Abstract
Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.
Highlights
We have recently demonstrated that the eukaryote-specific large subunit ribosomal protein eL42 assists catalysis of peptide bond formation at the peptidyl transferase center of 80S ribosomes in eukaryotic cells
The ribosomal protein eL42 presents the following characteristics: (i) it was recently shown to directly and actively contribute to the activity of 80S ribosomes at the elongation step of translation [13], suggesting that this rp might control the rate of protein biosynthesis in health and in disease; (ii) it was found to be overexpressed in human hepatocellular carcinoma as well as in several human tumor cell-lines, suggesting that its extraribosomal role might be related to tumor cell proliferation [14]; (iii) in the crystallographic structure of S. cerevisiae 80S ribosomes or of the 50S subunit of Haloarcula marismortui, most of the anticancer drugs were shown to target the eL42 protein [15 - 19]
We have explored the potential of the large subunit ribosomal protein eL42 as a therapeutic target in the human cancer-pertinent rp.eL42-p53-Mdm2 pathway
Summary
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. The ribosomal protein eL42 (rp eL42) presents the following characteristics: (i) it was recently shown to directly and actively contribute to the activity of 80S ribosomes at the elongation step of translation [13], suggesting that this rp might control the rate of protein biosynthesis in health and in disease; (ii) it was found to be overexpressed in human hepatocellular carcinoma as well as in several human tumor cell-lines, suggesting that its extraribosomal role might be related to tumor cell proliferation [14]; (iii) in the crystallographic structure of S. cerevisiae 80S ribosomes or of the 50S subunit of Haloarcula marismortui, most of the anticancer drugs were shown to target the eL42 protein [15 - 19] These observations point out a connection between protein synthesis on the ribosome and cancer cells growth.
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