In Vitro Analysis of Protein:Protein Interactions in the Human Cancer-Pertinent rp.eL42-p53-Mdm2 Pathway
Blanche Aguida1, 2, Tahar Bouceba3, Jean-Bernard Créchet1, Horrhus Hounguè1, 4, Callinice Capo-Chichi2, Jun-ichi Nakayama5, Soria Baouz1, Hélène Pelczar1, Anne Woisard1, Nathalie Jourdan6, *, Codjo Hountondji1, *
Identifiers and Pagination:Year: 2019
First Page: 64
Last Page: 80
Publisher ID: TOBIOCJ-13-64
Article History:Received Date: 30/05/2019
Revision Received Date: 15/09/2019
Acceptance Date: 17/09/2019
Electronic publication date: 30/09/2019
Collection year: 2019
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
We have recently demonstrated that the eukaryote-specific large subunit ribosomal protein (rp) eL42 assists catalysis of peptide bond formation at the peptidyl transferase center of 80S ribosomes in eukaryotic cells. Recently, several ribosomal proteins were shown to have extraribosomal functions independent of protein biosynthesis. Such functions include regulation of apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both Mdm2-p53-dependent and p53-independent mechanisms. Our objective is to demonstrate that overexpression of eL42 in tumor may incapacitate cell anti-tumor mechanism through interaction with the tumor suppressor protein p53 and its partner Mdm2.
Co-immunoprecipitation technique and the binding assays on Biacore were used to probe interactions between recombinant eL42, p53 and Mdm2 proteins in a so-called rp-p53-Mdm2 axis.
We demonstrate that the ribosomal protein eL42, the tumor suppressor protein p53 and the ubiquitin E3 ligase Mdm2 interact with each other in a ternary rp.eL42:p53:Mdm2 complex. Precisely, the interaction between eL42 and p53 is characterized by a strong binding affinity (KD value in the nanomolar range) that is likely to trigger the sequestration of p53 and the inhibition of its tumor suppressor activity. Furthermore, the p53:Mdm2 and eL42:Mdm2 complexes exhibit comparable binding affinities in the micromolar range compatible with Mdm2 being the enzyme which ubiquitinates both the p53 and eL42 substrates. Interestingly, pyridoxal 5'-phosphate (PLP), one of the active forms of vitamin B6, binds to eL42 and significantly inhibits the interaction between eL42 and p53, in accordance with the observation that vitamin B6 is associated with reduced risk of cancer.
Our study emphasized one more major mechanism of p53 downregulation involving its sequestration by eL42 upon the overexpression of this ribosomal protein. The mechanism described in the present report complemented the well-known p53 downregulation triggered by proteasomal degradation mediated through its ubiquitination by Mdm2.