Plant homeodomain finger protein 20 (PHF20) is a multidomain protein mainly involved in the activation of p53 and in the prevention of its ubiquitylation. Furthermore, it uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting that it plays a role in the cross-talk between lysine methylation and histone acetylation. Writer and eraser proteins have been the main focus of therapeutic development but over the past few years a relatively underexplored group of proteins, the readers, have emerged as promising targets operating at the interface of translating histone marks. While therapeutic potential is evident, there’s need to establish specific biochemical assay for drug discovery. We describe here the development of a high-throughput, nonradioactive bead-based assay that is suitable for screening applications to identify new PHF20 ligands. The Tudor domain of the protein was expressed in E.Coli and purified by affinity chromatography using the GST tag. Biotinylated peptide H4K20me2 was incubated with the protein, afterwards the addition of biotinilated donor beads and anti-GST acceptor beads allowed us to measure the activity of the protein. The optimization of the assay was performed varying assay buffer, reaction time, substrate and protein concentration. Overall, the results presented demonstrate that this novel homogenous and nonradioactive PHF20 assay could represent a powerful technology for measuring readers activity.

Development of a Non-Radioactive, No-Wash Biochemical Assay for High-Throughput Screening of Small Molecule Modulators of PHF20.

FEOLI, ALESSANDRA;Castellano, Sabrina;Tosco, Alessandra;Sbardella, Gianluca
2015-01-01

Abstract

Plant homeodomain finger protein 20 (PHF20) is a multidomain protein mainly involved in the activation of p53 and in the prevention of its ubiquitylation. Furthermore, it uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting that it plays a role in the cross-talk between lysine methylation and histone acetylation. Writer and eraser proteins have been the main focus of therapeutic development but over the past few years a relatively underexplored group of proteins, the readers, have emerged as promising targets operating at the interface of translating histone marks. While therapeutic potential is evident, there’s need to establish specific biochemical assay for drug discovery. We describe here the development of a high-throughput, nonradioactive bead-based assay that is suitable for screening applications to identify new PHF20 ligands. The Tudor domain of the protein was expressed in E.Coli and purified by affinity chromatography using the GST tag. Biotinylated peptide H4K20me2 was incubated with the protein, afterwards the addition of biotinilated donor beads and anti-GST acceptor beads allowed us to measure the activity of the protein. The optimization of the assay was performed varying assay buffer, reaction time, substrate and protein concentration. Overall, the results presented demonstrate that this novel homogenous and nonradioactive PHF20 assay could represent a powerful technology for measuring readers activity.
2015
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11386/4719094
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact