Ligase FBXW5 Exacerbates Nonalcoholic Steatohepatitis Through Ubiquinating Protein Kinase ASK1

ObjectiveNonalcoholic fatty liver disease(NAFLD)is a worldwide epidemic that includes simple fatty liver(SFL)and nonalcoholic steatohepatitis(NASH)due to causes other than alcohol and definite pathogenic factors.NASH may develop into cirrhosis and liver cancer at the end stage,and also it is one of the leading causes for liver transplantation.Current treatment of NASH,however,depends on lifestyle intervention,and medication for this disease is still in the stage of phase II and III trials with no specific drugs approved in China.Given the enormous burden it imposes on the individuals,families and societies,finding new treatments or therapeutic targets have become an important issue in current medical research.It was found in previous studies that ASK1 regulates inflammation activation,lipid accumulation,and fibrosis development in NASH,and the FBXWs subfamily of the SCF complex is involved in the regulation of ubiquitination and phosphorylation of ASK1.Our preliminary study showed that FBXW5 participated in ubiquitination-mediated ASK1 activation,and on this basis the mechanism of FBXW5 regulating ASK1 was further explored in the present study.Methods 1.Interaction between FBXW5 and ASK1 in L02 cells in which both proteins were simultaneously overexpressed was investigated by coimmunoprecipitation(CO-IP).At the same time,the activation of ASK1 and its downstream signaling pathway when FBXW5 was overexpressed and knocked out was determined by Western blot.2.Overexpression of different ubiquitination site-mutated and ASK1-site mutated proteins in L02 cells was detected by IP and Western blot to find out whether FBXW5 was involved in the ubiquitination-mediated activation of ASK1 under stimulatory or non-stimulatory conditions and to confirm the ubiquitination site(s)for FBXW5 and ASK1 interaction.3.Regions of FBXW5 involved in the ubiquitination of ASK1 were determined by expressing truncated FBXW5 proteins in L02 cells,and thus speculated the interaction pattern between FBXW5 and ASK1.4.C-terminal and N-terminal truncated FBXW5 proteins were overexpressed in cells to competitively inhibit the regions of FBXW5 participating in ASK1 ubiquitination activation,and lipid accumulation,inflammatory response,and fibrosis were detected by Western blot and real time PCR.Lipid accumulation was also visualized by oil red staining.5.C-terminal and N-terminal FBXW5 proteins were overexpressed in mice by injection of the corresponding vectors,and whether FBXW5 was a therapeutic target was determined by Western blot and real time PCR.Results 1.FBXW5 interacted with ASK1,and overexpression of FBXW5 activated ASK1 and its downstream pathways.2.FBXW5 promoted the ubiquitination of K63 in ASK1,and no promoting effect on ASK1 ubiquitination was observed when mutations at amino acid 1037 or(and)1046 were introduced in ASK1.3.Both N-and C-terminals of FBXW5 participated in the ubiquitination process.The N-terminal was involved in the transfer of ubiquitination chain and the C-terminal in the interaction with AKSK1.4.It was confirmed at the cellular and animal levels that the N-and C-terminals of FBXW5 were able to alleviate the steatosis of the liver and reduce the inflammatory response after being competitively inhibited.Conclusion FBXW5 activates ASK1 by K63 ubiquitination.FBXW5 participates in the transfer of ubiquitinated polypeptide chain via its N-terminal and the ubiquitination of ASK1 via it C-terminals by interacting with amino acids 1037 and 1046 of ASK1.In cellular and animal level experiments,functions of the N-terminal and C-terminal of FBXW5 are competitively inhibited by the N-terminal and C-terminal amino acid sequences to reduce the severity of NASH,suggesting that FBXW5(S1)and FBXW5(S3)are potential predrug for the treatment of NASH.