Ozleyen Adem, Duran Gizem Nur, Donmez Serhat, Ozbil Mehmet, Doveston Richard G, Tumer Tugba Boyunegmez
Leicester Institute for Structural and Chemical Biology, University of Leicester, Leicester, LE1 7RH, UK.
School of Chemistry, University of Leicester, Leicester, LE1 7RH, UK.
Sci Rep. 2025 Mar 14;15(1):8907. doi: 10.1038/s41598-025-89342-0.
NRF2 is a transcription factor responsible for coordinating the expression of over a thousand cytoprotective genes. Although NRF2 is constitutively expressed, its stability is modulated by the redox-sensitive protein KEAP1 and other conditional binding partner regulators. The new era of NRF2 research has highlighted the cooperation between NRF2 and PIN1 in modifying its cytoprotective effect. Despite numerous studies, the understanding of the PIN1-NRF2 interaction remains limited. Herein, we described the binding interaction of PIN1 and three different 14-mer long phospho-peptides mimicking NRF2 protein using computer-based, biophysical, and biochemical approaches. According to our computational analyses, the residues positioned in the WW domain of PIN1 (Ser16, Arg17, Ser18, Tyr23, Ser32, Gln33, and Trp34) were found to be crucial for PIN1-NRF2 interactions. Biophysical FP assays were used to verify the computational prediction. The data demonstrated that Pintide, a peptide predominantly interacting with the PIN1 WW-domain, led to a significant reduction in the binding affinity of the NRF2 mimicking peptides. Moreover, we evaluated the impact of known PIN1 inhibitors (juglone, KPT-6566, and EGCG) on the PIN1-NRF2 interaction. Among the inhibitors, KPT-6566 showed the most potent inhibitory effect on PIN1-NRF2 interaction within an IC range of 0.3-1.4 µM. Furthermore, our mass spectrometry analyses showed that KPT-6566 appeared to covalently modify PIN1 via conjugate addition, rather than disulfide exchange of the sulfonyl-acetate moiety. Altogether, such inhibitors would also be highly valuable molecular probes for further investigation of PIN1 regulation of NRF2 in the cellular context and potentially pave the way for drug molecules that specifically inhibit the cytoprotective effects of NRF2 in cancer.
NRF2是一种转录因子，负责协调一千多种细胞保护基因的表达。尽管NRF2是组成性表达的，但其稳定性受氧化还原敏感蛋白KEAP1和其他条件性结合伴侣调节因子的调控。NRF2研究的新时代突出了NRF2与PIN1在调节其细胞保护作用方面的合作。尽管有大量研究，但对PIN1-NRF2相互作用的理解仍然有限。在此，我们使用基于计算机、生物物理和生化方法描述了PIN1与三种不同的模拟NRF2蛋白的14聚体磷酸肽的结合相互作用。根据我们的计算分析，发现位于PIN1的WW结构域中的残基（Ser16、Arg17、Ser18、Tyr23、Ser32、Gln33和Trp34）对PIN1-NRF2相互作用至关重要。生物物理荧光偏振分析用于验证计算预测。数据表明，主要与PIN1 WW结构域相互作用的肽Pintide导致模拟NRF2的肽的结合亲和力显著降低。此外，我们评估了已知的PIN1抑制剂（胡桃醌、KPT-6566和表没食子儿茶素没食子酸酯）对PIN1-NRF2相互作用的影响。在这些抑制剂中，KPT-6566在0.3-1.4 μM的IC范围内对PIN1-NRF2相互作用表现出最有效的抑制作用。此外，我们的质谱分析表明，KPT-6566似乎通过共轭加成共价修饰PIN1，而不是通过磺酰乙酸部分的二硫键交换。总之，这些抑制剂对于进一步研究细胞环境中PIN1对NRF2的调节也将是非常有价值的分子探针，并可能为特异性抑制癌症中NRF2细胞保护作用的药物分子铺平道路。
