Research Highlight | Ubigene’s gene knockout cell line helps uncover the molecular mechanism of p53-BCL-2 interaction
Tumor suppressor gene TP53 is the most frequently mutated gene in the process of tumorigenesis, and TP53 gene mutations are detected in more than 50% of human tumors. P53 protein is activated under cellular stress conditions such as DNA damage, and regulates a variety of cellular processes, including cell cycle arrest, inhibition of cell proliferation, and promotion of apoptosis. P53 and B-cell lymphoma-2 (BCL-2) protein family play a crucial role in mediating apoptosis. P53 can not only regulate the expression of apoptosis related genes as a transcription factor, but also play a transcription independent role in regulating mitochondrial apoptosis through direct interaction with BCL-2 family proteins. However, at this stage, little is known about the molecular mechanism of the interaction between p53 and BCL-2 family proteins.
Recently, Yongheng Chen’s research group from Xiangya Hospital, Central South University published an original research paper entitled "Structures of p53/BCL-2 complex suggest a mechanism for p53 to antagonize BCL-2 activity" on Nature Communications (IF: 16.6). In this study, the HCT116 cell line with TP53 gene knocked out constructed by Ubigene was used. By analyzing the crystal structure of the complex of p53 and the anti-apoptotic protein BCL-2, and combining biochemical and cellular experiments, the molecular mechanism of how p53 and BCL-2 proteins regulate apoptosis through interaction was deeply elucidated .
In this study, the authors first used X-ray diffraction to obtain the crystal structure of p53 and BCL-2 complex with a resolution of 2.5 Å. The complex structure revealed that the two flexible loops of p53 were directly bound to the BH3 hydrophobic binding pocket on the surface of BCL-2 protein (Figure 1). Asp186, Leu188 and Leu201 of p53 protein located at the interaction interface and Phe104, Arg107, Tyr108 and Arg146 of BCL-2 protein played key roles in protein-protein interaction. GST pull-down and MST experiments related to mutations also verified this conclusion.
Figure 1. Structure of p53 and anti-apoptotic protein BCL-2 complex
Further structural analysis revealed that this BH3 hydrophobic binding pocket of p53 on the surface of BCL-2 is the main region of pro-apoptotic BCL-2 family members (such as the pro apoptotic protein Bax), and also the targeting position of clinical targeted drugs (such as Venetoclax). In order to further study how p53/BCL-2 interaction regulates apoptosis, the authors then verified that p53 can competitively bind BCL-2 with Bax and other pro-apoptotic proteins through fluorescence polarization experiments and microscale thermophoresis experiments, so that Bax and other pro-apoptotic proteins can be released to promote mitochondrial outer membrane permeabilization (MOMP) (Figure 2). In addition, the authors designed a p53 mutant (Ti-p53RRR) with defective interaction with BCL-2 on the basis of transcription defective p53 (Ti-p53). Flow cytometry experiments showed that p53-/-HCT116 cells transfected with Ti-p53RRR had significantly reduced apoptosis compared with Ti-p53 (Figure 3).
Figure 2. p53 competitively inhibits the interaction of BCL-2 with pro-apoptotic BCL-2 family proteins
Figure 3. Mutations in p53 interacting with BCL-2 reduce apoptosis
In summary, this study revealed a new mechanism of p53 interacting with BCL-2 protein and promoting apoptosis, that is, p53 forms a complex with BCL-2 by directly occupying the BH3-binding pocket of BCL-2, and antagonizes BCL-2 activity by releasing pro-apoptotic BCL-2 family proteins located in the pocket, thereby promoting apoptosis. These structural and functional data provide a new idea for further understanding the complex regulatory mechanism of p53-mediated mitochondrial apoptosis, and also provide an important basis for developing anticancer therapeutic strategies that target protein-protein interactions to activate apoptosis.
 Wei H, Wang H, Wang G, Qu L, Jiang L, Dai S, Chen X, Zhang Y, Chen Z, Li Y, Guo M, Chen Y. Structures of p53/BCL-2 complex suggest a mechanism for p53 to antagonize BCL-2 activity. Nat Commun. 2023 Jul 18;14(1):4300. doi: 10.1038/s41467-023-40087-2.