title: Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.
abstract: NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Kulikova
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia,; Petersburg Nuclear Physics Institute, National Research Centre Kurchatov Institute, Gatchina 188300, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Shabalin
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia,; St. Petersburg State University, St. Petersburg 199034, Russia.
authors.forename: Kirill
authors.initials: K
authors.lastname: Nerinovski
authors.affiliation: Department of Molecular Biology, University of Bergen, 5020 Bergen, Norway.
authors.forename: Christian
authors.initials: C
authors.lastname: Dölle
authors.affiliation: Department of Molecular Biology, University of Bergen, 5020 Bergen, Norway.
authors.forename: Marc
authors.initials: M
authors.lastname: Niere
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia,; Petersburg Nuclear Physics Institute, National Research Centre Kurchatov Institute, Gatchina 188300, Russia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Yakimov
authors.affiliation: School of Pharmacy, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: Redpath
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Khodorkovskiy
authors.affiliation: School of Pharmacy, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom.
authors.forename: Marie E
authors.initials: ME
authors.lastname: Migaud
authors.affiliation: Department of Molecular Biology, University of Bergen, 5020 Bergen, Norway,. Electronic address: mathias.ziegler@mbi.uib.no.
authors.forename: Mathias
authors.initials: M
authors.lastname: Ziegler
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia,; Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia. Electronic address: andrey.nikiforov@gmail.com.
authors.forename: Andrey
authors.initials: A
authors.lastname: Nikiforov
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways; D009243:NAD / Q000096:biosynthesis*; D009525:Niacin / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism; D010766:Phosphorylation; D011726:Pyridinium Compounds; D011994:Recombinant Proteins / Q000378:metabolism; D012263:Ribonucleosides / Q000096:biosynthesis* / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D016207:Cytokines; D011726:Pyridinium Compounds; D011994:Recombinant Proteins; D012263:Ribonucleosides; C018613:nicotinamide-beta-riboside; D009243:NAD; C030285:nicotinic acid ribonucleoside; D009536:Niacinamide; D009525:Niacin; D010430:Pentosyltransferases; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human; D015720:5'-Nucleotidase; C021864:nicotinate phosphoribosyltransferase
keywords: 5′-nucleotidase; NAD biosynthesis; molecular cell biology; nicotinamide; nicotinamide adenine dinucleotide (NAD); nicotinic acid; nicotinic acid riboside; nucleoside/nucleotide metabolism