Lim Esther W, Parker Seth J, Metallo Christian M
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Department of Radiation Oncology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA.
Methods Mol Biol. 2020;2088:51-71. doi: 10.1007/978-1-0716-0159-4_4.
Oxidation-reduction (redox) reactions are ubiquitous in biology and typically occur in specific subcellular compartments. In cells, the electron transfer between molecules and organelles is commonly facilitated by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). While often taken for granted, these metabolic reactions are critically important for maintaining redox homeostasis and biochemical potentials across membranes. While C tracing and metabolic flux analysis (MFA) have emerged as powerful tools to study intracellular metabolism, this approach is limited when applied to pathways catalyzed in multiple cellular compartments. To address this issue, we and others have applied H (deuterium) tracers to observe transfer of labeled hydride anions, which accompanies electron transfer. Furthermore, we have developed a reporter system for indirectly quantifying NADPH enrichment in specific subcellular compartments. Here, we provide a detailed description of H tracing applications and the interrogation of mitochondrial versus cytosolic NAD(P)H metabolism in cultured mammalian cells. Specifically, we describe the generation of reporter cell lines that express epitope-tagged R132H-IDH1 or R172K-IDH2 and produce (D)2-hydroxyglutarate in a doxycycline-dependent manner. These tools and methods allow for quantitation of reducing equivalent turnover rates, the directionality of pathways present in multiple compartments, and the estimation of pathway contributions to NADPH pools.
氧化还原反应在生物学中无处不在，通常发生在特定的亚细胞区室。在细胞中，分子和细胞器之间的电子转移通常由吡啶核苷酸促进，如烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和烟酰胺腺嘌呤二核苷酸（NADH）。虽然这些代谢反应常常被视为理所当然，但它们对于维持跨膜的氧化还原稳态和生化电位至关重要。虽然碳追踪和代谢通量分析（MFA）已成为研究细胞内代谢的强大工具，但当应用于多个细胞区室中催化的途径时，这种方法存在局限性。为了解决这个问题，我们和其他人应用了氢（氘）示踪剂来观察伴随电子转移的标记氢化物阴离子的转移。此外，我们开发了一种报告系统，用于间接定量特定亚细胞区室中的NADPH富集。在这里，我们详细描述了氢追踪应用以及对培养的哺乳动物细胞中线粒体与细胞质NAD(P)H代谢的研究。具体来说，我们描述了报告细胞系的生成，这些细胞系表达表位标记的R132H-IDH1或R172K-IDH2，并以多西环素依赖性方式产生（D）2-羟基戊二酸。这些工具和方法允许定量还原当量周转率、多个区室中存在的途径的方向性，以及估计途径对NADPH池的贡献。
