The tumor suppressor protein p53 is a crucial transcription factor that regulates physiological processes, including apoptosis, cell cycle progression, DNA damage stress and repair, and programmed cell death. The activity of the intracellular p53 protein is tightly regulated by MDM2 and MDMX proteins. Despite significant advances in studying the interactions among p53, MDM2, and MDMX, obtaining in situ information about these interactions in living cells remains highly challenging. In this study, we proposed a new method for investigating the interaction of p53, MDM2, and MDMX in living cells by combining fluorescence triple correlation spectroscopy (FTCS) with a protein fusion labeling technique. p53, MDM2, and MDMX were fluorescently labeled by gene engineering techniques, and FTCS was used to detect triple-labeled complexes. We achieved the first in situ observation of the formation of the p53-MDM2-MDMX ternary complex in living cells and quantified the concentration distribution of the protein complex in different cellular regions. An investigation was conducted into the influences of the MDMX structure on the formation of the p53-MDM2-MDMX ternary complex. We found that the core RING domain of MDMX significantly impacts the stability of the ternary complex. Furthermore, we investigated the dissociation kinetics of the p53-MDM2-MDMX ternary complex in the presence of p53 inhibitors and developed a novel method for evaluating the inhibitor efficacy within living cells. We confirmed that the potent inhibitor RO-5963 disrupts complex stability by driving an extremely fast dissociation rate constant. Our approach is of significant importance for elucidating the mechanisms and progression of tumorigenesis.
Lu et al. (Sun,) studied this question.