Fluorescent proteins (FPs) stand as pivotal tools extensively employed across diverse biological research endeavors in various model systems. However, long-standing concerns surround their use due to the numerous side effects associated with their expression. Recent investigations have brought to light the significance of hydrogen peroxide (H2O2) that is associated with the maturation process of green fluorescent protein (GFP) fluorophores. The structural and functional impairments associated with GFP expression are possibly linked to this amount of H2O2. In this study, we assess the impact of the GFP-based HyPer7 biosensor on cellular homeostasis and proteome changes, aiming to identify potential risks related to oxidative stress responses that potentially risks the application of such tools. Cells expressing genome-integrated HyPer7 demonstrated altered mitochondrial membrane potential (MMP), which was alleviated by the addition of antioxidants or culturing cells at physiological normoxia (5 kPa O2). Additionally, HyPer7-expressing cells also exhibited significant impairment in mitochondrial oxidative respiration, suggesting broader mitochondrial dysfunction. Through untargeted proteomics analysis, we identified 26 proteins exhibiting differential expression in HyPer7-expressing cells compared to respective control cells. Functional annotation analysis showed that the list of the delineated proteins is associated with cellular responses to stress and the regulation of antioxidant mechanisms. Our findings underscore the significance of caution and validation in ensuring a thorough comprehension of cellular responses when using fluorescent protein-based tools, thereby enhancing the reliability of the results.

中文翻译：

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基于 GFP 的生物传感器的慢病毒转导诱导的生物能量转移和蛋白质组学特征


荧光蛋白 （FPs） 是各种模型系统中各种生物学研究工作中广泛应用的关键工具。然而，由于它们的表达有许多副作用，人们长期以来一直担心它们的使用。最近的研究揭示了过氧化氢 （H2O2） 的重要性，它与绿色荧光蛋白 （GFP） 荧光团的成熟过程有关。与 GFP 表达相关的结构和功能损伤可能与该数量的 H 2 O 2 有关。在这项研究中，我们评估了基于 GFP 的 HyPer7 生物传感器对细胞稳态和蛋白质组变化的影响，旨在确定与氧化应激反应相关的潜在风险，这些风险可能会给此类工具的应用带来风险。表达基因组整合的 HyPer7 的细胞表现出线粒体膜电位 （MMP） 的改变，通过添加抗氧化剂或在生理常氧 （5 kPa O2） 下培养细胞来缓解这种情况。此外，表达 HyPer7 的细胞在线粒体氧化呼吸方面也表现出显著损害，表明更广泛的线粒体功能障碍。通过非靶向蛋白质组学分析，我们与各自的对照细胞相比，鉴定了 26 种在 HyPer7 表达细胞中表现出差异表达的蛋白质。功能注释分析表明，描绘的蛋白质列表与细胞对压力的反应和抗氧化机制的调节有关。我们的研究结果强调了谨慎和验证的重要性，以确保在使用基于荧光蛋白的工具时彻底理解细胞反应，从而提高结果的可靠性。