Genetics and Metabolism Section, Liver Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2024;2839:53-75. doi: 10.1007/978-1-0716-4043-2_4.
Iron forms essential cofactors used by many nuclear enzymes involved in genome maintenance. However, unchaperoned nuclear iron may represent a threat to the surrounding genetic material as it promotes redox toxicity that may affect DNA integrity. Safely handling intracellular iron implies metal transfer and cofactor assembly processes based on protein-protein interactions. Identifying those interactions commonly occurs via high-throughput approaches using affinity purification or proximity labeling coupled with mass spectrometry analysis. However, these methods do not identify the subcellular location of the interactions. The one-on-one confirmation of proposed nuclear interactions is also challenging. Many approaches used to look at protein interactions are not tailored for looking at the nucleus because the methods used to solubilize nuclear content are harsh enough to disrupt those transient interactions. Here, we describe step-by-step the use of Proximity Ligation Assay (PLA) to analyze iron-mediated protein-protein interactions in the nucleus of cultured human cells. PLA allows the subcellular visualization of the interactions via the in situ detection of the two interacting proteins using fluorescence confocal microscopy. Briefly, cells are fixed, blocked, permeabilized, and incubated with primary antibodies directed to target proteins. Primary antibodies are recognized using PLA probes consisting of one PLUS and one MINUS oligonucleotide-labeled secondary antibody. If the two proteins are close enough (<40 nm), the PLA probes are ligated and used as the template for rolling circle amplification (RCA) with fluorescently labeled oligonucleotides that yield a signal detectable using fluorescence confocal microscopy. A fluorescently labeled membrane-specific stain (WGA) and the DNA-specific probe DAPI are used to identify cellular and nuclear boundaries, respectively. Confocal images are then analyzed using the CellProfiler software to confirm the abundance and localization of the studied protein-protein interactions.
铁形成许多参与基因组维持的核酶所必需的辅助因子。然而，没有伴侣的核铁可能对周围的遗传物质构成威胁，因为它促进了可能影响 DNA 完整性的氧化还原毒性。安全处理细胞内铁意味着基于蛋白质-蛋白质相互作用的金属转移和辅助因子组装过程。通过使用亲和纯化或邻近标记与质谱分析相结合的高通量方法，通常可以识别这些相互作用。然而，这些方法不能确定相互作用的亚细胞位置。一对一确认所提出的核相互作用也具有挑战性。许多用于研究蛋白质相互作用的方法不适合研究细胞核，因为用于溶解核内容物的方法足够苛刻，足以破坏这些瞬时相互作用。在这里，我们逐步描述了使用邻近连接分析 (PLA) 来分析培养的人类细胞细胞核中铁介导的蛋白质-蛋白质相互作用。PLA 允许通过使用荧光共焦显微镜原位检测两个相互作用的蛋白质来对相互作用进行亚细胞可视化。简而言之，细胞被固定、阻断、透化，并与针对靶蛋白的一抗孵育。一抗通过由一个 PLUS 和一个 MINUS 寡核苷酸标记的二抗 PLA 探针识别。如果两个蛋白质足够接近（<40nm），则 PLA 探针被连接，并用作带有荧光标记的寡核苷酸的滚环扩增 (RCA) 的模板，产生可使用荧光共焦显微镜检测的信号。荧光标记的膜特异性染料 (WGA) 和 DNA 特异性探针 DAPI 分别用于识别细胞和核边界。然后使用 CellProfiler 软件对共焦图像进行分析，以确认所研究的蛋白质-蛋白质相互作用的丰度和定位。
