| Cell apoptosis,also known as programmed cell death.The Bcl-2 family proteins regulate the permeability of outer membrane of mitochondria through protein-protein interactions?PPI?,regulate cell apoptosis,and play a significant role in the development of tumors.Therefore,anti-apoptotic Bcl-2 protein is considered as an important target protein for cancer.In April2016,ABT-199 became the first bcl-2 inhibitor anticancer drug approved by the FDA.However,phosphorylation modification triggers changes in the conformation of Bcl-2family proteins and remodels the PPI network.So,the phosphorylated Bcl-2 protein is considered as a new target for cancer treatment.For example,phosphorylation modification of Bcl-2 protein in leukemia cells prevents Bcl-2 inhibitors from directly binding with Bcl-2protein and inhibiting Bcl-2 anti-apoptosis function and therefore becomes resistant to Bcl-2inhibitors such as ABT-199,Obatoclax,and S1 in clinical trials.The antagonize small molecules of phosphorylated Bcl-2 proteins are potential anticancer drugs.So far,the lack of studies on the structure of phosphorylated Bcl-2 proteins is a bottleneck for targeting phosphorylated Bcl-2 proteins to solve the research and development of drug-resistant small molecule drugs.In the structural studies of Bcl-2 protein,the loop region of Bcl-2 protein was replaced by the part of the loop region of Bcl-xl protein,a protein with higher solubility and stable structure.However,the loop region contains the phosphorylation site of the Bcl-2 protein.By analyzing the structure of phosphorylated Bcl-2 protein,we can accurately reveal the significance of Bcl-2 protein phosphorylation modification,which is of great value for drug discovery and pathological process of tumor development.In this paper,we constructed a simulated phosphorylated EEE-Bcl-2-EK?62-206??T69E,S70E,S87E?mutant with a loop region.Through three-dimensional nuclear magnetic resonance and fluorescence polarization experiments,we firstly discovered that the phosphorylation site pT69/pS70 in the loop region and the active site of the Bcl-2 protein drove the conformational changes of the BH3 binding grooves.At the same time,we demonstrated that phosphorylated Bcl-2 proteins enhanced their anti-apoptotic function by transfection Bcl-2 protein phosphorylation modified mutant physique particles and other cell biological studies.Based on the structure of phosphorylated Bcl-2 protein,our group designed an allosteric regulator S1-6e targeting Bcl-2 protein by installing a carboxylic acid group to phosphorylated Bcl-2 inhibitor S1-6 to intervene protein phosphorylation modifications.It was confirmed that S1-6e binds to the BH3 groove and carboxylic acid group can interact with R106/R109 by nuclear magnetic resonance and fluorescence polarization experiments.To sum up,S1-6e binds to Bcl-2 protein and disrupts the interaction network between the phosphorylation site and R106/R109,promoting the dephosphorylation of Bcl-2 protein.Therefore,the bifunctional s1-6e exhibits stronger killing activity in tumor cells compared with the traditional Bcl-2 inhibitors.In summary,for the first time this study found that the loop region of phosphorylated Bcl-2 protein regulates the conformation of active grooves BH3 grooves and the anti-apoptotic function of Bcl-2 protein,providing a structural basis for explaining the resistance of the Bcl-2 inhibitors.By analyzing the molecular basis of phosphorylated Bcl-2protein,we designed the allosteric modulator S1-6e targeting Bcl-2 protein,and clarified the binding mode of S1-6e and Bcl-2 protein,which further proved that Bcl-2 phosphorylation modifications intervenes in Bcl-2 phosphorylation modifications by influencing the allosteric regulation.The allosteric regulation and chemical intervention of Bcl-2 protein phosphorylation modification provide a scientific basis for us to understand the significance of protein phosphorylation,the identification of new anti-tumor targets,and new anti-tumor treatment strategies. |
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