Molecular and Biochemical Techniques for Deciphering p53-MDM2 Regulatory Mechanisms

Abstract

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

Keywords: ATM; DNA damage response; MDM2; MDMX; p53; p53 mRNA; post-translational modification; protein-RNA interactions; protein-protein interactions.

Figure 1

Illustrations of novel techniques used to detect interactions in the p53 pathway. (a) Fluorescence cross-correlation spectroscopy (FCS) used to study the p53-MDM2 interaction in living cells. FCS uses proteins that are labelled independently with different fluorescent probes. Analysis of the FCS data examines minute fluctuations in fluorescence intensity induced by a low number of diffusing labelled molecules, caused by spontaneous deviations from the mean in thermal equilibrium in a confocal configuration [68]. (b) Proximity ligation assay (PLA), used to study the interaction of the p53 mRNA in the RING domain of HDM2 and the formation of the N-terminal HDMX-HDM2 heterodimer. This technique consists of two primary antibodies generated in different species that recognize the proteins of interest and two secondary antibodies that carry oligonucleotide sequences that are ligated and amplified, allowing detection by fluorophores [14]. (c) Proximity ligation ELISA (PLE) set to investigate the trimeric interaction among MDM2 and the nascent p53 peptide on isolated p53 polysomes (via RPL5 or RPL11). PLE involves three primary antibodies. One capture antibody (i.e., from goat), targeting one binding factor and a set of primary antibodies (from mouse and rabbit), targeting two additional binding factors. Upon capturing the trimeric complex via the capture antibody, recognizing one of the binding partners, signal amplification, and detection is performed following the principles of PLA.

Figure 2

Different techniques have been used to unravel the interaction among of p53-MDM2-MDMX. A phosphorylation on Ser 395 of the MDM2 change its conformation and allow the binding of the p53 mRNA that results in increased p53 protein synthesis after DNA damage. On the other hand, under normal conditions, interaction of MDMX and/or MDM2 with p53 negatively controls the p53 levels. Techniques that allow us to study the interactions in-situ are shown in red.