Split-luciferase complementary assay of NLRP3 PYD-PYD interaction indicates inflammasome formation during inflammation
Split-luciferase complementary assay of NLRP3 PYD-PYD interaction indicates inflammasome formation during inflammation
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DOI：
10.1016/j.ab.2021.114510
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发表时间：
2022-02-01
影响因子：
2.9
通讯作者：
Hosseinkhani, Saman
中科院分区：
文献类型：
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作者：
Isazadeh, Mohsen;Amandadi, Mojdeh;Hosseinkhani, Saman
NLRP 3炎性体是先天免疫系统的关键大分子复合物，其响应于广泛的刺激而激活炎性信号级联。结构研究表明，细胞内胞质受体NLRP 3在刺激时寡聚化，并作为支架形成半胱氨酸天冬氨酸蛋白酶原-1活化所必需的ASC丝。尽管NLRP 3炎性体的结构证据丰富，但NLRP 3 Pyrin结构域的相互作用及其功能相关性知之甚少。在这项研究中，裂解荧光素酶互补测定被用作一种替代方法来研究NLRP 3（PYD）-NLRP 3（PYD）在炎性小体形成过程中的相互作用。由于同型NLRP 3相互作用主要基于静电相互作用，因此NLRP 3（PYD）相互作用界面处的磷酸模拟残基（S5）已被突变以显示对荧光素酶活性的破坏性作用。根据所呈现的结果，所设计的生物传感器能够在体外监测NLRP 3 PYD-NLRP 3 PYD相互作用。目前的报告基因分析不仅提供了一种特异性的NLRP 3（PYD）-NLRP 3（PYD）分析来研究PYD-PYD的体外相互作用，而且还提供了一种合适的系统来筛选化学品和药物以鉴定NLRP 3的激活剂和抑制剂。
The NLRP3 inflammasome is a key macromolecular complex of the innate immune system that activates the inflammatory signalling cascade in response to a wide range of stimuli. Structural studies have shown that the intracellular cytosolic receptor NLRP3 oligomerizes upon stimulation and serves as a scaffold to form the ASC filaments necessary for procaspase-1 activation. Despite the abundant structural evidences on NLRP3 inflammasome, the interactions of the NLRP3 Pyrin domain and its functional relevance are poorly understood. In this study, the split luciferase complementation assay is used as an alternative approach to investigate NLRP3(PYD)- NLRP3(PYD) interactions during inflammasome formation. Since the homotypic NLRP3 interaction is mainly based on electrostatic interactions, a phosphomimetic residue (S5) at the interface of the NLRP3(PYD)s interactions has been mutated to show a disruptive effect on luciferase activity. According to the results presented, the designed biosensor was able to monitor the NLRP3PYD-NLRP3PYD interaction in vitro. The current reporter assay not only provides a specific NLRP3(PYD)-NLRP3(PYD) assay to study the PYD-PYD interaction in vitro, but also provides a suitable system for screening chemicals and drugs to identify activators and inhibitors of NLRP3.