Probing Bim's Regulation Of Hsp70 Chaperone Function By Small Molecules

Protein homeostasis is necessary for cells to perform basic cellular functions normally.Molecular chaperone proteins are important regulators of protein homeostasis.The 70 k Da heat shock protein(Hsp70)is one of the molecular chaperones ubiquitous in cells.From prokaryotes to eukaryotes,Hsp70 plays an important role in the regulation of a wide range of basic cell functions,such as the folding of newly synthesized proteins,the entry of peptides into mitochondria,chloroplasts and endoplasmic reticulum,the decomposition of protein complexes and the regulation of protein activity.The molecular chaperone function of Hsp70 requires the regulation of co-chaperone proteins to complete.Hsp70 has many co-chaperone,including J proteins family and NEFs family.Our group reported for the first time that the BH3-only protein Bim is a co-chaperone protein of Hsp70.Bim binds to Hsp70 through the BH3 domain,stabilizes the ATP conformation of Hsp70,and improves the ATPase activity of Hsp70.But for a long time,Bim has been classified as an apoptosis activator: on the one hand,Bim can directly activate the Bcl-2 pro-apoptotic member Bax/Bak,and on the other hand,it can also antagonize the Bcl-2/Mcl-1 protein that resists apoptosis.Therefore,it is difficult to study the mechanism of Bim regulating Hsp70 in mammalian cells.There is no Bcl-2 family homologous protein in Saccharomyces cerevisiae.Bim can be expressed exogenously in yeast and maintain its activity without achieving its pro-apoptotic function.Therefore,Saccharomyces cerevisiae is a simple model that is very suitable for studying the pro-survival function of Bim.In addition,small molecules have the advantages of simple structure,clear function,and single target.They are ideal tools for studying protein functions.The first Hsp70/Bim small molecule inhibitor S1g-6(2-(6-cyclohexylthio-2-cyano-1-oxy-1H-phenalenylamino)ethyl Morpholine),which can directly and specifically bind to the binding site of Bim on the surface of Hsp70,is a molecular tool for studying the function of Bim as an auxiliary chaperone in regulating Hsp70.This paper firstly expressed and purified recombinant Hsc70 and J protein to study the regulation of Hsp70 by Bim in vitro.Through enzyme activity experiments and substrate thermal denaturation experiments,it was proved that Bim positively regulates the molecular chaperone function of Hsp70 in vitro.The ATPase activity of Hsp70 plays a co-chaperone function.Afterwards,the Bim-transformed yeast strain was treated with S1g-6,and the growth curve of the yeast was detected.From the yeast growth phenotype,it was preliminarily proved that the transfer of foreign protein Bim promoted the growth of yeast,while S1g-6 inhibited the growth of yeast.Co-immunoprecipitation of Hsp70,Western blot detection of the protein-protein interaction between Hsp70 and Bim proved that Bim promotes the growth of yeast by binding to Hsp70 in yeast,while S1g-6 inhibits the growth of yeast by dissociating this interaction.Under heat stress treatment conditions,the protein-protein interaction between Hsp70 and substrate protein was detected by detecting the yeast growth curve and Western blot,which proved that Bim improved the heat resistance and heat stress recovery ability of yeast.It is confirmed that Bim plays a positive co-chaperone function in yeast,promotes the molecular chaperone function of Hsp70,and demonstrates that Bim has a pro-survival function in addition to its pro-apoptotic function.In addition,through TMT technology(tandem mass spectrometry tag technology)to identify a series of Bim-regulated Hsp70 interacting proteins and S1g-6 dissociation of Hsp70/Bim dimers of Hsp70 interacting proteins,and the differential proteins are enriched in GO.Set analysis further confirmed that Bim promotes the growth of yeast by positively regulating the function of Hsp70 in yeast,revealing the survival-promoting function of Bim to a certain extent.In summary,this thesis proves that a certain concentration of Bim can positively regulate the function of Hsp70 molecular chaperones under different temperature conditions in vitro,and proves the pro-survival function of Bim in yeast cells without interference from Bcl-2family proteins.It provides a research direction for the further study of Bim,a new Hsp70co-chaperone protein,in mammalian cells.