An improved protein lipid overlay assay for studying lipid-protein interactions
An improved protein lipid overlay assay for studying lipid-protein interactions
复制标题
用于研究脂质-蛋白质相互作用的改进的蛋白质脂质叠加测定
DOI：
10.1186/s13007-020-00578-5
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发表时间：
2020-03-06
期刊：
影响因子：
5.1
通讯作者：
Yu, Xiang
中科院分区：
文献类型：
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作者：
Han, Xiuli;Yang, Yongqing;Yu, Xiang
背景脂质在细胞中执行多种功能，并且脂质-蛋白质相互作用在新陈代谢中发挥关键作用。尽管已经开发了各种技术来研究脂质-蛋白质相互作用，但与大多数脂质结合的相互作用的蛋白质伙伴仍然未知。蛋白质脂质覆盖 (PLO) 测定揭示了许多脂质-蛋白质相互作用，但其应用存在未解决的技术问题。结果在此，我们发现，如果在 PLO 测定中的孵育步骤之前进行单独的封闭步骤，则封闭蛋白质可能会干扰脂质与其结合蛋白之间的相互作用。为了克服这个问题，我们通过将孵育步骤与封闭步骤相结合来修改 PLO 测定。验证实验包括磷脂酰肌醇-3-磷酸 (PI3P) 及其市售相互作用蛋白 G302、C18:1、C18:2、C18:3 和拟南芥质膜 H+-ATP 酶 (PM H+-ATPase) AHA2 C 末端、磷脂酰甘油 (PG) 和 AtROP6，以及磷脂酰丝氨酸 (PS) 和 AHA2 C 末端。经典 PLO (CPLO) 测定中的脂质-蛋白结合信号较弱且不可重现，但改良的 PLO (MPLO) 测定显示出显着提高的灵敏度和重现性。结论这项工作确定了 CPLO 测定的局限性，并且改进的测定法的灵敏度和重现性均得到改善，这可以证明对于研究脂质 - 蛋白质相互作用更有效。
BackgroundLipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues.ResultsHerein, we found that blocking proteins may interfere with interactions between lipids and their binding proteins if a separate blocking step is carried out before the incubation step in the PLO assay. To overcome this, we modified the PLO assay by combining an incubation step alongside the blocking step. Verification experiments included phosphatidylinositol-3-phosphate (PI3P) and its commercially available interacting protein G302, C18:1, C18:2, C18:3 and the Arabidopsis plasma membrane H+-ATPase (PM H+-ATPase) AHA2 C-terminus, phosphatidylglycerol (PG) and AtROP6, and phosphatidylserine (PS) and the AHA2 C-terminus. The lipid-protein binding signal in the classical PLO (CPLO) assay was weak and not reproducible, but the modified PLO (MPLO) assay displayed significantly improved sensitivity and reproducibility.ConclusionsThis work identified a limitation of the CPLO assay, and both sensitivity and reproducibility were improved in the modified assay, which could prove to be more effective for investigating lipid-protein interactions.