| Apoptosis is a type of programmed cell death.The Bcl-2 family protein plays an important role in the intrinsic apoptosis pathway as apical regulator.Anti-apoptotic members in Bcl-2 family proteins interact with pro-apoptotic proteins via the BH3 motif,preventing pro-apoptotic proteins from altering the permeability of mitochondrial outer membrane(MOMP)to induce apoptosis.Overexpression of Anti-apoptotic proteins always correlates with development and progression of cancer.Many studies have shown that in addition to 16 classical Bcl-2 family proteins,there are many unknown non-Bcl-2 BH3 receptor containing BH3 groove in the organism,which can interact with Bcl-2 family proteins to affect apoptosis.The discovery of these proteins and revealing new mechanisms by which they regulate apoptosis in their pathways will provide new cancer targets for anti-tumor therapy and provide a basis for precise treatment.However,the BH3 motif-mediated protein-protein interaction(PPI)usually has the characteristics of weak binding,transient binding,and reversibility.Traditional biological methods such as co-immunoprecipitation are difficult to find new non-Bcl-2 BH3 receptor.In recent years,chemical proteomics technology has developed rapidly.The most widely used is the activity-based probes(ABPs).The core of ABPs is to design an active molecular probe that can covalently react with the active amino acid residues of proteins.The probe uses a protein inhibitor as the core of the structure,and on the basis of retaining the ability to inhibit the target protein,the function of in-situ recognition-capture inhibitor target protein is realized by photocrosslinking reaction,and is suitable for searching for new a class of Bcl-2 family proteins.The Bcl-2 protein inhibitor,a BH3 functional mimetic molecule,induces apoptosis by absorbing the BH3 groove of the target protein by mimicking the function of the BH3 motif.Therefore,ABPs constructed by Bcl-2 inhibitors are the best tools for capturing new BH3 receptor proteins.In this paper,a series of Bcl-2 inhibitor S1 derivatives have been obtained,which are linked to a "click chemistry" group and a photocrosslinking reaction group,thereby constructing a molecular library of ABPs.Screening of active probe molecules that interfere with the apoptotic phenotype in a variety of tumor cells,and in situ capture of a novel non-Bcl-2 BH3 receptor,Hsp70,at the proteome level.Discovered and demonstrated the new function of HSP70 and BH3-only protein Bim heterodimer.The new function reveals that Hsp70/Bim heterodimer plays an important role in regulating tumor cell apoptosis.In a variety of leukemia cell lines such as CML and AML,and solid tumor cell lines such as breast cancer and cervical cancer,screening of 10 S1 derivatives of ABPs based on apoptosis-inducing activity revealed that S1b-probe can be ten times higher than other derivatives(CML IC50 = 1~2 ?M;U937 etc IC50=10~20 ?M).The target protein captured,enriched and identified by ABPs technology is Hsp70.Hsp70 interfering RNA experiments further demonstrated that Hsp70 is a target for S1b-induced apoptosis.The Hsp70 and BH3 peptides were tested to interact with Kd=51-116 n M in vitro by FP detection and ITC experiments in vitro;using co-immunoprecipitation(Co-IP)assay in multiple CML cell lines The presence of Hsp70 and Bim heterodimer,Co-IP experiments with BH3 peptide competition confirmed that the interaction between Hsp70 and Bim was mediated through the BH3 motif of Bim.Further protein two-dimensional nuclear magnetic proof Bim BH3 motif binds to the nucleotide binding domain of Hsp70 protein;combined with immunobulation and other cell biology experiments,it is proved for the first time that Hsp70 is a new class of BH3 receptor protein.At the same time,Bim was first discovered as a function of Hsp70 co-chaperone.In vitro purified Hsp70 trypsin proteolysis,single-transform ATPase catalytic rate assay and other experiments proved that Bim can promote the ATPase activity of Hsp70 protein.After knocking down Bim by interfering RNA technology in CML cell line,the ATPase activity of Hsp70 under anti-Hsp70 antibody immunoprecipitation decreased,indicating that Bim is an auxiliary molecular chaperone that positively regulates Hsp70 function.Further,in the Bim knockdown CML cell line,we demonstrated that the levels of Hsp70 substrate proteins AKT and Raf that promote tumor survival are down-regulated,indicating that Hsp70/Bim dimer exerts anti-apoptotic function by increasing the stability of tumor oncogenic protein..In summary,by using ABPP and other methods,we discovered that Hsp70 is a new class of Bcl-2 protein for the first time,and uncovered the binding mode of Hsp70 and Bim,and reveals the molecular mechanism of Bim as a co-chaperone protein of Hsp70 involved in protein folding.It provides new targets and mechanisms for cancer therapy... |
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