Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase
Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase
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DOI：
10.1016/j.ab.2004.03.010
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发表时间：
2004-06-15
影响因子：
2.9
通讯作者：
Ohmiya, Y
中科院分区：
文献类型：
--
作者：
Otsuji, T;Okuda-Ashitaka, E;Ohmiya, Y
蛋白水解加工在生理和病理生理细胞功能中起着至关重要的作用，如肽的产生，细胞周期和凋亡。我们开发了一种新的生物物理生物发光共振能量转移（BRET）系统之间的分泌瓦古拉荧光素酶（Vluc）和增强型黄色荧光蛋白（EYFP）的细胞生物过程的可视化。融合蛋白（Vluc-EYFP）的生物发光光谱为双峰（λ（max）= 460 nm（Vluc）和525 nm（EYFP）），表明Vluc的激发态能量转移到EYFP（简称BRET）。BRET信号可以在培养基中测量，并在活细胞中定量产生两种神经肽，即伤害抑制素（NST）和伤害感受素/FQ中的N/OFQ（N/OFQ）。NST和N/OFQ位于同一前体上，但NST对N/OFQ诱导的中枢功能具有拮抗作用。在Vine EYFP中插入NST N/OFQ前体（Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly）的一部分使得融合蛋白在NG 108 -15细胞中在Lys-Arg处可裂解，并且前蛋白转化酶I增强这种消化。BRET信号的变化量化了融合蛋白的加工。我们的新的分子内BRET系统，使用分泌的荧光素酶是有用的研究在活细胞中的肽加工。(C)2004年爱思唯尔公司All rights reserved.
Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal ( lambda(max) = 460 nm (Vluc) and 525 nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST N/OFQ precursor (Glu-Gln-Lys-Gin-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vine EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase I enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells. (C) 2004 Elsevier Inc. All rights reserved.