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Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 3 was 1562 bp and included a 175 bp 5'-untranslated region (UTR), a 141 bp 3'-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, ChCas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of ChCas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, participates in both innate and adaptive immunity and regulates the apoptotic process. In this study, we observed that an ortholog of TRAF6 could inhibit the activity of p53 and suppress the apoptotic process in the Hong Kong oyster, Crassostrea hongkongensis. To investigate the possible molecular mechanism of the ChTRAF6-induced antiapoptotic effect, a GST pull-down screening assay was conducted, and ChPellino was found to physically interact with ChTRAF6. In addition, the interaction between them was confirmed by Co-immunoprecipitation. Furthermore, western blotting revealed that the phosphorylation level of ChPellino was decreased after the RNAi of ChTRAF6, demonstrating that ChTRAF6 may be an upstream regulator of Pellino activation. Furthermore, the apoptosis level of hemocytes increased after ChPellino knockdown, and ChPellino overexpression suppressed ChTRAF6-dependent p53 activation. Taken together, these results indicate that ChPellino plays a critical role in suppressing ChTRAF6-dependent anti-apoptosis in the hemocytes of Crassostrea hongkongensis., (Copyright © 2019. Published by Elsevier Ltd.)

Apoptosis has been primarily investigated in mammals, and little is known about apoptosis in mollusks. The proteins Bax and Bak play critical roles in the mitochondrial apoptosis pathway and in determining cell fate. In this study, ChBax and ChBak, homologs of the well-known Bax and Bak proteins, were identified from the oyster Crassostrea hongkongensis. The ChBax/Bak proteins consist of 207/232 amino acids with the typical domains found in BCL-2 family members. ChBax and ChBak mRNA expression were detected in all 8 of the selected oyster tissues and at the different stages of development. Fluorescence microscopy revealed that the full-length proteins of ChBax/Bak were located in the cytoplasm and mitochondrial outer membrane, of HEK293T cells, respectively. Furthermore, both of the genes' expression levels were found to increase in the hemocytes of oysters challenged with pathogens. The over-expression of ChBax or ChBak activates the p53-Luc reporter gene in HEK293T cells in a dose-dependent manner. These results indicate that ChBax and ChBak may play important roles in the mitochondrial apoptotic pathway in oysters., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Caspase-8, a member of the caspase family, is an initiating caspase and plays a crucial role in apoptosis. In this study, the full-length cDNA of caspase8-like (CASP8-like) was isolated from Crassostrea hongkongensis (C. hongkongensis) by RACE-PCR. ChCASP8-like contained a 1599-bp open reading frame (ORF) encoding 533 amino acids with two conserved death effector domains (DEDs) and a cysteine aspartase cysteine structural domain (CASc). Amino acid sequence comparison showed that ChCASP8-like shared the highest identity (85.4%) with CASP8-like of C. angulata. The tissue expression profile showed that ChCASP8-like was constitutively expressed in gills, hepatopancreas, mantle, adductor muscle, hemocytes and gonads, and was significantly upregulated in hemocytes, hepatopancreas and gills under hyper-salinity stress. The apoptosis-related genes, including ATR, CHK1, BCL-XL, CASP8-like, CASP9 and CASP3, were significantly activated by hyper-salinity stress, but were remarkably inhibited by ChCASP8-like silencing. The caspase 8 activity was increased by 1.7-fold after hyper-salinity stress, and was inhibited by 9.4% by ChCASP8-like silencing. Moreover, ChCASP8-like silencing clearly alleviated the apoptosis resulting from hyper-salinity stress. These results collectively demonstrated that ChCASP8-like played a crucial role in inducing apoptosis against hyper-salinity stress. [ABSTRACT FROM AUTHOR]

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dualfluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves. [ABSTRACT FROM AUTHOR]

Caspase-8, a member of the caspase family, is an initiating caspase and plays a crucial role in apoptosis. In this study, the full-length cDNA of caspase8-like (CASP8-like) was isolated from Crassostrea hongkongensis (C. hongkongensis) by RACE-PCR. ChCASP8-like contained a 1599-bp open reading frame (ORF) encoding 533 amino acids with two conserved death effector domains (DEDs) and a cysteine aspartase cysteine structural domain (CASc). Amino acid sequence comparison showed that ChCASP8-like shared the highest identity (85.4%) with CASP8-like of C. angulata. The tissue expression profile showed that ChCASP8-like was constitutively expressed in gills, hepatopancreas, mantle, adductor muscle, hemocytes and gonads, and was significantly upregulated in hemocytes, hepatopancreas and gills under hyper-salinity stress. The apoptosis-related genes, including ATR, CHK1, BCL-XL, CASP8-like, CASP9 and CASP3, were significantly activated by hyper-salinity stress, but were remarkably inhibited by ChCASP8-like silencing. The caspase 8 activity was increased by 1.7-fold after hyper-salinity stress, and was inhibited by 9.4% by ChCASP8-like silencing. Moreover, ChCASP8-like silencing clearly alleviated the apoptosis resulting from hyper-salinity stress. These results collectively demonstrated that ChCASP8-like played a crucial role in inducing apoptosis against hyper-salinity stress.

Oysters have evolved an effective and powerful innate immune system to recognize foreign substances and eliminate invaders through orchestrated immune responses such as programmed cell death (PCD), including apoptosis, autophagy, necroptosis, and pyroptosis. Both apoptosis and autophagy have been considered to be crucial mechanisms for oyster defence. However, the role of pyroptosis, a gasdermin (GSDM)-dependent programmed cell death pathway that is critical for defence against infection, in oysters remains unclear. In this study, we investigated the activity of Crassostrea hongkongensis GSDME (Ch GSDME), which is cleaved by inflammatory C.hongkongensis caspase-1 (Ch CASP1). qRT–PCR analysis showed that Ch CASP1 and Ch GSDME were widely expressed in oyster developmental stages and tissues, and responded to Vibrio coralliilyticus challenge. Meanwhile, hemocyte death was reduced after knockdown of Ch CASP1 or Ch GSDME by RNA interference (RNAi). Following injection of V. coralliilyticus , the cell death rate of the knockdown Ch CASP1 or Ch GSDME group was significantly lower than that of the control group. In vitro , coexpressing Ch CASP1 and Ch GSDME in HEK293T cells resulted in pyroptotic morphology, a bubble-like membrane, and a truncated protein (Ch GSDME N-terminal). Subsequently, immunofluorescence revealed that Ch GSDME-NT oligomerized at the cell membrane, inducing bubble-like membrane formation, thereby causing low cytotoxicity. Luciferase reporter gene analysis revealed that Ch CASP1 significantly upregulated Nuclear factor kappa-B (NF-κB) and modestly upregulated the p53 signalling pathway. Taken together, our study demonstrated that the pyroptosis-related genes Ch CASP1 and Ch GSDME mediate pyroptosis in HEK293T cells and play important roles in cell death during V. coralliilyticus infection in C. hongkongensis. • ChCASP1 and ChGSDME were first isolated and cloned from C. hongkongensis. • RNAi of ChCASP1 or ChGSDME resulted in decrease of hemocytes death rate in C. hongkongensis. • Overexpression of ChCh CASP1 and Ch GSDME produced Ch GSDME-NT and induced pyroptosis in HEK293T cells. • Ch GSDME-NT mediates pyroptosis in HEK293T cells. • Ch CASP1 significantly up-regulates NF-κB-Luc reporter and modestly up-regulated p53-Luc reporter in HEK293T cells. [ABSTRACT FROM AUTHOR]

