Expanding the set of genetically encoded tools for compartment-specific manipulation of redox metabolism in living cells

摘要 细胞所面临的代谢环境对细胞行为有着深远的影响。更是如此 还原-氧化（氧化还原）环境，但如何调节氧化还原代谢的许多方面， 它指导细胞的决定是知之甚少。为了系统地解决这些紧迫问题， 有必要拥有这样的工具，利用这些工具，细胞氧化还原环境的关键贡献者可以安全地， 以空间分辨率和最重要的时间分辨率直接调制。我们以前使用过H2O形成 来自短乳杆菌（LbNOX）的NADH氧化酶，以在异位时降低NADH/NAD+比率 在哺乳动物细胞的细胞质或线粒体中表达。此外，我们还设计了一种变体 对NADPH（TPNOX）具有严格特异性的酶。我们随后采用了LbNOX和 TPNOX作为基因编码工具，表明NAD+再生而不是ATP产生是一个关键的 哺乳动物细胞增殖的需要。在我们最初的MIRA ESI应用程序中，我们计划继续 开发进化启发的，基因编码的工具，用于时空调制关键细胞 氧化还原参数在项目1中，我们计划通过开发基因编码工具来扩展我们的工具包， 直接调节NADH还原应激（即增加的NADH/NAD+比率）。在项目2中，我们将阐明 不同背景下NADH还原应激的代谢和细胞后果。我们将使用 果蝇飞到直接测试是否在氧化或还原方向的氧化还原调节相关 with stress应力resistance抗性，healthspan健康and lifetime寿命.在项目3中，我们将结合联合收割机蛋白质工程和成像 技术来开发我们的工具版本，其中相应的酶活性由小分子控制。 分子或光刺激以实现相应氧化还原对的时间控制。使用我们的工具， 我们还将阐明各种氧化还原活性小分子的作用，包括系统线粒体， 复合物I抑制和相关的氧化还原失衡在帕金森病神经元丢失进展中的作用 （PD）。本管理补充申请要求获取BioTek Cytation C10共聚焦成像 阅读器，这将允许我们使用自动显微镜同时量化多个细胞参数， 包括细胞大小和形状，亚细胞结构的形态和功能变化，细胞器间 通信和基于荧光的生物传感器的图像。总之，使用BioTek Cytation C10 仪器将大大加速项目1-3中描述的实验。

Abstract The metabolic environment that cells face has profound effects on cellular behavior. This is especially true for the reduction-oxidation (redox) environment, but many aspects of how redox metabolism is regulated and how it directs cellular decisions are poorly understood. In order to systematically address these pressing questions, it is necessary to have tools with which key contributors to the cellular redox environment can be safely and directly modulated with spatial and, most importantly, temporal resolution. We previously used a H2O-forming NADH oxidase from Lactobacillus brevis (LbNOX) to decrease the NADH/NAD+ ratio when ectopically expressed in cytoplasm or mitochondria of mammalian cells. Furthermore, we engineered a variant of this enzyme with strict specificity towards NADPH (TPNOX). We subsequently employed both LbNOX and TPNOX as genetically encoded tools to show that NAD+ regeneration but not ATP production is a critical requirement of proliferation of mammalian cells. In our original MIRA ESI application, we plan to continue development of evolution-inspired, genetically encoded tools for spatiotemporal modulation of key cellular redox parameters. In Project 1, we plan to expand our toolkit by developing a genetically encoded tool for the direct modulation of NADH reductive stress (i.e. increased NADH/NAD+ ratio). In Project 2, we will elucidate the metabolic and cellular consequences of the NADH reductive stress in various backgrounds. We will use Drosophila flies to directly test whether redox modulation in either the oxidative or reductive direction is correlated with stress resistance, healthspan and lifespan. In Project 3 we will combine protein engineering and imaging techniques to develop versions of our tools where the corresponding enzymatic activity is controlled by small molecule or light stimulation to achieve temporal control of the corresponding redox pairs. Using our tools, we will also illuminate the role of various redox active small molecules, including systemic mitochondrial complex I inhibition and associated redox imbalance, in the progression of neuronal loss in Parkinson’s disease (PD). This Administrative Supplement requests the acquisition of a BioTek Cytation C10 confocal imaging reader, which would allow us to use automated microscopy to quantify multiple cell parameters simultaneously, including cellular size and shape, morphological and functional changes in subcellular structures, inter-organelle communication and to image fluorescence-based biosensors. In summary, access to a BioTek Cytation C10 instrument will significantly accelerate experiments described in Projects 1-3.