ABSTRACT
Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay-based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12-mer randomized peptide library consisting of 2.54 × 106 colony-forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet-I-PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet-I-PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 µM for PCNA-P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP-box containing protein involved in DNA replication and repair. P3 inhibited the PCNA-p1551-70 interaction with a half maximal inhibitory activity of 16.2 µM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.
Subject(s)
Escherichia coli , Peptide Library , Proliferating Cell Nuclear Antigen , Protein Binding , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Flow Cytometry , Drug Evaluation, Preclinical/methods , Cell Surface Display Techniques/methods , Humans , Peptides/metabolism , Peptides/genetics , Peptides/chemistry , Peptides/pharmacologyABSTRACT
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.
