The Functional Significance of McMafF_G_K in Molluscs: Implications for Nrf2-Mediated Oxidative Stress Response.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal regulator of antioxidant gene expression in mammals, forming heterodimer complexes with small Maf proteins through its BZip domain. However, the underlying mechanism of Nrf2 action in molluscs remains poorly understood. The thick shell mussel, Mytilus coruscus, represents a model organism for the marine environment and molluscs interaction research. In this study, we used in silico cloning to obtain a small Maf homologue called McMafF_G_K from M. coruscus. McMafF_G_K possesses a typical BZip domain, suggesting its affiliation with the traditional small Maf family and its potential involvement in the Nrf2 signaling pathway. Transcriptional analysis revealed that McMafF_G_K exhibited a robust response to benzo[a]pyrene (Bap) in the digestive glands. However, this response was down-regulated upon interference with McMafF_G_K-siRNA. Interestingly, the expression levels of Nrf2, NAD(P)H: quinone oxidoreductase (NQO-1), and Glutathione Peroxidase (GPx), which are key players in oxidative stress response, showed a positive correlation with McMafF_G_K in digested adenocytes of M. coruscus. Furthermore, in vitro analysis of antioxidant capacity in digestive gland cells demonstrated that Bap exposure led to an increase in reactive oxygen species (ROS) levels, accompanied by an elevation in total antioxidant capacity (T-AOC), potentially counterbalancing the excessive ROS. Strikingly, transfection of McMafF_G_K siRNA resulted in a significant rise in ROS level and a down-regulation of T-AOC level. To validate the functional relevance of McMafF_G_K, a glutathione S-transferase (GST) pull-down assay confirmed its interaction with McNrf2, providing compelling evidence of their protein interaction. This study significantly contributes to our understanding of the functional role of McMafF_G_K in the Nrf2 signaling pathway and sheds light on its potential as a target for further research in oxidative stress response.

Unraveling the protective role of Nrf2 in molluscs: Insights into mitochondrial and apoptosis pathways in the defense against Bap-induced oxidative stress.

Benzopyrene (Bap) is a major constituent of petroleum pollutants commonly found in aquatic environments, and its mutagenic and carcinogenic properties have adverse effects on aquatic organisms' development, growth, and reproduction. The antioxidant defense system element, NF-E2-related factor 2 (Nrf2), has been linked to the oxidative stress response in marine invertebrates exposed to toxic substances. In a previous study, a novel Nrf2 homologue, McNrf2, was identified in mussel Mytilus coruscus, a significant model marine molluscs in ecotoxicology studies. McNrf2 showed the potential to trigger an antioxidant defense against oxidative stress induced by Bap. Here, we employed an Nrf2 overexpression and inhibition model using SFN and ML385 as Nrf2 inducer and inhibitor, respectively. Next, immunofluorescence technique was used to evaluate the nuclear activation of Nrf2 induced by Bap-mediated oxidative stress. Transmission electron microscopy revealed that overexpression of Nrf2 could maintain the quantity and structural integrity of mitochondria, while flow cytometry analysis showed that Nrf2 could alleviate Bap-induced cellular apoptosis. These findings suggest that Nrf2 can protect molluscs from Bap-induced oxidative stress through the mitochondria and apoptosis pathways, providing a novel perspective on Nrf2's antioxidant function.

The Nrf2 molecule trigger antioxidant defense against acute benzo(a)pyrene exposure in the thick shell mussel Mytilus coruscus.

The NF-E2-related factor 2 (Nrf2), an ubiquitous, evolutionarily conserved transcription factor, acts as a major sensor of oxidative stress in cells. In the present study, a Nrf2 homolog was newly identified in the thick shell mussel Mytilus coruscus. Accordingly, its functional role in antioxidant defense in response to acute benzo(a)pyrene (Bap) exposure was assessed. The newly identified McNrf2 affiliated to traditional Nrf2 family through Blast, multiple alignment and phylogenetic analysis. After acute exposure to Bap, antioxidants including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathine reductase (GR) were significantly induced in gills and digestive glands at both mRNA and enzymatic levels, and the expression of McNrf2 mRNA was also up-regulated. The analysis of correlating the expression of McNrf2 and the mRNA levels of these antioxidant genes showed positive ties, indicating that Nrf2 was needed for protracted induction of such genes. Further, the recombinant McNrf2 was produced through pET-32a prokaryotic system. After 50 µg/L Bap exposure, ROS generation and LPO level in gills of Nrf2 over-expressed mussels significantly decreased compared to Nrf2 wild-type mussels, as well as reduced ROS production in digestive glands. Collectively, these results show that Nrf2 pathway can provide protection from oxidative stress triggered by Bap in the thick shell mussel.

