Misincorporations at Artificially Constructed Termination Stop Codons in the Presence of the Amino Glycoside Gentamicin

ABSTRACT: Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate the incorporation of amino acids at the stop codon and the production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. We report on the development of a human cell model that can be used to determine whether rules can be developed using mass spectrometry that defines the type of amino acid that is placed at a premature termination codon during readthrough mediated by an aminoglycoside. The first premature termination codon we analysed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Although we could detect a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered so we needed to create a more robust artificial premature stop codon model. P53 expression plasmids were developed that incorporate a string of single synthetic UGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 122-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-UGA129 mutation, treated with Gentamycin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-UGA129; as expected based on the two base pairing of the respective anticodons to UGA with R being the most abundant using all three codons. By contrast, incorporating the amber or ochre premature stop codons, UAA129 or UAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. The incorporation of these amino acids at codons 127, 128, or 129 generally results in a p53 protein that is predicted to be ‘unfolded’ or inactive as defined by Molecular Dynamic Simulations. As such, the data also highlight the need in the future to not only produce novel small molecules that can read through premature termination codons but also the need to design methods to insert the required amino acid at the position that could result in a ‘wild-type’ functional protein.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Lenka Hernychova  

LAB HEAD: Prof. Ted Hupp

PROVIDER: PXD039307 | Pride | 2024-10-29

REPOSITORIES: pride