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Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors.

ABSTRACT: Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type-specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells.

SUBMITTER: Abshire ET

PROVIDER: S-EPMC7606674 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors.

Abshire Elizabeth T ET Hughes Kelsey L KL Diao Rucheng R Pearce Sarah S Gopalakrishna Shreekara S Trievel Raymond C RC Rorbach Joanna J Freddolino Peter L PL Goldstrohm Aaron C AC

The Journal of biological chemistry 20200823 44

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type-specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and proc ...[more]

PMID: 32839274

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Dataset Information

Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors.

Publications

Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide adenine dinucleotide cofactors.

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