A FRET-Based Assay for the Identification of PCNA Inhibitors.
Int J Mol Sci
; 24(14)2023 Jul 24.
Article
en En
| MEDLINE
| ID: mdl-37511614
ABSTRACT
Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.
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Base de datos:
MEDLINE
Asunto principal:
Enfermedades Neurodegenerativas
/
Transferencia Resonante de Energía de Fluorescencia
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Int J Mol Sci
Año:
2023
Tipo del documento:
Article