Hydrolase genes play an important role in the immune defense system in mollusks. We found a new hydrolase gene in Crassostrea hongkongensis. To explore its function, we cloned and characterized the hydrolase gene (Chhd) from C. hongkongensis. The full-length cDNA of Chhd is 936 bp, which is composed of a 531 bp open reading frame (ORF), a 92 bp 5′ -untranslated region (UTR) and a 313 bp 3′ UTR, the ORF encodes a polypeptide of 176 amino acids which contains a hydrolase_2 domain. qRT-PCR results showed that Chhd was ubiquitously expressed in tissues of C. hongkongensis, and the expression was highest in the mantle. After bacterial challenge, the expression of Chhd in hemocytes was significantly up-regulated. Recombinant pEGFP-N1-Chhd was distributed in the cytoplasm of HEK293T cells. The results of prokaryotic expression showed that pET28a-Chhd protein could express in large quantities. The expression of Chhd in total protein of C. hongkongensis was detected by western blot assay. In addition, Chhd inhibited the activities of NF-κB/p21-Luc in HEK293T cells, and RNA interference of Chhd significantly reduced the apoptosis rate of C. hongkongensis hemocytes. Taken together, our finding indicated that Chhd was involved in immunity and apoptosis regulation of C. hongkongensis, and would provide basic data for understanding the immune mechanism of shellfish.

Hemocytes are the central organ of immune defense against pathogens by means of inflammation, phagocytosis, and encapsulation in mollusks. The well-functioning of the host immune system relies on the hemocytes’ task exertion and frequent renewal, but the underlying renewal mechanism remains elusive at the gene level. Here, we identified one transcription factor, LIM homeobox 9, in Crassostrea hongkongensis (ChLhx9) that could be involved in hemocyte apoptosis or renewal. ChLhx9 contains a homeodomain and two LIM domains. The expression profile of ChLhx9 showed that it was specific and had high expression in hemocytes, and it significantly increased under the bacterial challenge. RNA interference of ChLhx9 dramatically decreased the apoptosis rate of hemocytes when compared with a control group, which strongly implies its pro-apoptotic role in hemocytes. Furthermore, the genomic responses to the knockdown of ChLhx9 were examined through RNA-seq, which showed that multiple pathways associated with cell apoptosis, including the apoptosis pathway, hippo signal pathway and p53 signaling pathway, were significantly down-regulated. Meanwhile, seven of the key apoptotic genes were confirmed to be upregulated by ChLhx9, among which ChASPP1 (apoptosis stimulating protein of p53) was confirmed to induce hemocyte apoptosis strongly, which demonstrates that ChASPP1 was a downstream target mediated by ChLhx9 that caused apoptosis. In conclusion, tissue-specific transcription factor ChLhx9 induces hemocyte apoptosis through activating apoptotic genes or pathways, which could contribute to hemocyte renewal and immune defense in oysters.

TRIM proteins are a group of highly conserved proteins participating in a variety of biological processes such as regulation of development, apoptosis, and innate immunity. However, the functions of these proteins in the mollusk are still poorly understood. In the present study, a TRIM9 homolog (named ChTRIM9 ) was first identified from a transcript-ome library in the Hong Kong oyster Crassostrea hongkongensis . The full-length cDNA of ChTRIM9 is 2928 bp and has a predicted Open Reading Frame ORF) encoding 721 amino acids, encoding a putative 80.2 kDa protein. SMART analysis indicated that ChTRIM9 contains the three typical TRIM domains, a RING finger, two B-boxes, and a coiled-coil domain in the N-terminal region, whereas the C-terminal region contains a SPRY domain. qRT-PCR analysis revealed a ubiquitous presence of ChTRIM9 , with the highest expression in the gills. Upon bacterial challenge in vivo, the ChTRIM9 transcripts in hemocytes were significantly down-regulated, indicating its involvement in signal transduction in immune response of oysters. Furthermore, ChTRIM9 was found to be localized mainly in the cytoplasm, and its over-expression inhibited the transcriptional activity of the NF-κB gene in HEK293T cells, demonstrating its negative role in regulating NF-κB signaling. [ABSTRACT FROM AUTHOR]

