Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

Veronika Kulikova, Konstantin Shabalin, Kirill Nerinovski, Christian Dölle, Marc Niere, Alexander Yakimov, Philip Redpath, Mikhail Khodorkovskiy, Marie E Migaud, Mathias Ziegler*, Andrey Nikiforov

*Corresponding author for this work

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)27124-37
Number of pages14
JournalJournal of Biological Chemistry
Volume290
Issue number45
Early online date18 Sep 2015
DOIs
Publication statusPublished - 06 Nov 2015

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Niacin
NAD
Cells
5'-Nucleotidase
Nicotinamide Mononucleotide
Metabolism
Niacinamide
HEK293 Cells
Biosynthesis
Hep G2 Cells
Catalysis
HeLa Cells
Yeast
Culture Media
Cell Survival
Yeasts
nicotinamide-beta-riboside
nicotinate mononucleotide

Cite this

Kulikova, V., Shabalin, K., Nerinovski, K., Dölle, C., Niere, M., Yakimov, A., ... Nikiforov, A. (2015). Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells. Journal of Biological Chemistry, 290(45), 27124-37. https://doi.org/10.1074/jbc.M115.664458
Kulikova, Veronika ; Shabalin, Konstantin ; Nerinovski, Kirill ; Dölle, Christian ; Niere, Marc ; Yakimov, Alexander ; Redpath, Philip ; Khodorkovskiy, Mikhail ; Migaud, Marie E ; Ziegler, Mathias ; Nikiforov, Andrey. / Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells. In: Journal of Biological Chemistry. 2015 ; Vol. 290, No. 45. pp. 27124-37.
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Kulikova, V, Shabalin, K, Nerinovski, K, Dölle, C, Niere, M, Yakimov, A, Redpath, P, Khodorkovskiy, M, Migaud, ME, Ziegler, M & Nikiforov, A 2015, 'Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells', Journal of Biological Chemistry, vol. 290, no. 45, pp. 27124-37. https://doi.org/10.1074/jbc.M115.664458

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells. / Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey.

In: Journal of Biological Chemistry, Vol. 290, No. 45, 06.11.2015, p. 27124-37.

Research output: Contribution to journalArticle

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AU - Kulikova, Veronika

AU - Shabalin, Konstantin

AU - Nerinovski, Kirill

AU - Dölle, Christian

AU - Niere, Marc

AU - Yakimov, Alexander

AU - Redpath, Philip

AU - Khodorkovskiy, Mikhail

AU - Migaud, Marie E

AU - Ziegler, Mathias

AU - Nikiforov, Andrey

PY - 2015/11/6

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N2 - 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.

AB - 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.

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