Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

中文翻译：

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HiPLA：高通量成像邻近连接分析

蛋白质-蛋白质相互作用对于细胞结构和功能是必不可少的。为了描述蛋白质相互作用的复杂组装如何促进健康和疾病中的细胞过程，需要高度敏感且可以大规模应用的新方法。在这里，我们开发了 HiPLA（高通量成像邻近连接分析），这是一种在高通量成像筛选格式中采用成熟的基于抗体的邻近连接分析作为系统可视化蛋白质相互作用组的新方法的方法。使用 HiPLA 和靶向核蛋白的抗体库，我们在早衰症哈钦森-吉尔福德早衰综合征 (HGPS) 模型中探测了 60 种蛋白质和相关的翻译后修饰 (PTM) 与核层的相互作用。我们确定了与 HGPS 中的核层有差异地相互作用的蛋白质子集。将 HiPLA 与定量间接免疫荧光结合使用，我们发现大多数差异相互作用都伴随着相互作用蛋白表达的相应变化。总之，HiPLA 提供了一种新的方法来大规模探测细胞蛋白质-蛋白质相互作用，并揭示了对蛋白质复合物组装的机械见解。