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

扩展用于活细胞中氧化还原代谢的隔室特异性操作的基因编码工具集

• 批准号: 10582469
• 负责人: Valentin Cracan
• 金额: $ 24.65万
• 依托单位: SCINTILLON INSTITUTE FOR PHOTOBIOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582469
• 关键词: Address; Administrative Supplement; Biosensor; C10; Cells; Cellular Structures; Communication; Complex; Cytoplasm; Development; Disease; Drosophila genus; Engineering; Environment; Enzymes; Event; Evolution; Face; Image; Imaging Techniques; Lactobacillus brevis; Light; Longevity; Mammalian Cell; Metabolic; Metabolism; Methodology; Microscopy; Mitochondria; Morphology; NADH; NADH oxidase; NADP; Natural regeneration; Organelles; Oxidation-Reduction; Parkinson Disease; Physiology; Production; Protein Engineering; Reader; Resistance; Resolution; Role; Shapes; Specificity; Stress; Subcellular structure; Testing; Variant; base; cell behavior; confocal imaging; experimental study; fluorescence imaging; fly; healthspan; instrument; neuron loss; novel; small molecule; spatiotemporal; stress reduction; temporal measurement; tool

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.

抽象的 细胞面临的代谢环境对细胞行为有着深远的影响。 还原氧化（氧化还原）环境，但氧化还原代谢如何调节的许多方面以及如何 为了系统地解决这些紧迫的问题，人们对它指导的细胞决策知之甚少。 有必要拥有能够安全、可靠地对细胞氧化还原环境的关键贡献者进行保护的工具。 我们之前使用过 H2O 形成，直接以空间分辨率和最重要的是时间分辨率进行调制。 来自短乳杆菌 (LbNOX) 的 NADH 氧化酶可在异位时降低 NADH/NAD+ 比率 在哺乳动物细胞的细胞质或线粒体中表达。此外，我们设计了其变体。 我们随后使用了 LbNOX 和对 NADPH (TPNOX) 具有严格特异性的酶。 TPNOX 作为基因编码工具，表明 NAD+ 再生而非 ATP 产生至关重要 在我们最初的 MIRA ESI 应用中，我们计划继续进行。 开发受进化启发的基因编码工具，用于关键细胞的时空调节 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。 在项目 2 中，我们将阐明 NADH 还原应激的直接调节（即增加 NADH/NAD+ 比率）。 我们将使用不同背景下 NADH 还原应激的代谢和细胞后果。 果蝇直接测试氧化或还原方向的氧化还原调节是否相关 在项目 3 中，我们将结合蛋白质工程和成像。 开发我们工具版本的技术，其中相应的酶活性很小 使用我们的工具进行分子或光刺激来实现相应氧化还原对的时间控制， 我们还将阐明各种氧化还原活性小分子的作用，包括全身线粒体 复合物 I 抑制和相关氧化还原失衡在帕金森病神经元损失进展中的作用 (PD)。本行政补充要求获取 BioTek Cytation C10 共聚焦成像。 阅读器，这将使我们能够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、细胞器间的变化 总之，访问 BioTek Cytation C10。 仪器将显着加速项目 1-3 中描述的实验。