Apollo-NADP+: a spectrally tunable family of genetically encoded sensors for NADP+
Apollo-NADP+: a spectrally tunable family of genetically encoded sensors for NADP+
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DOI：
10.1038/nmeth.3764
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发表时间：
2016-04-01
期刊：
影响因子：
48
通讯作者：
Rocheleau, Jonathan V.
中科院分区：
文献类型：
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作者：
Cameron, William D.;Bui, Cindy V.;Rocheleau, Jonathan V.
NADPH依赖性抗氧化途径在清除氧化磷酸化产生的过氧化氢（H2 O2）中起着关键作用。不充分的清除导致H2 O2积累，并可能导致疾病。为了测量NADPH/NADP（+）氧化还原状态，我们探索了基于稳态荧光各向异性的遗传编码传感器，该稳态荧光各向异性是由于同源荧光蛋白（homoFRET）之间的FRET（荧光共振能量转移）;我们将这些传感器称为Apollo传感器。我们创建了NADP（+）的Apollo传感器（Apollo-NADP（+）），其利用酶失活的葡萄糖-6-磷酸脱氢酶（G6 PD）的NADP（+）依赖性同源二聚化。该传感器是可逆的，响应于葡萄糖刺激的代谢，并且光谱可调以与许多其他传感器兼容。我们使用Apollo-NADP（+）研究β细胞对氧化应激的反应，并通过用H2 O2传感器Hyperer对Apollo-NADP（+）的Cerulean标记版本进行成像，证明NADPH在H2 O2积累之前显著耗尽。
NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP(+) (Apollo-NADP(+)) that exploits NADP(+)-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP(+) to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean -tagged version of Apollo-NADP(+) with the H2O2 sensor HyPer.