Effects Of Palmitoylation On The Metabolism And Circulation Of CD82 And Its Molecular Mechanism

Background:On the surface of cell membranes,CD82,as a member of the Tetraspanin family,clusters with other tetraspanin family members,growth factor receptors,integrins,and gangliosides to form a membrane protein complex with specific functions(Tetraspanin Web),which regulates the expression,targeting,and function of cell surface molecules.Thus,it can affect the proliferation and metastasis of tumor cells.Palmitoylation is a posttranslational modification of proteins catalyzed by palmitoyl transferase,which is involved in the regulation a large number of biological processes and is crucial to the function of oncogenes and tumor suppressors.S-palmitoylation is reversible and proteins can cycle between palmitoylation and depalmitoylation forms.Dynamic palmitoylation increases the hydrophobicity of CD82 and regulates the localization,conformation,and stability of CD82 in membrane protein complexes.However,the molecular mechanisms of how palmitoylation affects CD82 membrane localization,metabolism and circulation in cells remain to be fully elucidated.We propose the following hypothesis: CD82 palmitoylation site mutation,incorrectly folded CD82 into the endosome,possibly through intracellular vesicles,lysosomes,circulating bodies,Golgi apparatus,endoplasmic reticulum,proteasome metabolism cycle.Objective:To investigate the effect of CD82 palmitoylation on CD82 cycle and metabolism in breast cancer cells and its molecular mechanism.Methods:1.The ABE(Acyl-biotinyl exchange)was used to detect the palmitoylation levels of CD82 in breast cancer MDA-MB-231 cells in the normal,wild and palmitoylation mutant group.2.Immunocolocalization method was used to detect the effect of CD82 palmitoylation mutation on the colocalization between CD82 and late endosomal marker(Rab7A),lysosome marker(LAMP1),circulating marker(Rab11A),Golgi marker(GM130),autophagosome marker(LC3B),proteasome marker(PSMB9),and endoplasmic reticulum marker(P4HB);3.Western blot was used to detect the effect of CD82 palmitoylation mutation on the recovery of lysosomes,circulating bodies,Golgi apparatus,autophagosomes,proteasome and endoplasmic reticulum inhibitors.4.The combination of CD82 and markers of intracellular metabolic pathway after CD82 palmitoylation mutation was detected by co-immunoprecipitation to further determine the cyclic metabolic pathway of CD82.Results:1.CD82 was palmitoylated.Wild-type CD82 has a higher palmitoylated level than the control group,and mutations at different palmitoylated sites of CD82 can reduce the palmitoylation level of CD82.2.Immunocolocalization results showed that after Cys5+74 mutation,CD82 was significantly co-located with lysosome marker LAMP1 and circulatory marker Rab11 A compared with the control group.After Cys5 or Cys74 mutation,CD82 and the Golgi apparatus marker GM130 were colocalized.3.The results of CHX assay showed that the palmitoylation mutation of CD82 at Cys5,Cys74,Cys5+74,accelerated the degradation of CD82.Inhibitor recovery experiments showed that CD82 degradation accelerated by mutation at Cys5+74 palmitoylation site could be restored by lysosome inhibitor CQ.The palmitoylation mutation of CD82 at the Cys5 site,Cys74 site accelerates the degradation of CD82 and can be restored by the Golgi apparatus inhibitor BFA.4.Co-immunoprecipitation results showed that CD82 had enhanced the interaction with lysosome marker LAMP1 after mutation at Cys5+74,and CD82 had direct interaction with Golgi marker GM130 after mutation at Cys5 and Cys74.Conclusion:1.The mutation of CD82 palmitoylation site reduced the palmitoylation degree of CD82;2.Palmitoylation affects the intracellular metabolic cycle of CD82.CD82 is metabolized through lysosome pathway after mutation at Cys5+74,and CD82 is metabolized through Golgi apparatus after mutation at Cys5 or Cys74.