Misincorporations of amino acids in p53 in human cells at artificially constructed termination codons in the presence of the aminoglycoside Gentamicin

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61389030%3A_____%2F24%3A00602392" target="_blank" >RIV/61389030:_____/24:00602392 - isvavai.cz</a>

Alternative codes found

RIV/00209805:_____/24:00080046

Result on the web

<a href="https://doi.org/10.3389/fgene.2024.1407375" target="_blank" >https://doi.org/10.3389/fgene.2024.1407375</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fgene.2024.1407375" target="_blank" >10.3389/fgene.2024.1407375</a>

Result language

angličtina

Original language name

Misincorporations of amino acids in p53 in human cells at artificially constructed termination codons in the presence of the aminoglycoside Gentamicin

Original language description

Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate incorporation of amino acids at the stop codon and production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. Herein, we report on the development a human cell model that can be used to determine whether rules can be developed using mass spectrometry that define the type of amino acid that is placed at a premature termination codon (PTC) during readthrough mediated by an aminoglycoside. The first PTC we analyzed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Despite of identifying 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, hence we constructed a more robust artificial PTC model. P53 expression plasmids were developed that incorporate a string of single synthetic TGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 121-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-TGA129 mutation, treated with Gentamicin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-TGA129, as expected based on the two-base pairing of the respective anticodons in the tRNA to UGA, with R being the most abundant. By contrast, incorporating the amber or ochre premature stop codons, TAA129 or TAG129 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. A reproducible non-canonical readthrough termination codon-skip event at the extreme C-terminus at codon 436 in the SBP-p53 fusion protein was detected which provided a novel assay for non-canonical readthrough at an extreme C-terminal PTC. The incorporation of amino acids at codons 127, 128, or 129 generally result in a p53 protein that is predicted to be 'unfolded' or inactive as defined by molecular dynamic simulations presumably because the production of mixed wild-type p53 and mutant oligomers are known to be inactive through dominant negative effects of the mutation. The data highlight the need to not only produce novel small molecules that can readthrough PTCs or C-terminal 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.

Czech name

—

Czech description

—

Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10603 - Genetics and heredity (medical genetics to be 3)

Project

<a href="/en/project/GA23-06884S" target="_blank" >GA23-06884S: Characterization of the ribosome translating pre-spliced mRNAs for the synthesis of antigenic peptide substrates for the MHC-I pathway</a><br>

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2024

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Frontiers in genetics

ISSN

1664-8021

e-ISSN

1664-8021

Volume of the periodical

15

Issue of the periodical within the volume

NOV 5

Country of publishing house

CH - SWITZERLAND

Number of pages

16

Pages from-to

1407375

UT code for WoS article

001356788600001

EID of the result in the Scopus database

2-s2.0-85209398276