Department of Medical Sciences, University of Turin, Turin, Italy.
Department of Clinical Sciences, Food and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy.
Antioxid Redox Signal. 2019 Nov 20;31(15):1150-1165. doi: 10.1089/ars.2019.7799.
Nicotinamide adenine dinucleotide (NAD) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD levels in live cells and to address the issue of the compartmentalization of NAD biosynthesis, we exploited a recently described genetically encoded NAD biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of A375 melanoma cells, a model of metastatic melanoma (MM). FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD-producing enzyme in MM cells, was used to monitor NAD depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Throughout this work, we validated the use of genetically encoded NAD biosensors to characterize subcellular distribution of NAD production routes in MM. The chance of real-time monitoring of NAD fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD pool manipulation in cancer and metabolic diseases.
烟酰胺腺嘌呤二核苷酸（NAD）在广泛的正常和病理条件下发挥核心作用。抑制 NAD 生物合成可以在癌症中（包括黑色素瘤）进行治疗性利用。为了获得活细胞中 NAD 水平的定量，并解决 NAD 生物合成的区室化问题，我们利用了最近描述的遗传编码 NAD 生物传感器（LigA-环状排列的 Venus），该传感器靶向 A375 黑色素瘤细胞（转移性黑色素瘤（MM）的模型）的细胞质、线粒体和细胞核。FK866 是烟酰胺磷酸核糖基转移酶（NAMPT）的特异性抑制剂，是 MM 细胞中主要的 NAD 产生酶，用于监测生物传感器转导的 A375 细胞中 NAD 耗竭动力学的亚细胞水平。此外，我们用 NAD 前体（包括烟酰胺、烟酸、烟酰胺核苷和喹啉酸）处理 FK866 阻断的 A375 细胞，突出了每种底物从 NAMPT 阻断中恢复的细胞器特异性能力。然后在分离的细胞器中对 NAD 生物合成酶进行了生化研究，结果表明 NAMPT 存在于所有三个细胞区室中，而烟酰胺磷酸核糖基转移酶主要存在于细胞质和线粒体中，烟酰胺核苷激酶存在于线粒体和核中。与生物传感器数据一致，喹啉酸磷酸核糖基转移酶的表达水平极低。在整个工作过程中，我们验证了遗传编码 NAD 生物传感器在 MM 中用于表征 NAD 产生途径的亚细胞分布的用途。在化学干扰后实时监测 NAD 波动的机会，以及对辅助因子生物合成区室化的更深入理解，为癌症和代谢疾病中 NAD 池操作的靶向策略奠定了基础。
