University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Pharmacampus, 48149 Münster, Germany.
University of Münster, Institute for Pharmaceutical Technology and Biopharmacy, Pharmacampus, 48149 Münster, Germany.
Int J Mol Sci. 2023 Jul 24;24(14):11858. doi: 10.3390/ijms241411858.
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 PCNA-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNA 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.
增殖细胞核抗原（PCNA）是人类 DNA 代谢的关键调节因子。其一个重要的相互作用伙伴是 p15，它参与 DNA 复制和修复。靶向 PCNA-p15 相互作用是一种有前途的抗癌治疗策略。在这里，开发了一种基于荧光共振能量转移（FRET）的 PCNA-p15 相互作用分析测定法。除了作为鉴定和表征 PCNA-p15 相互作用抑制剂的筛选工具之外，该测定法还适用于研究其亲和力的突变诱导变化。这对于在分子水平上分析与疾病相关的 PCNA 或 p15 变体特别有用。最近，PCNA 变体 C148S 与共济失调毛细血管扩张症样障碍 2 型（ATLD2）相关。ATLD2 是一种神经退行性疾病，其基础是由于 PCNA 受损导致的 DNA 修复缺陷。孵育时间依赖性 FRET 测量表明对 PCNA-p15 亲和力没有影响，但对 PCNA 稳定性有影响。通过本征色氨酸荧光、差示扫描荧光法（DSF）和非对称流场流分离（AF4）测量证实了 PCNA 稳定性受损和聚集行为增加。对疾病相关的 PCNA 变体的分析证明了所开发的相互作用测定法的多功能性。
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