The Mechanism Research Of Hsp70/Bim Heterodimer Regulating Autophagy By Using Small Molecules

Autophagy is an evolutionary mechanism of conservative and a process of precise coordination.In cancer,autophagy plays a dual role,which inhibit tumor initiation but support progress ——On the one hand,autophagy can increase the tumor cells in adverse environment to stress tolerance ability,including the tumorigenic environmental stresses and anti-cancer drug therapy to maintain its survival;On the other hand,autophagy can inhibit tumor production and metastasis at different stages of tumor development,and even become the death path of some apoptotic defective tumor cells.Although there is increasing clinical evidence that targeted autophagy can improve the efficacy of cancer therapy and inhibit tumor expansion alone or in combination,there is still a need to focus on selective tumor suppression of autophagy regulatory targets and corresponding specific autophagy regulatory small molecules.In 2020,our research group reported that heat shock protein 70(Hsp70)is a new bcl-2like protein,and found that Hsp70 has protein-protein interaction with BH3-only protein Bim,Moreover,Hsp70/Bim was an atopic tumor target of CML.Our research group also developed the first small molecule S1g-2 that specifically disrupt HSP70-Bim PPI.It was further found that the mortality rate of disrupt Hsp70/Bim dimer was higher in BCR-ABL TKI-independent patients than in TKI-sensitive patients,and gradually increased with the increase of BCR-ABL TKI-independent resistance.Interestingly,Hsp A1 L was found by si RNA regulation of Parkin translocus(Hsp70 member)may increase mitochondrial quality control,which is closely related to autophagy,Parkin and TOMM20 are also found in the S1g-2 influenced Hsp70 protein interaction proteome.Therefore,this paper uses the small molecule S1g-2,which specifically disrupts HSP70-Bim PPI,as a chemical tool,along with its analogue S1,which has been shown to specifically disrupt Bcl-2/Beclin-1 dimer.Based on vivo and vitro studies,we found the new function of Hsp70 and BH3-only protein Bim,revealing that HSP70-BIM PPI protects cells through mitochondrial autophagy and apoptosis,acting as a dual function “lifeguard”.Therefore,S1g-2 represents a new mechanism-based antitumor drug candidate that can guide autophagy and apoptosis.By detecting the expression levels of autophagy proteins under different conditions,it was found that S1 enhanced stress-induced autophagy and S1g-2 inhibited stress-induced autophagy in the absence of apoptosis induced interference of autophagy.Using chemical proteomics,the autophagy regulatory targets of S1g-2 were identified as Hsp70 protein,and the autophagy regulatory targets of S1 were Bcl-2 and Mc L-1.By simultaneously detecting the expression level of autophagy protein and the content of Hsp70/Bim interacting protein,it was found that the increase of autophagy level was mediated by Hsp70/Bim dimer By acridine orange staining and interfering RNA,it was further proved that S1g-2 inhibited HBSS induced autophagy through the dissociation of Hsp70/Bim dimer.By comparing S1g-2with the lysosomal inhibitor buffiamycin A1(Baf A1),it was found that HSP70-BIM PPI affected induced autophagy,but did not affect basic autophagy Respectively through the test in the mitochondria,endoplasmic reticulum and golgi apparatus organelles autophagy protein levels of proteins specifically expressed organelles detection and the level of autophagy protein LC3 positioning with mitochondria,found that Hsp70 / Bim dimers specificity to adjust mitochondrial autophagy,and the endoplasmic reticulum and golgi body autophagy level has no effect,is a selective autophagy targets.Through cell studies and in vitro ubiquitination analysis of stress-induced Hsp70 and Bim Parkin TOMM20 interaction proteins and TOMM20 ubiquitination levels,it was found that HSP70-BIM PPI disrupted Parkin and TOMM20 complex,which in turn promoted Parkin transfer to mitochondria By Bax/Bak double knockout(DKO)mouse fibroblast(MEF)assay and stable transfection of tetracycline induced Bim expression,the mechanism of ubiquitination of TOMM20 and promoting mitochondrial autophagy was found to be independent of Bax/Bak By detecting AMPK phosphorylation of S1g-2,we found that S1g-2selectively inhibited stress-induced autophagy without interfering with the AMPK pathway,suggesting that the mechanism was independent of energy.These findings suggest that HSP70-BIM PPI protects apoptosis through mitochondrial autophagy,acting as a double-function lifeguard.In summary,these results suggest that Hsp70-Bim PPI mediates mitochondrial autophagy,and S1g-2 inhibits autophagy activity by disrupting Hsp70-Bim PPI,suggesting that S1g-2 is the study of Hsp70-Bim PPI provides an ideal tool for its unknown molecular role in autophagy and an anticancer target based on autophagy.