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• W2787361799 abstract "CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism. Dynamic regulation of CoA in different tissues and organs supports metabolic flexibility. Two mammalian Nudix hydrolases, Nudt19 and Nudt7, degrade CoA in vitro. Nudt19 and Nudt7 possess conserved Nudix and CoA signature sequences and specifically hydrolyze the diphosphate bond of free CoA and acyl-CoAs to form 3′,5′-ADP and 4′-(acyl)phosphopantetheine. Limited information is available on these enzymes, but the relatively high abundance of Nudt19 and Nudt7 mRNA in the kidney and liver, respectively, suggests that they play specific roles in the regulation of CoA levels in these organs. Here, we analyzed Nudt19−/− mice and found that deletion of Nudt19 elevates kidney CoA levels in mice fed ad libitum, indicating that Nudt19 contributes to the regulation of CoA in vivo. Unlike what was observed for the regulation of Nudt7 in the liver, Nudt19 transcript and protein levels in the kidney did not differ between fed and fasted states. Instead, we identified chenodeoxycholic acid as a specific Nudt19 inhibitor that competed with CoA for Nudt19 binding but did not bind to Nudt7. Exchange of the Nudix and CoA signature motifs between the two isoforms dramatically decreased their kcat. Furthermore, substitutions of conserved residues within these motifs identified amino acids playing different roles in CoA binding and hydrolysis in Nudt19 and Nudt7. Our results reveal that the kidney and liver each possesses a distinct peroxisomal CoA diphosphohydrolase. CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism. Dynamic regulation of CoA in different tissues and organs supports metabolic flexibility. Two mammalian Nudix hydrolases, Nudt19 and Nudt7, degrade CoA in vitro. Nudt19 and Nudt7 possess conserved Nudix and CoA signature sequences and specifically hydrolyze the diphosphate bond of free CoA and acyl-CoAs to form 3′,5′-ADP and 4′-(acyl)phosphopantetheine. Limited information is available on these enzymes, but the relatively high abundance of Nudt19 and Nudt7 mRNA in the kidney and liver, respectively, suggests that they play specific roles in the regulation of CoA levels in these organs. Here, we analyzed Nudt19−/− mice and found that deletion of Nudt19 elevates kidney CoA levels in mice fed ad libitum, indicating that Nudt19 contributes to the regulation of CoA in vivo. Unlike what was observed for the regulation of Nudt7 in the liver, Nudt19 transcript and protein levels in the kidney did not differ between fed and fasted states. Instead, we identified chenodeoxycholic acid as a specific Nudt19 inhibitor that competed with CoA for Nudt19 binding but did not bind to Nudt7. Exchange of the Nudix and CoA signature motifs between the two isoforms dramatically decreased their kcat. Furthermore, substitutions of conserved residues within these motifs identified amino acids playing different roles in CoA binding and hydrolysis in Nudt19 and Nudt7. Our results reveal that the kidney and liver each possesses a distinct peroxisomal CoA diphosphohydrolase. CoA is an obligate cofactor utilized as an acyl carrier in hundreds of metabolic reactions. Numerous CoA thioesters are also involved in the post-translational modification of histones and thousands of other proteins (1Choudhary C. Weinert B.T. Nishida Y. Verdin E. Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling.Nat. Rev. Mol. Cell. Biol. 2014; 15 (25053359): 536-55010.1038/nrn374710.1038/nrm3841Crossref PubMed Scopus (892) Google Scholar2Hirschey M.D. Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation.Mol. Cell. Proteomics. 2015; 14 (25717114): 2308-231510.1074/mcp.R114.046664Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 3Resh M.D. Fatty acylation of proteins: The long and the short of it.Prog. Lipid Res. 2016; 63 (27233110): 120-13110.1016/j.plipres.2016.05.002Crossref PubMed Scopus (167) Google Scholar, 4Daniotti J.L. Pedro M.P. Valdez Taubas J. The role of S-acylation in protein trafficking.Traffic. 2017; 18 (28837239): 699-71010.1111/tra.12510Crossref PubMed Scopus (32) Google Scholar5Sabari B.R. Zhang D. Allis C.D. Zhao Y. Metabolic regulation of gene expression through histone acylations.Nat. Rev. Mol. Cell. Biol. 2017; 18 (27924077): 90-10110.1038/nrm.2016.140Crossref PubMed Scopus (491) Google Scholar). Three major subcellular CoA pools are found in the cytosol, mitochondria, and peroxisomes to support specific metabolic pathways (6Horie S. Ishii H. Suga T. Changes in peroxisomal fatty acid oxidation in the diabetic rat liver.J. Biochem. 