Native PAGE to study the interaction between the oncosuppressor p53 and its protein ligands

Abstract

In the present study, we investigated a new approach for studying the interaction between p53 and MDM2/X (where MDM is murine double minute protein). The method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and can be useful for analyzing the dissociation power exerted by other molecules on the p53-MDM2/X complex.

Optical Biosensors Based on Photonic Crystals Supporting Bound States in the Continuum

“…Absolute Ethanol, H 2 O 2 , and NaOH were purchased from Carlo Erba (Cornaredo, Italy), HEPES powder was purchased from Promega (Madison, WI, USA). The MDM2 and p53 protein fragments used in this work were heterologously expressed in Escherichia coli and characterized as previously reported [ 36 , 44 ]. Piranha solution was used according to the safety guidelines of University of Chicago Environmental Health and Safety (Chicago, IL, USA).…”

“…This modification is mainly ruled by the oncoprotein MDM2, which is the principal cellular antagonist of p53. Generally, this kind of fundamental interaction is investigated by using different methods, including NMR technology [ 33 , 34 ] and fluorescence polarization [ 35 ] (all techniques requiring complex tagging/derivatization procedures), and more recently electrophoresis [ 36 ]. In contrast to these techniques, our approach potentially allows the real-time recognition and label-free identification of the proteins’ interaction at nanomolar concentration, since the very intense optical field at the surface and the minimal loss of our transparent device make these PhC structures extremely sensitive to the external environment perturbation.…”

MDM2 oligomers: antagonizers of the guardian of the genome

“…24 The MDM2 p53-binding domain actually displays a three- to four-fold greater affinity for p53 than that of MDMX, which suggests that the presence of the MDM2 p53-binding domain in the Mdm2 C462A/C462A mice offers an advantage in transcriptional suppression over that of the MDMX p53-binding domain, which is also present in these mice. Physical MDM2 C462A –p53 interaction could contribute to the delay in death of mice that express the MDM2 p53-binding domain compared with mice that do not (for example, Mdm2 C462A/C462A mice vs MdmX knockout mice 103,104 ), although further experiments will be required for confirmation. Moreover, although it is possible that MDM2 C462A protein retains the ability to suppress p53 through direct binding of the p53 transactivation domain, whether the homooligomerization capacity of MDM2 is required remains to be determined in vivo .…”

Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds

“…Several methodological approaches have been proposed to study the dissociation of the p53·MDM2/X complex exerted by molecules with potential pharmacological activity, including NMR technology , surface plasmon resonance , and fluorescence polarization . We have recently proposed an electrophoretic method to study the interaction between p53 and its protein ligands , taking advantage from the different electrophoretic mobility displayed by p53 and MDM2/X. Here, we used this method to analyze the dissociative potency exerted by Nutlin‐3a, Nutlin‐3b, and RO‐5963, well known inhibitors of the p53·MDM2/X complex formation .…”

“…Stock solutions of the inhibitors were prepared in DMSO at 100 mM final concentration. The production of p53, MDM2, and MDMX protein fragments used in this work, the electrophoresis in nondenaturing conditions, and the densitometric analysis of stained protein bands were carried out as reported . Complex formation was attained by incubating protein fragments at a 1:1 molar ratio in the 8–40 μM range, for 30 min at 37°C.…”