NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG
NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG
复制标题
NanoLuc 生物发光驱动光动力激活胆囊收缩素 1 受体与基因编码蛋白光敏剂 miniSOG
DOI：
10.3390/ijms21113763
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发表时间：
2020-06-01
影响因子：
5.6
通讯作者：
Cui, Zong Jie
中科院分区：
文献类型：
--
作者：
Li, Yuan;Cui, Zong Jie
与激动剂的可逆激活相反，胆囊收缩素1受体（CCK 1 R）被光动力作用中产生的单线态氧永久激活，磺化铝酞菁或基因编码的微型单线态氧发生器（miniSOG）作为光敏剂。在这些工作中，卤素光源被用来驱动光动力作用。对于可能的光动力学CCK 1 R生理学的体内应用，实验动物携带连接到外部光源的笨重的光传递装置可能会干扰它们的行为。因此，在目前的工作中，生物发光驱动的miniSOG光动力CCK 1 R激活的可能性进行了检查，通过Fura-2钙成像监测。在平行实验中，发现在AR 4 - 2 J细胞中质膜（PM）定位表达miniSOG（PM）后，用蓝色发光二极管（LED）（450 nm，85 mWcm（-2），1.5 min）照射诱导持续的钙振荡，其被CCK 1 R拮抗剂devazepide 2 nM阻断。NanoLuc通过内部核糖体进入位点（IRES）序列与miniSOG（PM）双顺反子表达（pminiSOG（PM）-IRES-NanoLuc）。发现所得miniSOG（PM）-IRES-NanoLuc-AR 4 - 2 J细胞在添加NanoLuc底物腔肠素后产生强生物发光。引人注目的是，发现腔肠素5 μ M在灌注的miniSOG（PM）-IRES-NanoLuc-AR 4 - 2 J细胞中触发持久的钙振荡（永久性CCK 1 R激活的标志）。这些数据表明，NanoLuc生物发光可以驱动miniSOG（PM）光动力CCK 1 R激活，为其未来的体内应用奠定了基础。
In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOG(PM) in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mWcm(-2), 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOG(PM) via an internal ribosome entry site (IRES) sequence (pminiSOG(PM)-IRES-NanoLuc). The resultant miniSOG(PM)-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOG(PM)-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOG(PM) photodynamic CCK1R activation, laying the foundation for its future in vivo applications.