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Identification of a Novel PARP14 Site Motif and Glycohydrolase Specificity Using TLC-MALDI-TOF

Transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD(+)) to target proteins is mediated by a class of human poly-ADP-ribose polymerases, PARPs, and removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been iden...

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Detalles Bibliográficos
Autores principales: Javed, Zeeshan, Nguyen, Hannah H., Harker, Kiana, Mohr, Christian M., Vano, Pia, Wallace, Sean R., Silvers, Clarissa, Sim, Colin, Turumella, Soumya, Flinn, Ally, Carter-O’Connell, Ian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10055325/
https://www.ncbi.nlm.nih.gov/pubmed/36993563
http://dx.doi.org/10.1101/2023.03.22.533863
Descripción
Sumario:Transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD(+)) to target proteins is mediated by a class of human poly-ADP-ribose polymerases, PARPs, and removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the discovery and validation of ADPr site motifs. We identify a minimal 5-mer peptide sequence that is sufficient to drive PARP14 specific activity while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is sequence independent and occurs within hours. Finally, we use the ADPr—peptide to highlight differential activities within the glycohydrolase family and their sequence specificities. Our results highlight: 1) the utility of MALDI-TOF in motif discovery and 2) the importance of peptide sequence in governing ADPr transfer and removal.