Two novel CYP3A isoforms in marine mussel Mytilus coruscus: Identification and response to cadmium and benzo[a]pyrene.

CYP3A enzymes play a crucial role in metabolic clearance of a variety of xenobiotics. However, their genetic information and function remain unclear in molluscs. In the present study, two novel CYP3A genes i.e. McCYP3A-1 and McCYP3A-2 were identified and characterized from the thick shell mussel Mytilus coruscus, and their tissue distribution as well as the response to cadmium (Cd) and benzo[a]pyrene (B[α]P) exposure were addressed using real time quantitative RT-PCR (qRT-PCR) and erythromycin N-demethylase (ERND) assay. McCYP3A-1 and McCYP3A-2 possess typically domains of CYP family such as helix-C, helix-I, helix-K, PERF and the heme binding domain as well as the characteristic domains of CYP3s including six SRS motifs. McCYP3A-1 and McCYP3A-2 transcripts were constitutively expressed in all examined tissues with high expression level in digestive glands, hepatopancreas and gonads. Upon B[α]P exposure, McCYP3A-1 and McCYP3A-2 mRNA expression in digestive glands showed a pattern of up-regulation followed by down-regulation, while under Cd exposure, showed a time-dependent induction profile. In addition, ERND activity, generally used as an indicator of CYP3, increased in a time-dependent manner after exposure to Cd and B[α]P. These results collectively indicated that McCYP3A-1 and McCYP3A-2 are CYP3A family member and may play a potential role in metabolic clearance of xenobiotics. Meanwhile, the current results may provide some baseline data to support McCYP3A-1 and McCYP3A-2 as candidate biomarkers for monitoring of PAHs and heavy metal pollution.

A novel invertebrate toll-like receptor with broad recognition spectrum from thick shell mussel Mytilus coruscus.

Toll-like receptors (TLRs) are a category of most well recognized pattern recognition molecules that act on a vital role in both innate and adaptive immunity. In the present study, a novel toll-like receptor (McTLRw) was identified and characterized in thick shell mussel Mytilus coruscus. McTLRw possesses one intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain, one transmembrane region (TM), one leucine rich repeat N-terminal domain (LRR_NT) and a few of leucine-rich repeats (LRRs), which all are common in typical TLRs. McTLRw transcripts were constitutively expressed in all examined tissues with higher expression levels in immune related tissues, and were significantly induced in haemocytes with the challenges of live Vibrio alginolyticus, lipopolysaccharide (LPS), peptidoglycans (PGN) and ß-glucan (GLU), but not induced by polyinosinic-polycytidylic acid (poly I:C). rMcTLRw exhibited affinity to LPS, PGN and GLU while no affinity to poly I:C. Further, the downstream of TLR signaling pathway myeloid differentiation factor 88a (MyD88a), interleukin-1 receptor-associated kinase-4 (IRAK4) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were significantly repressed in McTLRw silenced mussels while challenged with LPS. These results collectively indicated that McTLRw is one member of TLR family and involved in immune response to against invaders by taking participate in TLR mediated signaling pathway.

Expression of zona pellucida 3 gene is regulated by 17α-ethinylestradiol in adult topmouth culter Culter alburnus.

Estrogen could lead to abnormal modulation or disruption of physical development, reproduction and sexual behavior in aquatic wildlife, especially in fish. Information on the toxicity of estrogens to native species in that can be used in site-specific risk assessments is scarce. In the present study, one zona pellucida 3 (ZP3) homologue termed CaZP3 was firstly identified from topmouth culter Culter alburnus, following its structural characteristics, tissue distribution and transcriptional modulation to 17α-ethinylestradiol (EE2) exposure were investigated. Meanwhile, vitellogenin (VTG) gene was employed to provide a comparison of the reactive ability to EE2 induction. The CaZP3 characterized with analogical functional domains such as ZP domain, SP, IHP, EHP, 12 cysteine residues, one N-linked glycosylation site and two conserved O-linked glycosylation sites and equal number of eight exons and seven introns with ZP3 counterparts of higher species. CaZP3 mRNA predominantly expressed in ovary, besides, highly expressed in female heart and male muscle and relatively high expressed in testis. CaZP3 has the lower reactive ability to EE2 induction in comparison with VTG, however, CaZP3 transcripts were significantly induced in gonads of both male and female culter by EE2 and could be used as an alternative biomarker to monitor EE2 activity. The present results supplement the database for toxicity of EE2, especially for fish species endemic to China and provide some useful information for the monitoring of EE2 activity in aquatic environment.

