PXD039307

PXD039307 is an original dataset announced via ProteomeXchange.

Title	Misincorporations at Artificially Constructed Termination Stop Codons in the Presence of the Amino Glycoside Gentamicin
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 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.
HostingRepository	PRIDE
AnnounceDate	2024-10-29
AnnouncementXML	Submission_2024-10-29_07:47:57.296.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Lenka Hernychova
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2023-01-06 06:58:27	ID requested
⏵ 1	2024-10-29 07:47:57	announced

10.3389/FGENE.2024.1407375;

submitter keyword: LC-MSMS, cancer, aminoglycoside,p53 protein, premature stop codon

Prof. Ted Hupp
contact affiliation	University of Edinburgh, Institute of Genetics and Molecular Medicine, Edinburgh, EH4 2XR
contact email	ted.hupp@ed.ac.uk
lab head
Lenka Hernychova
contact affiliation	Masaryk Memorial Cancer Institutre, Zluty kopec 7, Brno 656 53, Czech Republic
contact email	lenka.hernychova@mou.cz
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/10/PXD039307

PRIDE project URI

• PRIDE
  1. PXD039307
     1. Label: PRIDE project
     2. Name: Misincorporations at Artificially Constructed Termination Stop Codons in the Presence of the Amino Glycoside Gentamicin