Establishment Of SRL-PFAC Bioluminescence Method Based On Hsp90 And LRP-1 And Peptide Screening

The skin is an effective barrier for the body to defend against the environment.It plays an important role in resisting bacterial invasion,xenobiotics invasion,and dehydration.Therefore,its integrity is crucial for the human body.As we all know,wound healing is a complex physiological process,and factors such as diabetes,vascular disease,and advanced age will affect wound healing and eventually form chronic wound.Traditionally,growth factors are the main driving force for wound healing,but only human recombinant platelet-derived growth factor BB(PDGF-BB)(becaplermin gel)has been approved by the FDA to treat diabetic foot ulcers.However,its disadvantages such as poor effect,high cost,and carcinogenic risk are gradually revealed.The number of people with chronic wounds is increasing worldwide,so we urgently need to develop new drugs that promote wound healing with good effects and low prices.Heat shock protein 90(90 kDa heat shock protein,Hsp90)is a ubiquitous molecular chaperone in cells.It is highly conservative in evolution and its main role is maintaining intracellular protein stability.In recent years,more and more studies have shown that when the tissue is injured,the cells will secrete Hsp90? to the outside of the cell,affecting the migration of cells and promoting wound healing.Low-density lipoprotein receptor-related protein 1(LRP-1),also known as CD91,belongs to type I transmembrane protein that mainly mediates endocytosis and particapate in signaling pathways.In recent years,more and more studies have proved that extracellular Hsp90(eHsp90?)transmits the signal of cell migration through LRP-1.The Renilla Luciferase Bimolecular Complement(split renilla luciferase protein fragment-assisted complementation,SRL-PFAC)technology is a new bioluminescence method for studying protein-protein interactions.In the preliminary work in the laboratory,we constructed the SRL-PFAC bioluminescence method based on Hsp90 and Cdc37,and we demonstrated that the SRL-PFAC technology can specifically monitor the interaction between Hsp90 / Cdc37 and reflect the difference in Hsp90 / Cdc37 binding level sensitively and quantitatively,so it can be used to screen Hsp90 / Cdc37 inhibitors.Based on this,we plan to construct a SRL-PFAC method based on Hsp90 and LRP-1,verifying its specificity and use this method to screen peptides that can promoting cell migration.We first constructed two plasmids,pcDNA3.1(+)-S1-LRP-1 II and pcDNA3.1(+)-S1-LRP-1.Luciferase results showed that NRL-Hsp90 and S1-LRP-1 II have significant complementary effects on the cell membrane or in the cell,and NRL-Hsp90 and S1-LRP-1 can complement with each other on the cell membrane or in the cell significantly.Western blot results showed that the transfected cells expressed Hsp90 and LRP-1 on the membrane and intracellularly,but didn't secrete Hsp90 and LRP-1into DMEM.Since NRL-Hsp90 and S1-LRP-1 II can complement with each other better,we choose pcDNA3.1(+)-S1-LRP-1 II for the next experiment.Peptide KR-9(KPHAEVVLR),LR-6(LYAEER),IL-6(INFEKL),and LN-6(LRDILN),which have been verified in the laboratory in the early stage,have cell migration promoting effects and VK-7(VNAIVFK)can not promote cell migration.Luciferase results indicate that the fluorescence values of the peptide KR-9,LR-6,IL-6,LN-6 are higher than control while VK-7 are lower than control.This indicates that the SRL-PFAC method we constructed is specific and can be used for screening peptides.Next,we screened two peptides RE-7(RYPILPE)and ES-9(EAGREVVGS)using the SRL-PFAC method.Transwell results show that these two peptides can indeed promote the migration of HSF cells effectively.MTS results show that neither of two peptides can promote the proliferation of HSF cells,but they have no toxic effect.