.2019 May 30;10(1):2367.

doi: 10.1038/s41467-019-10125-z.

The metabolites NADP⁺ and NADPH are the targets of the circadian protein Nocturnin (Curled)

Michael A Estrella¹, Jin Du¹, Li Chen^{2 3}, Sneha Rath¹, Eliza Prangley¹, Alisha Chitrakar¹, Tsutomu Aoki¹, Paul Schedl¹, Joshua Rabinowitz^{4 5}, Alexei Korennykh⁶

Affiliations

¹ 216 Schultz Laboratory, Department of Molecular Biology, Princeton, NJ, 08544, USA.
² 285 Frick Laboratory, Department of Chemistry, Princeton, NJ, 08544, USA.
³ Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, 08544, USA.
⁴ 285 Frick Laboratory, Department of Chemistry, Princeton, NJ, 08544, USA. joshr@princeton.edu.
⁵ Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, 08544, USA. joshr@princeton.edu.
⁶ 216 Schultz Laboratory, Department of Molecular Biology, Princeton, NJ, 08544, USA. akorenny@princeton.edu.

PMID:31147539
PMCID: PMC6542800
DOI: 10.1038/s41467-019-10125-z

The metabolites NADP⁺ and NADPH are the targets of the circadian protein Nocturnin (Curled)

Michael A Estrella et al. Nat Commun.2019.

.2019 May 30;10(1):2367.

doi: 10.1038/s41467-019-10125-z.

Authors

Michael A Estrella¹, Jin Du¹, Li Chen^{2 3}, Sneha Rath¹, Eliza Prangley¹, Alisha Chitrakar¹, Tsutomu Aoki¹, Paul Schedl¹, Joshua Rabinowitz^{4 5}, Alexei Korennykh⁶

Affiliations

¹ 216 Schultz Laboratory, Department of Molecular Biology, Princeton, NJ, 08544, USA.
² 285 Frick Laboratory, Department of Chemistry, Princeton, NJ, 08544, USA.
³ Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, 08544, USA.
⁴ 285 Frick Laboratory, Department of Chemistry, Princeton, NJ, 08544, USA. joshr@princeton.edu.
⁵ Lewis-Sigler Institute for Integrative Genomics, Princeton, NJ, 08544, USA. joshr@princeton.edu.
⁶ 216 Schultz Laboratory, Department of Molecular Biology, Princeton, NJ, 08544, USA. akorenny@princeton.edu.

PMID:31147539
PMCID: PMC6542800
DOI: 10.1038/s41467-019-10125-z

Abstract

Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP⁺ into NAD⁺ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
Metabolite profiling to identify NOCT substrate.a Metabolite extraction and treatment outline. Full-length human NOCT and the catalytic domain of NOCT (residues 122–431) were compared against full-length human NOCT E195A, NOCT (122–431) E195A mutant, or the catalytic domain of CNOT6L (158–555). Liver experiment 1 and HEK293 experiment used the catalytic domain of NOCT. Liver experiment 2 used GST-tagged full-length NOCT immobilized on glutathione beads.b Metabolite depletion in three independent experiments (Supplementary Data Set 1). Fold is defined as the ratio of LC-MS intensities for samples with WT NOCT vs average for samples with NOCT E195A and CNOT6L.c LC-MS/MS analysis of purified NADP⁺ and NADPH following incubation with WT or E195A human NOCT (122–431; 10 μM, 1 h at r.t.).d The identified reactions catalyzed by human NOCT

**Fig. 2**
NADP(H) cleavage characterization.a Time course cleavage of pure NADPH (1 mM) and NADP⁺ (1 mM) by purified human full-length NOCT (0.5 μM) at 22 °C, monitored by ion-exchange chromatography (IEC).b 60-min NADPH incubation with the catalytic domains of PDE12, CNOT6L, and NOCT as ina.c Measurement of kinetic parameters for the catalytic domain of human NOCT (4.8 μM).d Western blot analysis of full-length human FLAG-NOCT (C-terminal tag) expressed in A549 cells for 18 h.e NADPH (1 mM) cleavage by FLAG-NOCT purified from human cells.f Nicotinamide metabolite composition in WT and NOCT^−/− cells (Supplementary Data 2). Six replicates from three WT samples are compared against four replicates from two independently generated NOCT^−/− clones. Error bars are standard error (S.E.). Source data are provided as a Source Data file

**Fig. 3**
Evidence for NOCT mitochondrial localization.a MitoFates analysis of NOCT and reference human proteins.b Western blot for FLAG-NOCT, full-length vs ΔMTS.c Subcellular fractionation and western blot analysis of FLAG-NOCT, WT, ΔMTS, and E195A active site mutant. Error bars show S.E. from the data inc and Supplementary Fig. 3a,P-values here and throughout were determined by Welch’s two-tailed unpairedt-test. PARP-1, Tubulin, and COX4 mark the nucleus, the cytosol, and the mitochondria, respectively.d Confocal microscopy of NOCT variants inc. DAPI stains for nuclei, MitoSOX stains mitochondria.e Running average for MitoFates scores for ~10,500 mRNAs ordered according to gene expression levels in WT vs NOCT^−/− cells, measured by RNA-seq (data are provided in Supplementary Data 3).P-values for top 2000 vs bottom 2000 mRNAs. Error bars represent S.E. RNA-seq data represent four RNA-seq experiments: two in WT cells and two in independently derived NOCT-KO clones.f GSEA enrichment results for the genes with ≥50 reads, ranked according to WT/NOCT^−/− expression as ine (Supplementary Data 3). Source data are provided as a Source Data file

**Fig. 4**
Structure of human NOCT•NADPH•Ca²⁺ complex.a Surface representation of the complex with NADPH bound to the active site pocket.b NOCT conservation from 351 non-degenerate sequences mapped onto the NOCT structure. Color scale shows a range from invariable (red) to random (blue).c NADPH position and the divalent metal ion coordination in the catalytic center.d NADPH position relative to the α5β5 motif.e NADPH cleavage by NOCT mutants designed to disrupt the enzymatic activity. NOCT catalytic domain (500 nM) and NADPH (0.5 mM) were used. Reactions were conducted at 22 °C for 60 min

**Fig. 5**
Biochemical characterization of Curled.a Structure of human NOCT colored by sequence conservation between fruit fly and human proteins. Color scale shows a range from invariable (red) to random (blue).b³²P-poly-A RNA cleavage assays usingD. melanogaster Curled, Curled E140A active site mutant, and human deadenylases CNOT6L and PDE12 (2 μM each). Faint bands at 10 min in WT Curled arise from a minor contaminating endonuclease. The E140A mutant corresponds to the E195A mutation in human NOCT (Figs. 2a and 4e).c IEC trace for cleavage of NADPH (1 mM) by Curled fromDrosophila Melanogaster (500 nM). The catalytic constant was calculated ask_obs•S₀/E₀ from (c) and Supplementary Fig. 5b. This value represents the lower estimate fork_cat.d The proposed model for NOCT integration in the circadian system based on the results of our work. Source data are provided as a Source Data file

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References

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The metabolites NADP⁺ and NADPH are the targets of the circadian protein Nocturnin (Curled)

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The metabolites NADP⁺ and NADPH are the targets of the circadian protein Nocturnin (Curled)

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