LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.
LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.
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DOI：
10.15252/msb.20178071
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发表时间：
2018-07-11
影响因子：
9.9
通讯作者：
Wanker EE
中科院分区：
文献类型：
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作者：
Trepte P;Kruse S;Kostova S;Hoffmann S;Buntru A;Tempelmeier A;Secker C;Diez L;Schulz A;Klockmeier K;Zenkner M;Golusik S;Rau K;Schnoegl S;Garner CC;Wanker EE
蛋白质相互作用(PPI)的信息对于研究复杂的生物系统和制定治疗策略至关重要。在这里，我们提出了一种基于双读出生物发光的双杂交技术，称为Luthy，当测试二元相互作用时，它在一个实验过程中提供两个定量分数。首先通过生物发光共振能量转移(BRET)定量监测细胞中的PPI，并在细胞裂解后通过基于发光的共沉淀(LUC)再次定量评估PPI。与传统的单一读出方法相比，双读出程序检测相互作用具有更高的灵敏度，并且广泛适用于检测小分子或致病突变对PPI的影响。应用Luthy在聚焦屏幕上的应用，我们确定了突触前伴侣Cspα的42个相互作用，导致成人起病的神经元蜡样脂褐素沉着症(ANCL)，一种进行性神经退行性疾病。在研究致病错义突变L115R和∆L116在CSPα伴Luthy中的作用时，发现近50%的PPI受到影响。我们的研究提供了一个强大、灵敏的研究工具，用于研究疾病相关突变损害生物系统中蛋白质活性的分子机制。
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.