Recently, the Institute of Synthetic Biology of Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, The Shenzhen Institute of Synthetic Biology Innovation Gan Haiyun Research Group, Li Nan's Research Group and Mayo Medical Center Wang Zhiquan teamed up to publish "Defining Proximity Proteomics of Histone Modifications by" in the journal Genomics, Proteomics & Bioinformatics Antibody-mediated Protein A-APEX2 Labeling" article, co-corresponding authors by Researcher Haiyun Gan, Associate Researcher Li Nan and Professor Wang Zhiquan. Li Xinran, Zhou Jiaqi, Zhao Wenjuan and Wen Qing are the co-first authors of this article. The study developed a new technique that can be used to identify adjacent markers of proteins around modified histones – AMAPEX (Antibody-mediated protein A- ascorbate peroxidase 2 labeling).
(Source: Screenshot of the first page of the article)
Many biological processes are performed and regulated through molecular interactions between proteins and nucleic acids, including protein-protein interactions, protein-RNA interactions, and protein-DNA interactions. The imbalance of these interactions can lead to the occurrence of various human diseases, such as cancer, immune disorders and neurodegenerative diseases. Therefore, methods of studying the interactions between these molecules in cells provide a favorable tool for understanding the biological processes of human disease and their treatment.
Traditional affinity purification and yeast double hybridization methods have been widely used to discover potential molecular interactions. Antibody-based affinity purification binds to mass spectrometry-based proteomics to enrich and identify other protein molecules that interact stably with specific protein molecules. The development of these technologies has expanded our understanding of the networks of protein interactions in various systems. Affinity purification can also be combined with crosslinking and nucleic acid sequencing to identify protein-nucleic acid interactions, such as chromatin immunoprecipitation sequencing (ChIP-seq) and RNA immunoprecipitation sequencing (RIP-seq). However, the main limitation of affinity purification is that weak or transient interactions are often lost during cell lysis and subsequent washing steps. To overcome this, affinity purification can be combined with crosslinking, but false positives are higher after crosslinking. In addition, affinity purification is challenging in applying protein bait to insoluble targets or lacking high affinity antibodies. Yeast double hybridization and other protein complementary experiments represent another way to map protein interactions between proteins, proteins and RNA, and proteins and DNA in living cells. These methods are typically high-throughput and capable of screening thousands of potential molecular interactions. However, many protein complementary analyses have limitations, such as yeast double hybridization, which cannot study membrane proteins.
The development of proximity labeling (PL) complements traditional methods of studying intermolecular interactions in living cells, which typically use CRISPR gene editing or plasmid-based expression to fuse adjacent biotinases with bait proteins in cells for expression. After the addition of exogenous biotin, the proteins adjacent to the bait proteins are biotinylated, and these biotinylated proteins can be enriched by streptomyces-coupled magnetic beads, which can then be analyzed and identified by mass spectrometry. The most commonly used nearby biotinylases include ascorbate peroxidase APEX/APEX2, horseradish peroxidase HRP and biotin ligase BioID, BASU, TurboID, miniTurbo and the like.
Engineered ascorbic acid peroxidase (APEX2) is an engineered ascorbate peroxidase derived from plants. APEX2 is a very rapid proximity labeled peroxidase that can be catalyzed with biotin phenol to form biotin-phenoxy radicals catalyzed by H2O2, which react with specific amino acids rich in electrons (such as Tyr, Trp, Cys, and His) so that biotin is covalently attached to the protein. Due to the short half-life of phenoxy (
As mentioned above, although APEX2 has great advantages as an adjacent marker enzyme, current existing research methods rely on gene editing techniques that require engineered enzymes to express exogenous fusion proteins within cells, which limits its application in cell lines, primary cells, tissues, and pathological samples that are difficult to transfect, and cannot be applied to post-translational modified proteins (e.g., histone modifications). In order to solve these constraints, Gan Haiyun's research group has developed a new pA-APEX2 proximity labeling technology (AMAPEX) mediated by antibodies. This technique binds Protein A to the modified ascorbic acid peroxidase APEX2 to form pA-APEX2, which targets pA-APEX2 to the modified histone protein by specific antibodies. When substrate biotin phenol and hydrogen peroxide are present in cells, APEX2 can label biotin to all adjacent proteins within a radius of about 20 nm from the protein of interest. Later, due to the strong affinity between biotin and the avidin protein streptavidin, the biotin-tagged protein can be purified using streptavidin magnetic beads, and finally the protein around the target protein can be identified by LC-MS/MS analysis, and then the protein-protein interaction relationship in mammalian cells can be studied. This is shown in Figure 1.
Fig. 1 Antibody-mediated pA-APEX2 proximity labeling method construction process
By mass spectrometry, the histone H3K27me3-related PRC1 and PRC2 subunit complexes in mouse embryonic fibroblasts (MEF) can be accurately identified, and compared with the existing two methods (BAC-GFP[1] and ChromID[2]), the identification range and accuracy have been greatly improved. In addition, the interaction between NSD2 and H3K27me3 was verified by CO-IP, which proved the practicality of the method (Figure 2).
Overall, AMAPEX is a very effective tool that allows us to identify proteins adjacent to modified histones, study protein interactions, expand our understanding of interprotective networks, and help us explore the mechanisms of various diseases caused by regulatory network disorders, thus laying the foundation for disease treatment.
Figure 2 Identification of histone H3K27me3 adjacent proteome by AMAPEX method
The research completed by The team of researchers Haiyun Gan was funded by the National Key Research and Development Program of the National Foundation of China, the National Natural Science Foundation of China, the National Natural Science Foundation of China, the Guangdong Provincial Key Laboratory of Synthetic Genomics, and the Shenzhen Synthetic Biology Innovation Research Institute.
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