Human apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein crucial for DNA repair, apoptosis regulation, and genome stability maintenance. Its subcellular localization and diverse functions have made it a target of intense research, contributing to understanding its role in diseases such as neurodegeneration and cancer. However, due to the barrier of the nuclear membrane and the complex composition of the cellular environment, it is challenging to dynamically observe endogenous APE1 in the nuclei of live cells.
On March 30, 2024, the research group led by Professor Meiping Zhao from the College of Chemistry and Molecular Engineering at Peking University published a research paper entitled "Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe" in Nucleic Acids Research. The study reported a novel DNA fluorescent probe escorted by protein chaperones, which achieved targeted localization of the probe in the cell nucleus while ensuring specific response to APE1 and provided the first dynamic visualization of APE1 in different regions of the live cell nucleus.
In earlier studies, the authors found that biotinylated DNA containing abasic sites (AP-DNA) naturally resisted cleavage by various nucleases when assembled onto the surface of avidin (AVD)-modified silica-coated magnetic nanoparticles but could be efficiently cleaved by APE1. Subsequent research demonstrated that AVD bound to biotin exhibited even greater affinity for APE1 than unbound AVD itself. Based on the synergistic interaction between the APE1-AVD-DNA ternary system, the research team further developed a novel protein chaperone strategy to protect the DNA probe(ACP) (Fig.1).
Fig.1 Construction of the ACP fluorescent probe, selectivity test, and quantification of APE1 level in the cytoplasmic, mitochondrial, and nuclear extracts of different types of cells by using ACP.
The specificity of the ACP probe was validated using specific inhibitors, immunoprecipitation, and gene knockout cell lines, and the APE1 levels in protein extracts from the cytoplasm, nucleus, mitochondria, and exosomes of different cell types were quantitatively compared(Fig.2). The results indicated significant differences in nuclear APE1 levels among different cell types, whereas differences in cytoplasmic APE1 levels were relatively minor.
Afterwards, the authors covalently modified AVD with phenylboronic acid to obtain probes (PB-ACP), which were verified to target the nucleus in various cell lines via the importin α/β pathway. The study revealed that APE1 exhibited higher activity in the more loosely organized, transcriptionally active euchromatin and nucleolus regions, while its distribution in heterochromatin regions was relatively low.
Fig.2 Visualization APE1 in the nucleus and nucleolus by using the chaperone@DNA fluorescent probe(PB-ACP) constructed with phenylboronic acid modified AVD.
APE1 plays different roles in various regions within the cell, and its distribution and dynamic translocation in the nucleus are closely associated with its diverse functions. The authors further demonstrated the changes in APE1 activity and distribution in response to different types of stimuli in live cells (Fig.3).
This study provides a novel and effective molecular tool for comprehensively understanding the dynamic processes and molecular mechanisms of nucleic acid damage repair at the cellular level. This advancement holds promising implications for delving deeper into the mechanisms underlying cell aging, disease development, including cancer, and the potential for targeted therapies. Moreover, the proposed strategy of utilizing protein chaperones to assist nucleic acid probes opens avenues for the future development of highly intelligent probes in a completely new mode.
Fig.3 Monitoring of the changes in APE1 activity and distribution in live cells under different stimulus conditions.
Dr. Xiangjian Cao (a Ph.D. graduate), Jinghui Zheng and Ruilan Zhang (both Ph.D. candidates) from the College of Chemistry and Molecular Engineering, Peking University are co-first authors of the article. Prof.Meiping Zhao from the College of Chemistry and Molecular Engineering at Peking University is the corresponding author. Dr. Yan Guan and Dr. Wen Zhou from the Analytical Instrumentation Center in Peking University (PKUAIC) provided significant assistance in Optical Spectroscopy measurement and MALDI-TOF MS analysis. The research was supported by the National Natural Science Foundation of China and the Beijing National Laboratory for Molecular Sciences.
Link to the original article: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkae202/7637892
