Nudt8 is a novel CoA diphosphohydrolase that resides in the mitochondria

Abstract

CoA regulates energy metabolism and exists in separate pools in the cytosol, peroxisomes, and mitochondria. At the whole tissue level, the concentration of CoA changes with the nutritional state by balancing synthesis and degradation; however, it is currently unclear how individual subcellular CoA pools are regulated. Liver and kidney peroxisomes contain Nudt7 and Nudt19, respectively, enzymes that catalyze CoA degradation. We report that Nudt8 is a novel CoA-degrading enzyme that resides in the mitochondria. Nudt8 has a distinctive preference for manganese ions and exhibits a broader tissue distribution than Nudt7 and Nudt19. The existence of CoA-degrading enzymes in both peroxisomes and mitochondria suggests that degradation may be a key regulatory mechanism for modulating the intracellular CoA pools.

Keywords: Coenzyme A; Nudix hydrolase; cell compartmentalization; metabolic regulation; mitochondria.

Fig. 1

Expression and characterization of mouse Nudt8. (A) Alignment of the protein sequences of mouse and human Nudt8 and Nudt7. Invariant residues are highlighted in red, conserved residues in yellow, and groups of conserved residues are boxed. The Nudix box (blue stars) and the CoA box (pink circles) overlap at a glycine highlighted in black. Mm, Mus musculus; Hs, Homo sapiens; Genbank GI numbers for the aligned proteins are: MmNudt8, 13384950; HsNUDT8, 344217763; MmNudt7, 165972342; HsNUDT7, 157785656. Sequence alignments were obtained using ESPript 3 [44]. AUTHOR: Please check if Figure 1 has been reproduced from Ref. [44].. Please add the statement “Figure reproduced from Ref. [44]” to the end of figure caption if Figure 1 has been reproduced from published sources. Please also confirm if written permission has been obtained from the publisher/author if Figure 1 has been reproduced. Please ignore this query if figure parts described as being modified from other publications are sufficiently different and do not require permission statements for reproduction from the previous publisher/author. Please indicate in the query sheet as "Ignore" if this author query can be ignored(B) Coomassie blue-stained protein gel showing purified Nudt8. M, molecular weight marker. (C) Elution profile of purified, His-tagged Nudt8 from a Superdex 75 10/300 GL column. The inset shows the calibration curve used to estimate the apparent molecular weight of Nudt8. The calculated molecular weight of the recombinant protein, based on the amino acid composition, is 24.3 kDa. (D) High-performance liquid chromatography (HPLC) elution profile of the degradation product co-migrating with a 3′,5′-ADP standard formed in reaction mixtures containing Nudt8 and free CoA. * marks a solvent impurity.

Fig. 2

Divalent cation requirement and substrate specificity of recombinant Nudt8. (A) The ability of different divalent cations to support the hydrolytic activity of Nudt8 was tested in reaction mixtures containing 6 μg of Nudt8, 250 μm of free CoA and 0.5 mm of each cation introduced as the chloride salt. (B) Nudt8 and Nudt7 protein curves in the presence of the indicated concentrations of MgCl₂ and MnCl₂. Linear regression of the curves, obtained using three independently purified Nudt8 batches in duplicate, was used to calculate the specific activity of the enzymes ± standard error. In the presence of the optimal concentration of MnCl₂, the specific activity of Nudt8 (2.4 ± 0.1 pmoles·min⁻¹·ng⁻¹ of protein) was comparable to the specific activity of Nudt7 with either MgCl₂ (3.0 ± 0.1 pmoles·min⁻¹·ng⁻¹ of protein) or MnCl₂ (2.8 ± 0.1 pmles·min⁻¹·ng⁻¹ of protein). (C) CoA diphosphohydrolase activity of Nudt8 against multiple CoA substrates. Data in panels A and C are plotted as the mean (bars) of experiments conducted in duplicate using three independently purified Nudt8 batches (circles) ± SEM.

Fig. 3

Relative abundance and tissue distribution of Nudt8 mRNA and protein. (A) Relative abundance of mouse Nudt8, Nudt7, and Nudt19 transcripts in selected tissues. Data are reported as the mean (bars) of measurements on individual mice (circles) ± SEM. (B–D) HEK293T cells were transfected with either an empty plasmid or plasmids carrying the coding sequences for the FLAG-tagged versions of Nudt8, Nudt7, and Nudt19. Representative western blots showing that (B) the Nudt8 antibody did not cross-react with Nudt7 or Nudt19, while (C) the FLAG antibody detected all isoforms. (D) Quantification of the Nudt8 and FLAG signals from three independent transfections (circles) ± SEM. (E) Levels of Nudt8 protein in mouse tissues as quantified by western blot analysis. Data are reported as the mean (bars) of measurements of the Nudt8 signal in tissues from individual mice (circles) ± SEM. Inset, representative western blot. GAPDH was used to normalize the Nudt8 and FLAG signals in (B) and (C) and to normalize the tissue Nudt8 signal in (E).

Fig. 4

Nudt8 expression in the liver, kidneys and heart of fed and fasted mice. (A) Nudt8 mRNA in the liver, kidneys and heart of mice fed ad libitum or fasted for 24 h. (B) Western blot analysis showing that Nudt8 protein levels do not change with the nutritional state. Total protein loading is shown under each immunoblot. (C) Quantification of the western blots in (B) normalizing to total protein loading and expressing the corrected intensity relative to the fed state. Data in (A) and (C) are shown as the mean (bars) of measurements on individual mice (circles) ± SEM.

Fig. 5

Mitochondrial localization of Nudt8. HEK293 cells were transfected with plasmids encoding Nudt8 with a C-terminal FLAG tag, fixed, and analyzed by confocal microscopy. (A, C, D, F) Nudt8, visualized with the FLAG antibody, is in green. (B-C) Mitochondria, visualized using Mitotracker Orange CMTMRos, are in red. (E-F) Endogenous PMP70, used as a peroxisomal marker, is in red. Cell nuclei were stained with DAPI, in blue. Insets in merged images show details at higher magnification. The results are representative of at least two independent experiments. (G-H) Subcellular fractionation of mouse livers. Nudt8 exhibited a sedimentation profile similar to the mitochondrial marker citrate synthase. Catalase and Nudt7 were used as peroxisomal markers. The results are representative of four independent experiments quantified in (H) relative to the input in the Percoll layer, P. H, liver homogenate; F1–8, fractions 1–8.AUTHOR: Figure captions are extracted from the PDF source file. Please check if this is okay