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具有深远的影响。尤其如此 还原氧化（氧化还原）环境，但如何调节氧化还原代谢以及如何 它指导细胞决策知之甚少。为了系统地解决这些紧迫的问题， 有必要使用工具，可以安全地使用蜂窝氧化还原环境的关键贡献者 直接通过空间和最重要的是临时分辨率调节。我们以前使用了H2O形成 乳酸杆菌（LBNOX）的NADH氧化酶在生态上降低NADH/NAD+比率 在哺乳动物细胞的细胞质或线粒体中表达。此外，我们设计了这种变体 对NADPH（TPNOX）的严格特异性酶。随后我们既有LBNOX，又工作 TPNOX是一般编码的工具，以表明NAD+再生而不是ATP生产是关键的 哺乳动物细胞增殖的要求。在我们的原始Mira ESI应用中，我们计划继续 开发以进化为灵感的，一般编码的工具，用于钥匙蜂窝的空间时间调制 氧化还原参数。在项目1中，我们计划通过开发一个通用编码工具来扩展工具包 NADH的直接调节减轻应力（即NADH/NAD+比率增加）。在项目2中，我们将阐明 NADH的代谢和细胞后果减少了各种背景的应力。我们将使用 果蝇苍蝇直接测试氧化或还原方向上的氧化还原调节是相关的 具有压力性，健康范围和寿命。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，其中相应的酶活性由小型控制 使用我们的工具， 我们还将照亮各种氧化还原活性小分子的作用，包括全身线粒体 复杂的I抑制作用和相关的氧化还原不平衡，帕金森氏病神经元丧失的进展 （PD）。这种行政补充要求获得Biotek Cytation C10共聚焦成像 读者，这将使我们能够使用自动显微镜简单地量化多个单元参数， 包括细胞大小和形状，亚细胞结构的形态和功能变化，轨道间结构 通信和图像基于荧光的生物传感器。总之，访问Biotek Cytation C10 仪器将在项目1-3中显着加速实验。