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: 10272745
• 负责人: Valentin Cracan
• 金额: $ 31.32万
• 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/10272745
• 关键词: Acute; Address; Aging; Animal Model; Antioxidants; Automobile Driving; Bioenergetics; Cell physiology; Cells; Chronic; Consumption; Dependence; Development; Diabetes Mellitus; Disease; Drosophila genus; Electrons; Energy Metabolism; Engineering; Environment; Enzymes; Etiology; Goals; Homeostasis; Life; Light; Link; Longevity; Maintenance; Malignant Neoplasms; Membrane Potentials; Metabolic; Metabolism; Methodology; Mitochondrial Diseases; NADH; NADP; Nerve Degeneration; Oxidases; Oxidation-Reduction; Oxides; Pathology; Process; Production; Reaction; Resistance; Resolution; Role; Signal Transduction; Stress; System; Technology; Testing; Therapeutic; Time; Variant; base; cofactor; exhaust; healthspan; improved; metabolic abnormality assessment; mitochondrial membrane; novel; screening; small molecule; spatiotemporal; stress reduction; tool

Abstract One of the most important organizing principles in all life forms is the uninterrupted flow of electrons through reactions that involve reduction-oxidation (redox) changes. Not surprisingly, an imbalance in this fundamental cellular process, i.e. redox homeostasis, has been attributed to numerous diseases, including mitochondrial disorders, cancer, diabetes, neurodegeneration and the aging process itself. The redox cofactors, NADH and NADPH, and their oxidized forms are key contributors to the cellular redox environment, but it is unclear whether perturbations in their metabolism contribute directly to disease etiology or is simply a reflection of ongoing pathology. For most of these conditions, it is not known whether the observed redox imbalance is linked to altered bioenergetic efficiency or to a cellular process that is neither linked to ATP production nor to maintenance of the mitochondrial membrane potential. Another major challenge is that some of these redox reactions are redundant, i.e. have overlapping substrate dependency (towards NAD(P)H) or are found in more than one cellular compartment. To systematically address these pressing questions, methodology to modulate the steady-state concentrations of the NADH and NADPH cofactors is needed. Recently, we have developed genetically encoded tools to selectively decrease the NADH/NAD+ and NADPH/NADP+ ratios in live cells that are based on the heterologous expression of native or engineered versions of bacterial H 2O- forming NAD(P)H oxidases. In this proposal, 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) (Project 1). Preliminary screening of several bacterial enzymes has furnished promising candidates for driving NADH overproduction in different cellular compartments. The development of compartment-specific tools will enable studies to elucidate how overproduction of reducing equivalents in one cellular compartment is communicated to another and how NADH reductive stress remodels cellular metabolism (Project 2a). Multiple lines of evidence indicate that NAD(P)H-consuming redox cycling agents at low concentrations mildly exhaust antioxidant systems and that the resulting pro-oxidative shift promotes stress resistance and improves heathspan in several model organisms. We are using Drosophila as a model organism, to directly test whether redox modulation in either the oxidative or reductive direction are correlated with stress resistance, healthspan and lifespan (Project 2b). A third goal is to develop variants of our genetically encoded tools that are controlled by small molecules or by light to afford greater spatiotemporal control (Project 3). The latter is especially important as many redox processes crucial for redox signaling or energy metabolism and dysregulated in pathologies, occur rapidly (on an acute time scale). The successful completion of our studies will lead to enabling technologies for modulating the redox environment, which will be widely useful for metabolic studies on an acute or chronic time scale at the resolution of subcellular compartments.

抽象的 所有生命形式中最重要的组织原则之一是电子不间断地流过 涉及还原氧化（氧化还原）变化的反应。毫不奇怪，这一基本面的不平衡 细胞过程，即氧化还原稳态，已归因于许多疾病，包括线粒体疾病 疾病、癌症、糖尿病、神经退行性疾病和衰老过程本身。氧化还原辅助因子 NADH 和 NADPH 及其氧化形式是细胞氧化还原环境的关键贡献者，但尚不清楚是否 新陈代谢的扰动直接导致疾病病因，或者只是持续存在的反映 病理。对于大多数这些条件，尚不清楚观察到的氧化还原不平衡是否与 改变了生物能效率或与 ATP 产生无关或与 ATP 产生无关的细胞过程 维持线粒体膜电位。另一个主要挑战是其中一些氧化还原 反应是多余的，即具有重叠的底物依赖性（朝向 NAD(P)H）或在 不止一个细胞室。为了系统地解决这些紧迫的问题，方法论 需要调节 NADH 和 NADPH 辅因子的稳态浓度。最近，我们有 开发了基因编码工具来选择性降低 NADH/NAD+ 和 NADPH/NADP+ 比率 基于细菌 H 2O- 的天然或工程版本的异源表达的活细胞 形成 NAD(P)H 氧化酶。在本提案中，我们计划通过开发基因技术来扩展我们的工具包 用于直接调节 NADH 还原应激（即增加 NADH/NAD+ 比率）的编码工具（项目 1）。对几种细菌酶的初步筛选为驱动 NADH 提供了有希望的候选者 不同细胞区室的过度生产。隔间专用工具的开发将使 研究阐明一个细胞室中还原当量的过量产生是如何传达的 另一个问题是 NADH 还原应激如何重塑细胞代谢（项目 2a）。多行 有证据表明，低浓度的 NAD(P)H 消耗氧化还原循环剂会轻微耗尽 抗氧化系统以及由此产生的促氧化转变可促进抗应激能力并改善健康寿命 在几种模式生物中。我们使用果蝇作为模式生物，直接测试氧化还原是否 氧化或还原方向的调节与应激抵抗力、健康寿命和 寿命（项目 2b）。第三个目标是开发我们的基因编码工具的变体，这些工具由 小分子或光来提供更大的时空控制（项目 3）。后者尤其是 重要的是，许多氧化还原过程对于氧化还原信号或能量代谢至关重要，并且在 病理，发生迅速（在急性时间尺度上）。成功完成我们的学习将导致 调节氧化还原环境的技术，将广泛用于代谢研究 亚细胞区室分辨率的急性或慢性时间尺度。