright © 2020. Published by Elsevier Inc.)

Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four β-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (β-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H 2 O 2 -induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interests, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum resident protein 44 (ERp44) is a protein disulfide isomerase (PDI) and a member of thioredoxin family, which is involved in several functions such as oxidative folding and polymerization of molecules, carrier protein activity, regulation of the Ca 2+ ion levels in the endoplasmic reticulum (ER), cellular signaling, and maintenance of lumen redox homeostasis. In this study, ERp44 from Hippocampus abdominalis, commonly known as the big belly seahorse, was characterized. ERp44 possessed three PDI-like domains and one thioredoxin fold with a CXXC conserved motif. The open reading frame consisted of 1233 bp encoding 410 amino acids. Additionally, it contained a C-terminal RDEL motif, which suggests a localization of ERp44 to the endoplasmic reticulum. ShERp44 showed highest mRNA expression in the ovary, brain, and gills. Temporal expression of ShERp44 in blood showed significant upregulation against bacterial, LPS, and PolyI:C stimuli at 24 and 72 h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24 h in response to lipopolysaccharides and Streptococcus iniae and at 72 h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122 ± 0.0075 μmol/s protein/μg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5 min. Moreover, rShERp44-treated fathead minnow cells showed significant cell survival against 2-hydroxyethyl disulfide and thus exhibited capability to resist oxidative stress. Taken together, these findings provide insight into teleost defense mechanisms and functional properties of ERp44 in controlling redox homeostasis at the molecular level., (Copyright © 2019. Published by Elsevier Inc.)