Green fluorescent proteins induce oxidative stress in cells: A worrisome new wrinkle in the application of the GFP reporter system to biological systems?
Green fluorescent proteins induce oxidative stress in cells: A worrisome new wrinkle in the application of the GFP reporter system to biological systems?
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DOI：
10.1016/j.redox.2017.03.019
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发表时间：
2017-08
期刊：
影响因子：
11.4
通讯作者：
Zielonka J
中科院分区：
文献类型：
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作者：
Kalyanaraman B;Zielonka J
在发表于Redox Biology第12卷的论文“Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells”中，Ganini等人报道了荧光蛋白如增强型绿色荧光蛋白（eGFP）的表达诱导细胞中的氧化应激[1]。作者提出，活性氧（ROS）如超氧化物（O2·−）和过氧化氢（H2 O2）的形成增加可以解释先前在哺乳动物和细菌细胞以及过表达荧光蛋白的动物中报告的细胞毒性和组织异常[2-6]。重要的是，O2·−和H2 O2均诱导氧化还原信号传导机制，导致参与细胞增殖、细胞分化和细胞死亡的细胞调节蛋白的基因表达改变[7-10]。因此，本文中报告的发现可能对常规使用荧光蛋白标签的许多研究结果的解释产生重大影响[11-13]。Ganini等人对在纯化的eGFP或TagRFP存在下烟酰胺腺嘌呤二核苷酸磷酸（NAD（P）H）依赖性O2·-形成提出了非常强有力和令人信服的案例。使用自旋捕获技术，他们明确地显示了5，5-二甲基吡咯啉N-氧化物（DMPO）的O2·−和羟基自旋加合物的形成。DMPO的O2·−和羟基加合物的形成依赖于NADH，被超氧化物歧化酶（SOD）消除，并且不被添加的过氧化氢酶抑制，表明这两种加合物都来自O2·-。来自eGFP的NADH依赖性O2·-产生也通过SOD可降解的细胞色素c还原来证实。O2·-形成的结果伴随着使用FOX测定法监测H2 O2产生和NADH消耗速率，从而得出H2 O2形成速率等于NADH消耗速率的结论。此外，作者证明，在过量NADH的存在下，H2 O2的形成是催化的，表明eGFP的氧化还原循环活性。重要的是，这种活性归因于eGFP的成熟中间体之一，而不是成熟蛋白。由于在无细胞测定中使用的eGFP和NAD（P）H的浓度与细胞中报道的浓度相当，作者假设eGFP氧化还原循环活性发生在GFP表达细胞中，其中预期蛋白质的稳定合成和成熟。研究人员使用Amplex Red测定来估计稳定表达GFP的HeLa细胞中释放的细胞外H2 O2。Mason的实验室之前研究了光对Amplex Red氧化为试卤灵的影响，并且充分了解Amplex Red的光诱导氧化[14-16]。这些实验在黑暗中进行以避免光的影响，并且所有实验都涉及存在过氧化氢酶的对照样品以确认检测到的氧化剂的身份。与对照细胞相比，稳定表达GFP的细胞表现出增加的H2 O2产生。
In the paper “Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells” published in Volume 12 of Redox Biology, Ganini et al. report that expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP) induces oxidative stress in cells [1]. The authors propose that increased formation of reactive oxygen species (ROS) such as superoxide (O2•−) and hydrogen peroxide (H2O2) can explain the cytotoxicity and tissue abnormalities reported previously in mammalian and bacterial cells and animals overexpressing fluorescent proteins [2–6]. Importantly, both O2•− and H2O2 induce redox signaling mechanisms, leading to altered gene expression of cell regulatory proteins involved in cell proliferation, cell differentiation, and cell death [7–10]. Thus, the findings reported in this paper could have a major influence on the interpretation of results obtained from numerous studies that routinely use fluorescent protein tags [11–13]. Ganini et al. make a very strong and convincing case for nicotinamide adenine dinucleotide phosphate (NAD (P) H)-dependent O2•–formation in the presence of purified eGFP or TagRFP. Using the spin-trapping technique, they unequivocally show formation of O2•− and hydroxyl spin adducts of 5, 5-dimethyl-pyrroline N-oxide (DMPO). Formation of O2•− and hydroxyl adducts of DMPO was dependent on NADH, abrogated by superoxide dismutase (SOD) and not inhibited by added catalase, indicating that both adducts derive from O2•–. NADH-dependent O2•–production from eGFP was also confirmed by SOD-inhibitable cytochrome c reduction. The results on O2•–formation were accompanied by the monitoring of H2O2 generation using a FOX assay and of the rates of NADH consumption, leading to the conclusion that the rate of H2O2 formation is equal to the rate of NADH consumption. Furthermore, the authors demonstrate that, in the presence of excess NADH, H2O2 formation is catalytic, indicating the redox cycling activity of eGFP. Importantly, this activity was attributed to one of the maturing intermediates of eGFP, rather than to the mature protein. Because the concentrations of eGFP and NAD (P) H used in the cell-free assays were comparable to those reported in cells, the authors hypothesized that eGFP redox cycling activity occurs in GFP-expressing cells, where a steady synthesis and maturation of the protein is expected. The investigators used the Amplex Red assay to estimate extracellular H2O2 released in HeLa cells stably expressing GFP. Mason's laboratory previously investigated the effect of light on oxidation of Amplex Red to resorufin and is well aware of the light-induced oxidation of Amplex Red [14–16]. These experiments were conducted in the dark to avoid the effect of light, and all experiments involved control samples with catalase present to confirm the identity of the oxidant detected. Cells stably expressing GFP exhibited increased H2O2 production when compared with control cells.