Neuroproteomics, Max Delbrück Center for Molecular Medicine and Berlin Institute of Health, Berlin, Germany.
Synaptopathy, German Center for Neurodegenerative Diseases, Berlin, Germany.
Mol Syst Biol. 2018 Jul 11;14(7):e8071. doi: 10.15252/msb.20178071.
Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.
蛋白质-蛋白质相互作用（PPIs）的信息对于研究复杂的生物系统和开发治疗策略至关重要。在这里，我们提出了一种双读数基于生物发光的双杂交技术，称为 LuTHy，当测试二进制相互作用时，它在一个实验程序中提供两个定量分数。首先通过生物发光共振能量转移（BRET）的定量来监测细胞中的 PPI，并且在细胞裂解后，通过基于发光的共沉淀（LuC）再次进行定量评估。与传统的单读数方法相比，双读数程序具有更高的检测灵敏度，并且广泛适用于检测小分子或致病突变对 PPI 的影响。在聚焦筛选中应用 LuTHy，我们鉴定了 42 种与 CSPα的突触前伴侣蛋白有关的相互作用，CSPα 是导致成年发病的神经元蜡样脂褐质沉积症（ANCL）的原因，这是一种进行性神经退行性疾病。当使用 LuTHy 研究 CSPα 中的致病错义突变 L115R 和 ∆L116 对 PPI 的影响时，发现近 50%的相互作用受到影响。我们的研究提出了一种稳健、敏感的研究工具，对于研究与疾病相关的突变如何在生物系统中损害蛋白质活性的分子机制具有很高的实用价值。
