Department of Molecular Biology, University of Bergen, PB 7803, 5020 Bergen, Norway.
J Biol Chem. 2011 Jun 17;286(24):21767-78. doi: 10.1074/jbc.M110.213298. Epub 2011 Apr 19.
NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.
NAD 是一种重要的氧化还原载体，其降解是重要调节途径的关键要素。NAD 介导的功能是区室化的，必须通过特定的生物合成途径来提供燃料。然而，人们对不同的途径、它们的亚细胞分布以及在人类细胞中的调节知之甚少。特别是，产生线粒体 NAD（最大的亚细胞池）的途径仍然未知。为了可视化细胞中细胞器 NAD 的变化，我们将聚（ADP-核糖）聚合酶活性靶向线粒体基质。这种活性根据线粒体 NAD 的可用性合成可免疫检测的聚（ADP-核糖）。基于这个新的检测系统、详细的亚细胞酶定位和药理学抑制剂，我们确定了细胞外 NAD 前体、它们的细胞质转化以及线粒体 NAD 生成的途径。我们的结果表明，除了烟酰胺和烟酸外，只有相应的核苷容易进入细胞。核苷酸（如 NAD 和 NMN）在外周发生降解，导致形成可渗透的前体。这些前体都可以转化为细胞质和线粒体 NAD。对于线粒体 NAD 的合成，前体在细胞质中转化为 NMN。当被摄取到细胞器中时，NMN（与 ATP 一起）作为 NMNAT3 的底物形成 NAD。NMNAT3 被明确定位在线粒体基质中，是唯一已知存在于这些细胞器中的 NAD 合成酶。因此，我们全面剖析了哺乳动物 NAD 生物合成，为理解 NAD 介导的过程的调节以及这个基本分子的机体稳态奠定了基础。
