Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION
Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION
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DOI：
10.1074/jbc.m110.213298
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发表时间：
2011-06-17
影响因子：
4.8
通讯作者：
Ziegler, Mathias
中科院分区：
文献类型：
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作者：
Nikiforov, Andrey;Dolle, Christian;Ziegler, Mathias
NAD 是一种重要的氧化还原载体，其降解是重要调控途径的关键要素。 NAD 介导的功能是分开的，并且必须由特定的生物合成途径提供动力。然而，人们对人类细胞中的不同途径、它们的亚细胞分布和调节知之甚少。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。该活性根据线粒体 NAD 的可用性合成了免疫可检测的聚 (ADPribose)。基于这种新颖的检测系统、详细的亚细胞酶定位和药理学抑制剂，我们鉴定了细胞外 NAD 前体、它们的胞质转化以及线粒体 NAD 生成的途径。我们的结果表明，除了烟酰胺和烟酸之外，只有相应的核苷容易进入细胞。核苷酸（例如 NAD 和 NMN）经历细胞外降解，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 合成，前体在细胞质中转化为 NMN。当被细胞器吸收时，NMN（与 ATP 一起）作为 NMNAT3 的底物形成 NAD。 NMNAT3 最终定位于线粒体基质，并且是这些细胞器内唯一已知的 NAD 合成酶。因此，我们对哺乳动物 NAD 生物合成进行了全面的剖析，为理解 NAD 介导过程的调节以及这一基本分子的有机稳态奠定了基础。
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(ADPribose) 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.