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

摘要 在所有生命形式中，最重要的组织原则之一是电子不间断地流动 涉及还原-氧化(氧化还原)变化的反应。不足为奇的是，这种基本面的不平衡 细胞过程，即氧化还原动态平衡，已被归因于许多疾病，包括线粒体 疾病、癌症、糖尿病、神经退化和衰老过程本身。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因素，但目前尚不清楚 他们新陈代谢的紊乱直接导致了疾病的病因，或者仅仅是正在进行的 病理学。对于大多数这些情况，尚不清楚观察到的氧化还原失衡是否与 改变的生物能量效率或与细胞过程无关，既不与ATP的产生有关，也不与 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 不止一个细胞隔间。为了系统地解决这些紧迫的问题，方法 需要调节NADH和NADPH辅因子的稳态浓度。最近，我们有 开发了有选择地降低NADH/NAD和NADPH/NADP比率的遗传编码工具 基于细菌水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。在这项提案中，我们计划通过开发一种遗传基因来扩展我们的工具包 用于直接调节NADH还原应力(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提供了有前景的候选者。 不同细胞隔间的生产过剩。车厢专用工具的开发将使 研究阐明一个细胞隔间中还原当量的过量产生是如何传递的 以及NADH还原应激如何重塑细胞代谢(项目2a)。多行 有证据表明，低浓度下消耗NAD(P)H的氧化还原循环剂轻度排放 抗氧化剂系统和由此产生的促氧化转变增强了抗应激能力并改善了健康寿命 在几个模型生物中。我们正在用果蝇作为模式生物，直接测试氧化还原 氧化或还原方向的调节与抗逆性、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，以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氧化还原信号或能量代谢至关重要，并在 病理，发生迅速(在一个急性时间尺度上)。成功完成我们的研究将导致 能够调节氧化还原环境的技术，这将在代谢研究中广泛使用 亚细胞间隔的一种急性或慢性时间标度。

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.