1981; 90 (7334004): 1691-169610.1093/oxfordjournals.jbchem.a133645Crossref PubMed Scopus (142) Google Scholar, 7Van Broekhoven A. Peeters M.C. Debeer L.J. Mannaerts G.P. Subcellular distribution of coenzyme A: evidence for a separate coenzyme A pool in peroxisomes.Biochem. Biophys. Res. Commun. 1981; 100 (7259752): 305-31210.1016/S0006-291X(81)80097-6Crossref PubMed Scopus (43) Google Scholar). These include, among others, fatty acid synthesis in the cytosol, the TCA cycle and oxidation of long-chain and medium-chain fatty acids in the mitochondria, and bile acid conjugation and oxidation of very long-chain and branched-chain fatty acids in the peroxisomes. A dedicated pool of acetyl-CoA is also found in the endoplasmic reticulum, where it is involved in protein quality control and autophagy (8Peng Y. Puglielli L. N-lysine acetylation in the lumen of the endoplasmic reticulum: a way to regulate autophagy and maintain protein homeostasis in the secretory pathway.Autophagy. 2016; 12 (27124586): 1051-105210.1080/15548627.2016.1164369Crossref PubMed Scopus (21) Google Scholar), whereas nuclear acyl-CoAs, which freely equilibrate with the cytosolic pool across the nuclear pores, contribute to the regulation of gene expression (5Sabari B.R. Zhang D. Allis C.D. Zhao Y. Metabolic regulation of gene expression through histone acylations.Nat. Rev. Mol. Cell. Biol. 2017; 18 (27924077): 90-10110.1038/nrm.2016.140Crossref PubMed Scopus (491) Google Scholar). Tight control over the concentration of CoA in different organs is essential to maintain normal metabolism and organ function. Indeed, elevated CoA levels in the liver of diabetic mice promote excessive gluconeogenesis and hyperglycemia (9Leonardi R. Rock C.O. Jackowski S. Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.Diabetologia. 2014; 57 (24781151): 1466-147510.1007/s00125-014-3245-5Crossref PubMed Scopus (26) Google Scholar), whereas a supraphysiological concentration of CoA in skeletal muscle leads to decreased ATP levels and exercise performance (10Corbin D.R. Rehg J.E. Shepherd D.L. Stoilov P. Percifield R.J. Horner L. Frase S. Zhang Y.M. Rock C.O. Hollander J.M. Jackowski S. Leonardi R. Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.Mol. Genet. Metab. 2017; 120 (28189602): 350-36210.1016/j.ymgme.2017.02.001Crossref PubMed Scopus (8) Google Scholar). Adverse consequences are also associated with lower-than-normal CoA levels. Decreased synthesis of this cofactor in the nervous system is associated with a rare neurological disorder in humans (11Dusi S. Valletta L. Haack T.B. Tsuchiya Y. Venco P. Pasqualato S. Goffrini P. Tigano M. Demchenko N. Wieland T. Schwarzmayr T. Strom T.M. Invernizzi F. Garavaglia B. Gregory A. et al.Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.Am. J. Hum. Genet. 2014; 94 (24360804): 11-2210.1016/j.ajhg.2013.11.008Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Zhou B. Westaway S.K. Levinson B. Johnson M.A. Gitschier J. Hayflick S.J. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden–Spatz syndrome.Nat. Genet. 2001; 28 (11479594): 345-34910.1038/ng572Crossref PubMed Scopus (619) Google Scholar), whereas decreased synthesis of CoA in the liver leads to fasting hypoglycemia and hepatic triglyceride accumulation (13Leonardi R. Rehg J.E. Rock C.O. Jackowski S. Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.PLoS One. 2010; 5 (20559429)e1110710.1371/journal.pone.0011107Crossref PubMed Scopus (75) Google Scholar). Dynamic regulation of CoA within the normal physiological range is important to support the metabolic reprogramming that underlies the capacity to respond to changes in the metabolic state. For example, the increase in CoA that characterizes the fed-to-fasted transition in the liver is driven by the activation of the biosynthetic pathway and is required to sustain the high rates of hepatic fatty acid oxidation and gluconeogenesis under fasting conditions (13Leonardi R. Rehg J.E. Rock C.O. Jackowski S. Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.PLoS One. 2010; 5 (20559429)e1110710.1371/journal.pone.0011107Crossref PubMed Scopus (75) Google Scholar, 14Jackowski S. Leonardi R. Deregulated coenzyme A, loss of metabolic flexibility and diabetes.Biochem. Soc. Trans. 2014; 42 (25110012): 1118-112210.1042/BST20140156Crossref PubMed Scopus (21) Google Scholar). On the other hand, the net decrease in the concentration of CoA observed upon refeeding after a fast requires both inhibition of de novo CoA synthesis and active degradation of the cofactor accumulated in the fasted state. The existence of a CoA-degradation pathway in the liver and kidneys is supported by the fact that mice treated with an inhibitor of the CoA biosynthetic pathway exhibit a dramatic reduction in hepatic and renal CoA, indicating rapid CoA turnover in these tissues (15Zhang Y.M. Chohnan S. Virga K.G. Stevens R.D. Ilkayeva O.R. Wenner B.R. Bain J.R. Newgard C.B. Lee R.E. Rock C.O. Jackowski S. Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.Chem. Biol. 2007; 14 (17379144): 291-30210.1016/j.chembiol.2007.01.013Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Liver and kidneys contain high transcript levels of two Nudix hydrolases, Nudt7 and Nudt19, respectively, which exhibit CoA diphosphohydrolase activity in vitro (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar, 17Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357 (11415433): 33-3810.1042/0264-6021:357003310.1042/bj3570033Crossref PubMed Scopus (79) Google Scholar). Nudix hydrolases hydrolyze the diphosphate bond of a variety of substrates consisting of a nucleoside diphosphate linked to another moiety, x (18Bessman M.J. Frick D.N. O'Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271 (8810257): 25059-2506210.1074/jbc.271.41.25059Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 19Carreras-Puigvert J. Zitnik M. Jemth A.S. Carter M. Unterlass J.E. Hallström B. Loseva O. Karem Z. Calderón-Montaño J.M. Lindskog C. Edqvist P.H. Matuszewski D.J. Ait Blal H. Berntsson R.P.A. Häggblad M. et al.A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family.Nat. Commun. 2017; 8 (29142246)154110.1038/s41467-017-01642-wCrossref PubMed Scopus (71) Google Scholar). All members of the Nudix superfamily contain the conserved Nudix box motif G1N[5X]E7N[7X]R15NE16NXXE19NE20NXG22NU (where U is a hydrophobic residue, X is any residue, and superscript N denotes the position of a residue of the signature motif within a specific protein sequence) (20McLennan A.G. The Nudix hydrolase superfamily.Cell. Mol. Life Sci. 2006; 63 (16378245): 123-14310.1007/s00018-005-5386-7Crossref PubMed Scopus (455) Google Scholar, 21de la Peña A.H. Suarez A. Duong-Ly K.C. Schoeffield A.J. Pizarro-Dupuy M.A. Zarr M. Pineiro S.A. Amzel L.M. Gabelli S.B. Structural and enzymatic characterization of a nucleoside diphosphate sugar hydrolase from Bdellovibrio bacteriovorus.PLoS One. 2015; 10 (26524597)e014171610.1371/journal.pone.0141716Crossref PubMed Scopus (6) Google Scholar). Additionally, Nudix hydrolases that specifically hydrolyze CoA also contain a putative CoA binding motif, (L/M)(L/F)TXR(S/A)[3X](R/K)[3X]G[3X]FPGG (PROSITE accession number PS01293, formerly UPF0035), upstream of the Nudix box motif (22Cartwright J.L. Gasmi L. Spiller D.G. McLennan A.G. The Saccharomyces cerevisiae PCD1 gene encodes a peroxisomal nudix hydrolase active toward coenzyme A and its derivatives.J. Biol. Chem. 2000; 275 (10922370): 32925-3293010.1074/jbc.M005015200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The crystal structure of human NUDT7 has recently been released (PDB 3The abbreviations used are: PDBProtein Data BankCDCAchenodeoxycholic acidmBBmonobromobimanePTS1peroxisome targeting signal type 1ITCisothermal titration calorimetryKOMPKnock-out Mouse Project. code 5T3P). Similar to the structure of the bacterial homolog from Deinococcus radiodurans (24Kang L.W. Gabelli S.B. Bianchet M.A. Xu W.L. Bessman M.J. Amzel L.M. Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family.J. Bacteriol. 2003; 185 (12837785): 4110-411810.1128/JB.185.14.4110-4118.2003Crossref PubMed Scopus (34) Google Scholar), the crystal structure of the human enzyme does not contain CoA bound. Thus, the contribution of the CoA motif to CoA binding and the organization of the CoA-binding site in these enzymes are currently unknown. Protein Data Bank chenodeoxycholic acid monobromobimane peroxisome targeting signal type 1 isothermal titration calorimetry Knock-out Mouse Project. Independent studies and approaches have demonstrated the localization of Nudt7 in the peroxisomes (17Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357 (11415433): 33-3810.1042/0264-6021:357003310.1042/bj3570033Crossref PubMed Scopus (79) Google Scholar, 25Shumar S.A. Fagone P. Alfonso-Pecchio A. Gray J.T. Rehg J.E. Jackowski S. Leonardi R. Induction of neuron-specific degradation of coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.PLoS One. 2015; 10 (26052948)e013001310.1371/journal.pone.0130013Crossref PubMed Scopus (19) Google Scholar). Nudt7 mRNA and protein levels in the liver are regulated by the nutritional state and are inversely correlated with the tissue concentration of CoA (9Leonardi R. Rock C.O. Jackowski S. Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.Diabetologia. 