The Mechanism Of Cell Uptake Of Exogenous Glutathione S-transferases Pi(GSTpi) Protein

Glutathione S transferase(GSTs)is polygenic isozyme family,which catalyzes glutathione(GSH)nucleophilic S atoms with the substrate molecule electrophilic group for nucleophilic addition reaction.On the difference of the amino acid sequences,the mammalian GSTs are divided into six classes:?,?,?,?,? and ?,among these isozymes,glutathione S-transferase pi(GSTpi)is the most primary one in mammalian cells.GSTpi is determinant in cellular response to oxidative stress and protects tumor cells from apoptosis elicited by a variety of cytotoxic agents.The anti-inflammatory effect of endogenous GSTpi was first discovered in our group,and the GSTpi protein was effectively expressed in vitro by recombinant technique.The experiments further showed that injection of recombinant GSTpi protein can obviously reduce mortality of sepsis and endotoxin shock model in mice and the release of a variety of proinflammatory factors,which both paved the way for the research and development of recombinant protein GSTpi.We have observed the phenomenon of restructuring GSTpi protein into the cell in vitro,its mechanism is still unclear,and the mechanism is an important pharmacodynamics content of recombinant GSTpi proteinsCell endocytosis broadly divided into phagocytosis and pinocytosis,the latter includes macropinocytosis,clathrin-dependent endocytosis,caveolin-dependent endocytosis and the clathrin-and caveolin-independent endocytosis which need different protein molecules,such as flotillin-1,etc.In this study,we expressed and purifyied the GSTpi protein,and then with FITC fluorescent tags to the protein.After the use of various tools medicine,we used flow cytometry and laser confocal microscopy to detect and observe the mechanism of GSTpi protein into cells.In addition,the frozen section technique was used to detect the main tissue distribution of GSTpi protein in animalsBased on the past experiments,we have identified the recombinant GSTpi protein can enter RAW264.7 cells,THP-1 and mouse peritoneal macrophages,and the GSTpi entered the cells with the activity of transferase.It was reported that CD and LB are now recognized as the reagents which change actin polymerization and actin microfilament.CD and LB could combine with the actin and the growth of F-actin,inhibit the polymerization reaction,induct actin depolymerization,thus undermining phagocytosis and inhibiting the endocytosis of foreign materials.The results showed that inhibition of macropinocytosis by CD and LB did not influence the internalization of recombinant GSTpi protein,which indicated that FITC-GSTpi protein is not dependent on phagocytosis.FITC-dextran often used as a fluorescent marker to show the process of macropinocytosis formation.EIPA inhibits macropinocytosis by inhibiting the exchange of Na+/H+.The FITC-dextran control experiment showed that the macropinocytosis pathway in RAW264.7 cells is active,but the EIPA treatment did not affect the endocytosis of the FITC-GSTpi protein,that is,it did not depend on the macrophages.Clathrin-dependent is an another form of pinocytosis.CPZ is a kind of cationic amphiphilic drug by reversing clathrin transposition and regulating proteins from plasma membrane to intracellular vesicles to suppress the formation of clathrin pits,thus inhibiting clathrin-mediated endocytosis.CPZ reduced the amount of FITC-GSTpi protein into cells,suggesting that the FITC-GSTpi protein was connected with the clathrin-dependent endocytosis.According to the reviews,the cell surface of RAW264.7 does not express the caveolin,so the endocytosis of FITC-GSTpi protein is caveolin-independent.Clathrin-and caveolin-independent are also part of extracellular substance into the cell as flotillin-1-dependent,the specific mechanism and process are not very clear at present,but it mainly depends on flotillin-1 protein.After the flotillin-1 siRNA(m)interfered with the expression of flotillin-1 protein,study the effect on FITC-GSTpi protein endocytosis.Interfering with the expression of flotillin-1 protein did not affect the endocytosis of FITC-GSTpi which suggests the protein was independent of this pathway.There are overlaps between the endocytosis pathways,and some structures and substances are associated with a variety of endocytosis pathways.Microtubules can affect the cell morphology and intracellular transport.Noc and TX both reduce the amount of FITC-GSTpi protein,indicating that the FITC-GSTpi protein is connected to the microtubule.M?CD is a kind of cyclic oligomers of glucoside,it could reversibly extracted cholesterol from plasma membrane to inhibit some endocytic pathways that involve with cholesterol,such as caveolin-dependent and clathrin-dependent endocytosis.RAW264.7 cell membrane don't express caveolin,what's more,water soluble cholesterol reverses its effect on FITC-GSTpi protein,illustrating FITC-GSTpi protein into the cell associated with cholesterol,clathrin-dependent endocytosis.According to the above results,we concluded that FITC-GSTpi is clathrin-dependent endocytosis,involved with microtubule and cholesterol,unrelated with phagocytosis,micropinocytosis or flotillin-1 dependent endocytosis.