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in thick shell mussel, Mytilus coruscus.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Despite of the well study in vertebrates, there is few data ascribe to this TRAF member in invertebrates, especially in bivalves. In the present study, a novel TRAF6 homologue termed McTRAF6 was firstly characterized in Mytilus coruscus. Like its counterparts in mammals, McTRAF6 shared the domain topology containing one RING domain, two zinc finger domains, one coiled-coil region and a MATH domain. McTRAF6 transcripts predominantly expressed in gills, digestive glands and hemocytes in M. coruscus, and were significantly up-regulated in hemocytes after challenge with lipopolysaccharide (LPS) and polyinosine-polycytidylic acid (poly I:C). Further, the subcellular localization in cytoplasm and the activation of Nk-κB or ISRE luciferase reporter by overexpressed McTRAF6 were identified in HEK293T cells. These results collectively indicate that McTRAF6 is a member of TRAF6 subfamily and plays a potential role in immune defense system against pathogenic agents invasions in thick shell mussel. To our knowledge, this is the first report on component of TLR signaling pathway in thick shell mussel, providing further evidence for the existence of TLR pathway in M. coruscus and contribute to clarify the innate immune system of thick shell mussel.

The complement factor H (CFH) and its related protein 2 (CFHR2) mediating immune response in large yellow croaker Larimichthys crocea.

Complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. Complement regulators are crucial to prevent the injudicious production of these mediators and potential injury to self tissues. Here, we identified the complement factor H (CFH) and its related gene 2 (CFHR2) homologs from large yellow croaker (Larimichthys crocea), named LcCfh and LcCfhr2, respectively. The deduced LcCfh and LcCfhr2 proteins shared significant structural similarities and identified codes for a polypeptide consisting of various numbers of highly conserved SCR domains. LcCfh, LcCfhr1 and LcCfhr2 genes were detected in all examined tissues with predominantly expressions in liver, spleen and kidney, and their expressions all increased upon Vibrio alginolyticus challenge. In vitro assays showed that recombinant LcCfh was likely to act as a cofactor of CFI and played a negative regulation role in complement system, when recombinant LcCfhr2 seemed to play mechanisms independent of the activity of CFH. Both recombinant LcCfh and LcCfhr2 took participate in inflammatory reaction despite of the inequal ability to mediate pro-inflammation response. These data provide a new insight into the functional activities of teleost complement system.

Molecular characterization and expression analysis of a complement component C3 in large yellow croaker (Larimichthys crocea).

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. Complement component 3 is a key molecule in the complement system, whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the large yellow croaker (Larimichthys crocea), which was high similarity to other complement C3. We reported the primary sequence, tissue expression profile, polypeptide domain architecture and phylogenetic analysis of L. crocea complement component C3 (L.c-C3) gene. Its open reading frame (ORF) is 4962 bp and encodes for 1653 amino acids with a putative signal peptide of 23 amino acid residues. The deduced amino acid sequence showed that L.c-C3 has conserved residues and domains known to be crucial for C3 function. Phylogenetic analysis showed that L. crocea was closely related to Miichthys miiuy. The mRNA expressions of L.c-C3 was detectable at different tissues. L.c-C3 was expressed in a wide range of adult tissues, it showed highest expression in the liver. But the different developmental stages from fertilized egg to newborn larvae of the large yellow croaker the highest expression levels of L.c-C3 gene were not found. Bacterial challenge experiments showed that the levels of L.c-C3 mRNA expression were up-regulated in the liver, spleen and brain of adult large yellow croaker respectively. The results showed that L.c-C3 mRNA expression in the large yellow croaker is influenced by bacterial stress and L.c-C3 might play an important role in immunity mechanisms. This study will further increase our understanding of the function of L.c-C3 and molecular mechanism of innate immunity in teleosts.