Prostaglandin E receptor 4 (PTGER4) is an essential receptor that can detect various physiological and pathological stimuli and has been implicated in a wide variety of biological processes, including the regulation of immune responses, cytokine production, and apoptosis. In this report, the first mollusk PTGER4 , referred to as ChPTGER4 , was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis . Its full-length cDNA is 1734 bp in length, including 5′- and 3′-untranslated region (UTRs) of 354 bp and 306 bp, respectively, and an open reading frame (ORF) of 1074 bp. Ch PTGER4 comprises 357 amino acids and shares significant homology with its vertebrate homologs. The results of phylogenetic analysis revealed that Ch PTGER4 clusters with PTGER4 from the Pacific oyster. In addition, quantitative real-time PCR analysis revealed that ChPTGER4 was constitutively expressed in all tissues examined and that its expression was significantly up-regulated in hemocytes and gills following challenge by pathogens ( Vibrio alginolyticus , Staphylococcus haemolyticus and Saccharomyces cerevisiae ) and pathogen-associated molecular patterns (PAMPs: lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, fluorescence microscopy analysis revealed that Ch PTGER4 localized to the membrane, and its overexpression significantly enhanced NF-κB reporter gene activation in the HEK293T cell line. In summary, this study provides the first experimental evidence of a functional PTGER4 in mollusks, which suggests its involvement in the innate immune response in oyster. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis plays a key role in many biological processes, including homeostasis within the immune system. A family of cysteine proteases, the caspases, constitutes the core of the apoptotic machinery. We have characterized the first bivalve caspase-8 ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChCaspase-8). The full-length cDNA is 1945 bp in length encoding a putative protein of 557 amino acids that contains two N-terminal DED domains, and a CASc domain at the C-terminus. ChCaspase-8 is ubiquitously expressed in oysters, with highest expression levels in the gonad and labial palps. Following microbial infection, the expression of ChCaspase-8 increased in hemocytes from 12 to 72 h post-challenge. When expressed in HeLa cells, ChCaspase-8 is located in the cytoplasm, while over-expression of ChCaspase-8 in HEK293T cells activates the transcriptional activities of NF-κB. These results indicate that ChCaspase-8 might play an important role in the immune and apoptotic responses of oysters. [Copyright &y& Elsevier]

MDM2 (mouse double-minute) and p53 form a negative feedback loop and play a prominent role in preventing the induction of uncontrolled apoptosis. To better understand their potential roles in oyster Crassostrea hongkongensis , MDM2 and p53 homologs were first isolated and cloned in C. hongkongensis (named Ch MDM2 and Ch p53), and their mRNA expression patterns in tissues and developmental stages were analyzed. Multiple sequence alignment analysis and phylogenetic analysis of Ch MDM2 and Ch p53 displayed a high degree of homology and conservation. In addition, exposure to Vibrio coralliilyticus resulted in DNA damage and apoptosis in the hemocytes of C. hongkongensis , and found that the mRNA expression level of Ch MDM2 was decreased, while the relative expression of Ch p53 was significantly increased in the hemocytes and gills. Furthermore, fluorescence from Ch MDM2-EGFP and Ch p53-Red were found to be distributed in the nucleus of HEK293T cells. Besides, dual-luciferase reporter assays showed that Ch MDM2 antagonized with Ch p53 and participates in p53 signaling pathway. In addition, the interaction between Ch MDM2 and Ch p53 was confirmed strongly by Co-immunoprecipitation assays. Furthermore, the results of RNAi showed that Ch MDM2 and Ch p53 participated in apoptosis which induced infection of V. coralliilyticus. Taken together, our results characterized the features of Ch MDM2 and Ch p53, which played a critical role in apoptosis of C. hongkongensis. • Ch MDM2 and Ch p53 were first isolated and cloned from C. hongkongensis. • Ch MDM2 antagonized with Ch p53 and participates in p53 signaling pathway. • Ch MDM2 and Ch p53 were located in the nucleus of HEK293T cells. • Ch MDM2 and Ch p53 participated in apoptosis during V. coralliilyticus infection. [ABSTRACT FROM AUTHOR]