Elucidation Of Mitochondrial Apoptosis Pathway And Functional Characterization Of Voltage-dependent Anion Channel 2(VDAC2) In Pacific Oyster

Industries related to oyster,an important aquaculture species,are widely distributed over the world.Pacific oyster(Crassostrea gigas)is an estuarine and intertidal zone animal with sessile behavior,exposed to variable salinity,fluctuating temperature,toxic metals,desiccation and microbial pathogens,which are all adversity stresses that threaten the survival of this sedentary organism.Pacific oyster has evolved complex mechanisms to adapt the complex and inconstant circumstance.In recent years,it has been reported that apoptosis,as a vital part of the environmental adaptation system,gets involved in response of oyster to various stresses.So far,however,little is known about the exact mechanism of apoptosis in oyster,and even in all the mollusks.Studies in this field are urgently required to help us reveal the mechanism of mitochondrial apoptosis in C.gigas,which will further be helpful to understand how oyster acclimates to the fickle environment.With the publication of genome information of Pacific oyster,it is time to comprehensively investigate the molecular regulation mechanism of C.gigas.Several forms of apoptosis have been found up to now.Among them,mitochondrial apoptosis pathway is a very major one and has received more attention.The mitochondrial apoptosis has been demonstrated to be involved in the response of organisms to various environmental stresses,making organisms surviving.There are much more studies on the exact mechanisms of mitochondrial apoptosis in vertebrate than invertebrate.Base on the existing evidences,it could be suggested that mitochondrial apoptosis pathway is quite conserved throughout the entire vertebrate.But in invertebrate,although numerous DNA sequences of apoptosis-related homologous genes have been found in recent years,only a few have been done on the study of mitochondrial apoptosis mechanism in invertebrate as yet(less has been done in mollusks).Besides,the pathways seemed to be diverse in different invertebrates,according to the few existing evidence.Thus,more studies are needed to reveal the diversity of mitochondrial apoptosis in invertebrates and further understand the evolution of the pathway in animals.In this study,we systematically investigated the molecular regulatory mechanism of mitochondrial apoptosis in C.gigas,using the reverse genetics.UV irradiation was chosen to be used as the apoptosis inducer as we found UV irradiation could induce the apoptosis in the hemocytes of C.gigas.We found that UV irradiation lead to the increase of mitochondrial outer membrane permeabilization(MOMP)and the release of cytochrome c from mitochondria to cytosol in C.gigas.Based on this result,we considered that mitochondrial apoptosis pathway might involve MOMP and release of cytochrome c from mitochondria to cytosol,which was consistent with the pathway in vertebrate.Thereafter,UV irradiation was found to activate Cgcaspase 9 and Cgcaspase 3 in hemocytes of C.gigas.In addition,cytochrome c was also found to activate Cgcaspase 9 and Cgcaspase 3 in cytoplasmic extract of C.gigas.The results here suggested that Cgcaspase 9 and Cgcaspase 3 were both involved in mitochondrial apoptosis and the activation of them might be associated with the release of cytochrome c.The activity level of Cgcaspase 3 decreased once Cgcaspase 9 was inhibited,implying that Cgcaspase 9 could activate Cgcaspase 3 in mitochondrial apoptosis pathway.However,as we didn't find any APAF-1 homologue containing all the major domains in C.gigas,the mechanism of cytochrome c-mediated Cgcaspase 9 activation is still unclear.Additionally,we did some work to investigate the role of Pacific oyster Bcl-2 family.Due to the missing of BH3-only subfamily,only members of anti-apoptotic subfamily and pro-apoptotic subfamily were studied herein.They are CgBcl-2 and CgBcl-xL from the anti-apoptotic subfamily,and CgBak and CgBax from the pro-apoptotic subfamily.We obtained the full-length cDNA sequence of them and performed domain analysis on the predicted proteins encoded by the four genes.Then,the expression patterns of the genes under several types of stresses,including virus infection,vibrio infection,air exposure stimulation and heat shock treatment were analyzed using qPCR.The results showed that the genes responded to all types of stresses in various degrees.Besides,the expression levels of the four genes in hemocytes all changed significantly upon UV irradiation,implying their involvement in mitochondrial apoptosis of C.gigas.Then we conducted some more experiments and found that Pacific oyster members regulated the death or survival of the yeast(Saccharomyces cerevisiae)and influenced the apoptotic level of HEK293 T cells.Moreover,it was observed that the hemocytes in which CgBak was silenced by siRNA,displayed a lower apoptotic percentage after UV