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	Label-free high-precise nanopore detection of endopeptidase activity of anthrax lethal factor regulated by diverse conditions
	Li, Minghan1,2,3,4; Chen, Shanchuan1,2; Wang, Yunjiao1,2; Zhang, Shaoxia1,2,5; Song, Dandan1,2,5; Tian, Rong1,2; Geng, Jia3,4; Wang, Liang1,2
	2023
摘要	Endopeptidase activity of anthrax lethal factor (aLF) prevents the destroy of anthracis spore intracellularly by host macrophages, meanwhile disables the signaling pathways extracellularly that leads to host lethality. Hence, inhibitory of this activity is expected to be an alternative option to cure anthrax infection. Herein, we fabricated a nanopore platform via transmembrane pore construction in vitro, which allows precise mimics, monitoring of intercellular proteinic transport and enables the quantitative detection of aLF endopeptidase activity towards MAPKK signaling protein at single molecule level. Next, we inhibited the aLF activity via screening approaches of protein-metal ion acquisition and other condition controlment (proton/hydroxide strength, adapted tempera-ture, ionizing irradiation), which were identified by nanopore electrokinetic study. Upon the results, we found that Ca2+, Mg2+, Mn2+, Ni2+ collaborating with Zn2+ promote aLF activity efficiently. In contrary, Cd2+, Co2+, Cu2+ have great inhibitory effect. Result further revealed that, the speed of aLF endopeptidase activity with different ions functions as the nanopore signal frequency in linear manner, which enables evident distinction of those divalent ions using this proteinase assay. We also found the higher strength of the proton or hydroxide, the higher the inhibitory to aLF activity. Besides, adapted temperature and gamma-ray also play integral roles in inhibiting this activity. Our results lay experimental basis for accurate detection of aLF activity, meanwhile provide new direction to screening novel stimuli-responsive inhibitors specific to aLF.
关键词	Anthrax lethal factor MAPKKs protein Endopeptidase activity Toxin inhibitor Nanopore signaling
DOI	10.1016/j.bios.2022.114800
发表期刊	BIOSENSORS & BIOELECTRONICS
ISSN	0956-5663
卷号	219页码:8
通讯作者	Wang, Liang(wangliang83@cigit.ac.cn)
收录类别	SCI
WOS记录号	WOS:000879531300005
语种	英语