| Objective: Ubiquitin ligase RNF8 plays an important role in DNA damage repair,cell cycle regulation,neurodegenerative diseases,spermatogenesis and other fields.We recently found out that RNF8 also plays an important role in regulating lipid metabolism.In this study,we discovered the specific regulatory mechanism of RNF8 on the ubiquitination modification of ACSL4 and its effect on lipid metabolism,the aim is to reveal the way of lipid metabolism and provide a new entry point for the diagnosis and treatment of related metabolic diseases.Methods: Knockout of RNF8 mice were used to investigate the effect of RNF8 gene deletion on fat metabolism in mice by liver HE staining,oil red O staining and blood lipid detection;establish a high-fat diet model,and explore the effect of RNF8 deletion on fat metabolism in mice fed with high-fat diet by evaluating the liver lipid deposition,serum lipid level,content of fat metabolites,expression level of key proteins of liver fat metabolism pathway,and TEM observation of liver tissue.By infecting HL-7702 cells with lentivirus,establish a stable transfected cell line with knockdown and overexpression of RNF8,and a key protein in the lipid metabolism pathway,ACSL4,was found through conjoint analysis of multi-omic,the relationship between RNF8 and ACSL4 expression level was detected by Western blotting;Ubiquitin-proteasome pathway inhibitor test and protein half-life period test verifies the effect of RNF8 on ACSL4 content;the interaction between RNF8 and ACSL4,and it`s type of ubiquitination was detected by the co-immunoprecipitation and plasmid transfection,to further clarify the regulatory relationship between RNF8 and ACSL4.Results: 1.Compared with WT mice,RNF8 gene deficient mice are smaller in size,lower in body weight,lesser in abdominal fat,liver lesions and lipid deposition,and the expression level of liver lipid metabolism pathway regulatory proteins ACSL4,PPAR α,PGC-1 and CPT1 A have increased;After high-fat diet,compared with WT mice,RNF8 gene deficient mice had a smaller increase in body weight,lower increase in blood lipid levels,milder progression of liver lesions and lipid deposition,higher proportion of normal hepatocyte mitochondrial morphology,and the content of lipid metabolite AA was increased;There is a negative correlation between RNF8 and ACSL4 protein expression levels,also there is an interaction between the two proteins;ACSL4 was ubiquitinated by RNF8 through K48 pathway,and RNF8 mediated its degradation.Conclusion: The deletion of RNF8 can lead to an increase in liver lipid catabolism in mice,alleviating hepatic lipid accumulation and cell damage caused by the high-fat diet.The mechanism is that RNF8 modifies ACSL4 by ubiquitination via K48 pathway and mediates its degradation through ubiquitin-proteasome pathway,thus regulate the lipid metabolism in hepatocyte. |
