The Mechanism Research Of Hsp70 And Bcl-2 Family Proteins Synergistic Regulation On Apoptosis

Bcl-2 family proteins regulate apoptosis through BH3 domain-mediated protein-protein interaction(PPI),and anti-apoptotic Bcl-2-like proteins are important targets for many cancer therapies.More and more studies have found that in addition to the known Bcl-2 family Bcl-2-like proteins,there are also some proteins that can also form PPI with BH3-only protein through BH3 domain.The discovery of new Bcl-2-like proteins gradually improves the apoptotic network,which also means that new PPI mediate new apoptotic regulation mechanisms and new cancer targets need to be discovered urgently.In 2020,our group reported that heat shock protein 70(Hsp70)is a new Bcl-2-like protein.Bcl-2 family pro-apoptotic protein Bim is bound to the NBD region of Hsp70 through BH3 groove,and is a new co-chaperone of Hsp70,which helps Hsp70 fold and stabilize the oncogene proteins to protect tumor cells from apoptosis.Hsp70 is a large molecular chaperone that tumor cells and normal cells depend on for survival.It is involved in cancer related multiple signaling pathways to promote the development of tumor,including cell apoptosis,cell senescence and cell autophagy,cellular immunity and so on.But it's just a“non cancer” dependent protein on tumor cells,not as a tumor target.A growing body of evidence suggests that the heat shock family may exert tumor-specific functions through PPI.For example,the PPI of Hsp70-Bag3 has been reported as a possible anti-tumor target of the Hsp70 inhibitor MKT-077 and its analogs in breast cancer cell.Therefore,targeting Hsp70 related complexes has become a new research hotspot in the field of cancer therapy.Previous study of our group found that the binding interface of Hsp70-Bim PPI was similar to that of Bcl-2-Bim PPI.Therefore,we used a Bcl-2 inhibitor S1 as the initial small molecule to synthesize a series of derivatives and establish a molecular library for screening specific dissociation of Hsp70-Bim PPI.In this paper,a small molecule S1 g that can specifically dissociate Hsp70-Bim PPI was identified in vitro by fluorescence polarization experiment and isothermal titration calorimetry,etc.And the specificity of its target was also demonstrated in cells.The results of anti-tumor activity of S1 g in chronic myeloid leukemia(CML)cell lines,multiple solid tumor cell lines and normal cell lines showed that S1 g specifically bound to Hsp70 in CML cells lines,and its binding affinity was positively correlated with its ability to induce apoptosis.We further used S1 g and other known inhibitors of Hsp70 to reveal that eukaryotic promoters(EIFs)and ribosomal proteins(RPSs)are substrate proteins of Hsp70 in CML cells,demonstrating that S1 g reduced the stability of oncogenic substrate proteins by specifically dissociating Hsp70-Bim PPI to induce apoptosis in CML cells.In addition,S1 g showed good anti-tumor activity in xenograft model of CML in nude mice,suggesting that S1 g may be an anti-cancer drug candidate for the treatment of CML.In addition,as a Bcl-2-like protein,Hsp70 can interact with Bim,Bax and Bak through the BH3 domain,which are pro-apoptotic proteins of Bcl-2 family.Therefore,there may be an interaction mechanism between Hsp70 and Bcl-2 family proteins to regulate apoptosis.By using Bcl-2 inhibitors S1 and S1 g,we found that S1 and S1 g could induce endogenous apoptosis synergistically in leukemia cell lines K562,U937 and HL-60.When one of the Hsp70 or Bcl-2-like proteins is inhibited,the pro-apoptotic proteins Bim,Bax and Bak transfer between them,and Bim is an important member of the synergistic anti-apoptotic effect of Hsp70 and Bcl-2-like proteins.The release amount of Bim protein from Hsp70 or Bcl-2-like proteins was positively correlated with the ability to induce apoptosis of S1 or S1 g.In summary,this paper identified a small molecule S1 g that can specifically disrupt Hsp70-Bim PPI,and confirmed that Hsp70-Bim PPI is a specific target of CML,which provids a new tumor targeted therapy strategy for CML.On this basis,we used small molecule inhibitors S1 and S1 g prove that Bim transfer is the synergistic anti-apoptotic mechanism between Hsp70 and Bcl-2 like proteins in leukemia cells.Finally we preliminarily revealed the “crosstalk” of Hsp70 and Bcl-2 family proteins.