irradiation than the normal ones.It was suggested by the results here that Bcl-2 family of C.gigas might probably take charge in the regulation of mitochondrial apoptosis.Investigations on how Pacific oyster regulated mitochondrial apoptosis were then performed.We overexpressed CgBak and CgBax in He La cells before irradiated the cells with UV light.It turned out that CgBak protein and CgBax protein in some cells translocated from cytoplasm to mitochondria after the irradiation.Similar results were observed in HEK293 T cells overexpressing CgBak and CgBax,indicating the translocation of CgBak and CgBax from cytoplasm to mitochondria upon the occurrence of apoptosis.Thereafter,we incubated the mitochondria isolated from Pacific oyster hemocytes with CgBak protein or CgBax protein,and detected the release of cytochrome c from mitochondria.Based on these results,it was suggested that once mitochondrial apoptosis occurred,CgBak and CgBax would translocated from cytoplasm to mitochondria and induced the mitochondria to release cytochrome c to cytoplasm.In addition,we demonstrated the direct interaction between the proteins from pro-apoptotic subfamily and the ones from anti-apoptotic subfamily using yeast two hybrid and co-immunoprecipitation.The results obtained by the two assays also indicated that,it was the BH3 domain that mediated the direct interaction.CgBcl-2 and CgBcl-xL,the proteins from anti-apoptotic subfamily,seemed to inhibit the pro-apoptotic activities of CgBak and CgBax through the direct interaction.Subsequently,we focused on the regulatory factors at the upstream part of Bcl-2 family.The Cgp53 was chosen and investigated to reveal its role in mitochondrial apoptosis.We conducted qPCR analysis and found that the mRNA level of Cgp53 displayed a significant uptrend upon UV irradiation.Besides,once the expression of Cgp53 was inhibited,the hemocytes showed a significant lower apoptotic percentage upon UV irradiation,signifying the involvement and probable pro-apoptotic role of Cgp53 in mitochondrial apoptosis of C.gigas.A dual-luciferase reporter assay was performed and revealed that Cgp53 might activate the transcription of CgBak and CgBax in a dose-dependent manner.Moreover,the expression level of CgBak increased later upon UV irradiation in Cgp53-inhibited hemocytes than normal hemocytes,confirming the activation of Cgp53 to the transcription of CgBak,which further lead to apoptosis.We then assayed the direct interaction between Cgp53 and the four Bcl-2 family proteins studied in our work,using co-immunoprecipitation.The demonstration of the direct interaction implied the possibility that Cgp53 might regulate Bcl-2 family in a transcription-independent way.Taken together,a conclusion could be drawn that Cgp53 might regulate the activities of Pacific oyster Bcl-2 family in two distinct ways,the transcription-dependent way and the transcriptionindependent way,and thus regulated(induced)mitochondrial apoptosis of C.gigas.However,we could not find any member of BH3-only subfamily in Pacific oyster and even the whole mollusks.It will definitely be quite interesting to study how the missing of this crucial subfamily would impact on the adaptation of oyster to the intertidal circumstance.In addition,we did some research on Pacific oyster voltage dependent anion channel 2(VDAC2),revealing its function and association with mitochondrial apoptosis.We firstly obtained the full-length cDNA sequence of CgVDAC2 and analyzed its expression pattern in distinct tissues,at different developmental stages and upon virus infection,the result of which suggested the response of CgVDAC2 to OsHV-1 infection.Then we found that the overexpression of CgVDAC2 in HEK293 T cells reduced the UV-irradiation-induced apoptosis level.Moreover,Pacific oyster hemocytes in which CgVDAC2 was silenced by siRNA displayed a higher apoptotic percentage upon UV irradiation.Besides,the direct interaction between CgVDAC2 and CgBak was confirmed using yeast hybrid assay and co-immunoprecipitation assay.The results above indicated that CgVDAC2 might involve in regulation of mitochondrial apoptosis and inhibit the pro-apoptotic activity of CgBak.In summary,we elucidated the mechanism of mitochondrial apoptosis in C.gigas,which included p53,Bcl-2 family,mitochondria,cytochrome c,caspase 9,caspase 3 and so on.This is the first work systematically studying the mechanism of apoptosis in mollusks,which filled in a gap in this field and provided new evidence for better understanding the evolution and origin of mitochondrial apoptosis in invertebrate.This work would also be helpful for better understanding how oyster adapted to the complex and changeable circumstance.In addition,the investigation on the function of CgVDAC2 is the first one in mollusks.The results updated our knowledge on the relationship between VDAC2 and mitochondrial apoptosis,and provided a new idea in understanding the infection process of Os HV-1 to oyster.