A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes

Eustache, Stephanie; Créchet, Jean‐Bernard; Bouceba, Tahar; Nakayama, Jun‐ichi; Tanaka, Mayo; Suzuki, Mieko; Woisard, Anne; Tufféry, Pierre; Baouz, Soria; Hountondji, Codjo

doi:10.2174/1874091x01711010008

Cited by 7 publications

(5 citation statements)

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“…In the ribosome, RPL36AL contacts both the CCA end of P-site bound tRNA and the translation termination factor eRF1 (Baouz et al, 2009;Hountondji et al, 2014). Most interestingly, RPL36A carries 7 and RPL36AL 6 monomethylated residues, among them Lys38, the one variable amino acid between the paralogs (Eustache et al, 2017), and the contact with the tRNA and eRF1 relies on the methylation of Lys35 (Eustache et al, 2017;Hountondji et al, 2012). In addition, there is evidence for fractional methylation of RPL36A(L) at positions Gln51 and Lys53, part of a highly conserved motif, which gave rise to speculations that such an extensive methylation pattern would likely be used for the regulation of translation or ribosome activity (Hountondji et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Ribosomal RNA 2′-O-methylation dynamics impact cell fate decisions

et al. 2023

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Section: Discussionmentioning

confidence: 99%

Ribosomal RNA 2′-O-methylation dynamics impact cell fate decisions

et al. 2023

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“…Since the resonance unit (RU) that is the reflect of the refractive index is much greater for the analyte with several sites of interaction on the ligand leading to a large exchange surface between the two partners, the K D values defining the affinity between eL42 and translation factors or tRNA or rRNA are expected to be much lower (in the pM or nM range) [23] than that of small molecules. In accordance with this observation, the only probable sites of interaction between UCK-36 and the rP eL42 are the NH 2 end of the Lys-53 residue and the thio carbonyl (C= S) function of the thiosemicarbazone (UCK-36).…”

Section: Discussionmentioning

confidence: 99%

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2017

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Background: Thiosemicarbazones are a family known as excellent chelators of enzyme cofactors or molecules with multiple biological activities including antitumor activity [1,2]. Recent studies by Hountondji's team has revealed that the large subunit ribosomal protein eL42 (molecular weight of 12 kDa, pHi: 10,59) plays a functional role during the elongation step of translation [3] and is over expressed in most cancers [4][5][6]. Method:The method used was the one developed by Hountondji et al. [7] which converts the OH functional groups at the positions 2' and 3' of adenine sugar of the CCA tRNA into a dialdehyde group by oxidation with NaIO 4 . Each aldehyde function can form a Schiff base with the amino group of the side chain of K53 residue of eL42, which results in a covalent bond after reduction with NaBH 4 (crosslinking). Results and Discussion:The results of different experiments (the radioactivity, and the Western Blot technique) performed in this study show that 4 molecules (citral-4-phenylthiosemicarbazone, piperitone-4-phenylthiosemicarbazone, the benzoin-4-phenylthiosemicarbazone and 4-chlorobenzophenone-4-phenylthiosemicarbazone) out of 29 thiosemicarbazones tested showed activity by inhibiting the crosslinking reaction between tRNAox and eL42. These results suggest that the inhibition produced by thiosemicarbazones consist in masking the amino group of the side chain of Lys-53 located in the catalytic site of protein eL42, preventing Schiff base formation and the crosslinking reaction. These results are in accordance with those of Schneider-Poetsch et al. [8] who found that certain compounds for example cycloheximide bind to the eL42 protein through their carbonyl function. The weaker effects of benzoin-4-phenylthiosemicarbazone and 4-chlorobenzophenone-4-phenylthiosemicarbazone as compared with citral-4-phenylthiosemicarbazone and piperitone-4-phenylthiosemicarbazone are likely to reflect the inaccessibility of the NH 2 of Lys-53 of eL42 to the thiocarbonyl groups (C=S) of these thiosemicarbazones because of steric hindrance. Finally, binding assays on Biacore 3000 confirmed interactions between active molecules and the human eL42 protein. Conclusion:The inhibitory action of thiosemicarbazones on the interaction between the protein eL42 and tRNAox prevents formation of a Schiff base between the ε-amino group of Lys-53 and an aldehyde group of tRNAox. The Lys-53 residue of eL42 which contributes to the catalysis of peptide bond formation that represents the key step in the biosynthesis of proteins is a target of choice for small molecule inhibitors. Therefore, thiosemicarbazones can be used to reduce the rate of protein synthesis in order to block the hyper-proliferation of tumor cells (Figure 1).

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“…The surface plasmon resonance (SPR) biosensor experiments were performed on a Biacore 3000 instrument (GE Healthcare) of the Platform of Molecular Interactions of the Institute of Biology Paris Seine (IBPS, Sorbonne University). All experiments were performed in triplicate, as described previously [21,22]. The CM5 sensor chip was used.…”

Section: Surface Plasmon Resonancementioning

confidence: 99%

“…7B). We suspected that rp eL42 interferes with a component of the incubation mixture for the poly(U)-dependent poly(Phe) synthesis activity such as the tRNA substrate, because we had previously demonstrated that the interactions between eL42 and the tRNA molecules are characterized by strong binding affinities (in the nanomolar range) [22]. If this is the case, then the decrease in the poly(U)-dependent poly(Phe) synthesis activity would reflect the sequestration of the substrate [ 14 C]Phe-tRNA Phe that would prevent the poly(U)-directed [ 14 C]Phe incorporation into the poly(Phe) chain.…”

Section: Other Putative Roles For the Overexpression Of El42 In Cancementioning

confidence: 99%

In Vitro Analysis of Protein:Protein Interactions in the Human Cancer-Pertinent rp.eL42-p53-Mdm2 Pathway

Aguida¹,

Bouceba²,

Créchet³

et al. 2019

TOBIOCJ

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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.

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A Functional Role for the Monomethylated Gln-51 and Lys-53 Residues of the 49GGQTK53 Motif of eL42 from Human 80S Ribosomes

Cited by 7 publications

References 41 publications

Ribosomal RNA 2′-O-methylation dynamics impact cell fate decisions

Ribosomal RNA 2′-O-methylation dynamics impact cell fate decisions

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In Vitro Analysis of Protein:Protein Interactions in the Human Cancer-Pertinent rp.eL42-p53-Mdm2 Pathway

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