E3 Ubiquitin Ligase NEDD4 In The Formation Of Goose Fatty Liver And Its Mechanismresearch

As a high-grade food material,goose fatty liver is an important waterfowl product.It is produced by artificial overfeeding in a short period of time.Its weight can be 8-10 times the normal liver weight,containing a large amount of fat.However,unlike mammalian nonalcoholic fatty liver disease(NAFLD),goose fatty liver hardly has any obvious pathological changes,such as inflammation and fibrosis,which suggests that goose fatty liver has a unique tolerance mechanism.Uncovering this mechanism may provide a useful reference for improving the production of goose fatty liverand studying NAFLD of other livestock and poultry.It is known that ubiquitination is the main pathway of protein degradation,which is involved in a variety of cell biological processes,including cell growth and proliferation,survival and death,and immune response.In this study,the differentially expressed genes(DEGs)between normal liver and fatty liver of Landes goose were identified by transcriptome sequencing analysis.Among the DEGs,66were related to ubiquitination/deubiquitination.In particular,the mRNA expression level of neural precursor cell expressed,developmentally down-regulated 4(NEDD4)gene in fatty liver was significantly higher than that of normal liver at three time points of overfeeding,i.e.,77,84 and 89 d old of age.NEDD4,as an E3 ubiquitin ligase,plays an important role in liver regeneration and lipid metabolism via its target proteins including PTEN and IGF1R.However,it is unknow whether NEDD4 is involved in the development of goose fatty liver via its target proteins.To address this problem,the quantitative quantitative PCR and immunoblotting were used in this study to determine the expression of NEDD4 mRNA and protein in goose fatty liver and normal liver,immunoprecipitation and immunoblotting were used to detectthe physical correlation between NEDD4 and target proteins,and NEDD4 overexpressionwas used to verify the influence of NEDD4 on the degradation of target proteins in goose primary hepatocytes.This study may lay a foundation for further research on the role of NEDD4 in the formation of goose liver.The major results are as follow1.Transcriptome sequencing analysis identified 458,875 and 1733 DRGs in goose fatty liver vs.normal liver at three time points of overfeeding(77,84 and 89 d old of age)respectively,including 66 genes related to ubiquitination and deubiquitination.In particular,the mRNA expression level of NEDD4 gene in goose fatty liver was significantly high than that in normal liver at the three time points(P<0.05).2.NEDD4 and its potential target genes PTEN and IGF1R are involved in the formation of goose fatty liver:when feeding for 24 days,the mRNA and protein expression of NEDD4 in goose fatty liver is significantly higher than that in normal liver(P<0.05),It is suggested that the feeding induction of NEDD4 is mainly at the transcription level.For the potential target genes PTEN and IGF1R of NEDD4 during the formation of goose fatty liver,fluorescence quantitative PCR and immunoblotting analysis showed that compared with the control group,the mRNA expression of PTEN and IGF1R in goose fatty liver increased significantly(P<0.05)The protein expression of PTEN decreased significantly(P<0.05),and the protein expression of IGF1R showed a decreasing trend(P=0.07).These results indicate that NEDD4 and its target proteins PTEN and IGF1R are involved in the formation of goose fatty liver.3.Whether there is a physical relationship between NEDD4 and PTEN protein in foie gras,and the expression level of ubiquitination of PTEN is regulated by ubiquitination.To determine whether there is a physical relationship between NEDD4 and PTEN protein in foie gras,NEDD4 antibody is used Co-immunoprecipitation of foie gras tissue-like immunoprecipitation and immunoblot analysis of NEDD4 and PTEN.The results showed that there was a physical association between NEDD4 gene and PTEN gene in the goose liver tissues fed for 24 days.In order to determine whether PTEN was modified by ubiquitination during the formation of goose fatty liver,the lysates of goose fatty liver and normal liver tissue were used for PTEN immunoprecipitation,and the precipitate was subsequently used for ubiquitin immunoblotting analysis.The sample loading of the sample is the same.The results showed that the protein level of PTEN in goose liver tissue was regulated by ubiquitination,and the ubiquitination level in goose fatty liver was significantly higher than that in the control group.4.Overexpression of NEDD4 promotes the degradation of PTEN and IGF1R protein levels:Overexpression of NEDD4 in goose primary hepatocytes and detection of PTEN and IGF1R mRNA and protein expression levels to verify the targeting relationship between NEDD4 and PTEN and IGF1R.The results showed that the overexpression ofNEDD4 resulted in a significant down-regulation of PTEN and IGF1R and protein expression(P<0.05),but PTEN mRNA was not significantly affected.These results further confirm that NEDD4 can promote the degradation of PTEN and IGF1R in the formation of goose fatty liver.In summary,during the formation of goose fatty liver,the increased expression of NEDD4 promotes the ubiquitination and degradation of PTEN and IGF1R,and may protect the goose fatty liver from the damage caused by severe steatosis and promote goose through its target protein and fatty liver's growth.