The Molecular Mechanism Of GP73 Affecting Autophagy Of Hepatocellular Carcinoma By Regulating Rab23

Ojective: In our previous study,we found that GP73 has a TBC-like GAP motif.Based on this,it was discussed whether GP73 had GAP enzyme activity,and the molecular mechanism of the interaction between GP73 and Rab23 was preliminarily explored.It was further studied whether GP73 affected the hydrolysis of Rab23 through its GAP enzyme activity,thus revealing the main biological function of GP73's GAP enzyme activity.And to clarify its role in the occurrence and development of liver cancer and drug resistance,provide a theoretical basis for the clinical application of GP73 and clinical treatment research.Methods: 1.Rab protein interacting with GP73 was screened by Western blot and Co-IP technique.2.The GP73 TBC structure-like mutant vector was constructed by gene recombination technology and confirmed to be expressed in eukaryotic cells.3.The hydrolysis of Rab23 by GP73 and its mutants was studied by GAP hydrolase assay and GTP activity assay.4.The effect of Rab-GAPs mutants of GP73 and GP73 on autophagy was studied by Westernblot and fluorescence immunofluorescence staining.Results: 1.Screening of 15 Rab family molecules revealed that GP73 interacts with Rab23.2.Through motif analysis,it was found that GP73 has a very conserved TBC-like GAP enzyme motif.The amino acid sequences of GP73 were mutated at amino acid positions 248 and 310,and the GP73 GAPs enzyme mutant was constructed.The GP73(R248K)and GP73(Q310A)mutants were successfully expressed.3.The purified Rab23 protein was used as the reaction substrate.The GP73 and GP73(R248K)proteins were used as the reaction enzymes to dynamically monitor the hydrolysis of Rab23 by GP73.The results showed that GP73 could significantly promote the hydrolysis of Rab23..The purified Rab23 protein was mixed with GP73,GP73(R248K)purified protein,and the hydrolysis reaction was monitored dynamically.The results showed that the efficiency of GP73 protein for Rab23 hydrolysis was significantly higher than that of GP73 mutant protein.4.The mutant and unmutated plasmids were transfected into cells.It was found that under the stimulation of rapamycin,GP73 inhibited the autophagy of hepatoma cells.GP73 GAP mutants could not inhibit the autophagy of hepatoma cells.occur.Conclusion: GP73 has a GAP enzyme activity,can hydrolyze Rab23 protein,inhibit the occurrence of autophagy in hepatoma cells.Provide a reference for the study of the occurrence,development,treatment and prognosis of liver cancer.