2014; 57 (24781151): 1466-147510.1007/s00125-014-3245-5Crossref PubMed Scopus (26) Google Scholar, 26Reilly S.J. Tillander V. Ofman R. Alexson S.E. Hunt M.C. The nudix hydrolase 7 is an Acyl-CoA diphosphatase involved in regulating peroxisomal coenzyme A homeostasis.J. Biochem. 2008; 144 (18799520): 655-66310.1093/jb/mvn114Crossref PubMed Scopus (45) Google Scholar). Additionally, overexpression of a catalytically active, cytosolic form of Nudt7 has also recently been shown to cause a decrease in CoA levels in vivo (25Shumar S.A. Fagone P. Alfonso-Pecchio A. Gray J.T. Rehg J.E. Jackowski S. Leonardi R. Induction of neuron-specific degradation of coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.PLoS One. 2015; 10 (26052948)e013001310.1371/journal.pone.0130013Crossref PubMed Scopus (19) Google Scholar). Significantly less is known about Nudt19. Nudt19 exhibits modest sequence similarity to Nudt7 (∼30%), is larger than a typical Nudix hydrolase, including Nudt7, and contains a unique 45–49-amino acid insertion of unknown function within its Nudix box (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar). This enzyme, also known as RP2p, was originally reported to be a kidney protein whose transcript levels were robustly up-regulated by androgens (27Rheaume C. Barbour K.W. Tseng-Crank J. Berger F.G. Molecular genetics of androgen-inducible RP2 gene transcription in the mouse kidney.Mol. Cell. Biol. 1989; 9 (2710112): 477-48310.1128/MCB.9.2.477Crossref PubMed Scopus (14) Google Scholar). Later, recombinant Nudt19 was shown to specifically hydrolyze CoA species (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar), although modest activity against capped RNA has also been detected (28Song M.G. Bail S. Kiledjian M. Multiple Nudix family proteins possess mRNA decapping activity.RNA. 2013; 19 (23353937): 390-39910.1261/rna.037309.112Crossref PubMed Scopus (89) Google Scholar). Based on proteomics studies, Nudt19 localization has been reported in both peroxisomes and mitochondria (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar, 29Calvo S.E. Clauser K.R. Mootha V.K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins.Nucleic Acids Res. 2016; 44 (26450961): D1251-D125710.1093/nar/gkv1003Crossref PubMed Scopus (854) Google Scholar); however, it is currently unclear whether the enzyme has dual localization or exclusively resides in one of the two subcellular compartments, and which one. Given the presence of dedicated CoA pools and complementary metabolic processes in peroxisomes and mitochondria, this distinction is important to gain insight into the physiological role of Nudt19, even in relation to the other mammalian isoform, Nudt7. Indeed, it is presently unclear whether Nudt7 and Nudt19, which seem to be differentially expressed in liver and kidneys, differ in key aspects of their function. We have investigated the effect of Nudt19 deletion on CoA levels, the localization of this enzyme in intact cells, and its regulation by selected metabolites. Additionally, we used a combination of computational modeling, substrate docking, and site-directed mutagenesis to identify residues involved in CoA binding and hydrolysis in Nudt19 and analyzed the role of equivalent residues in Nudt7. We show that Nudt19 is a peroxisomal enzyme that contributes to the regulation of kidney CoA levels in vivo and provide evidence supporting the conclusion that the CoA-binding sites of Nudt19 and Nudt7 are different. Previous work has shown that Nudt19 hydrolyzes free CoA and acyl-CoAs to 3′,5′-ADP and 4′-(acyl)phosphopantetheine in vitro (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar). We confirmed these findings by measuring the activity of recombinant Nudt19 against several acyl-CoAs and comparing it to the better characterized Nudt7 (17Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357 (11415433): 33-3810.1042/0264-6021:357003310.1042/bj3570033Crossref PubMed Scopus (79) Google Scholar, 25Shumar S.A. Fagone P. Alfonso-Pecchio A. Gray J.T. Rehg J.E. Jackowski S. Leonardi R. Induction of neuron-specific degradation of coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.PLoS One. 2015; 10 (26052948)e013001310.1371/journal.pone.0130013Crossref PubMed Scopus (19) Google Scholar) (Fig. 1, A and B). We determined that the optimal concentration of MgCl2 for Nudt7 and Nudt19 was 4 and 10 mm, respectively, when free CoA was used as the substrate (data not shown). However, in experiments where we tested multiple acyl-CoAs, it was necessary to keep the concentration of MgCl2 at 4 mm for both Nudt7 and Nudt19 to prevent the precipitation of lauroyl- and stearoyl-CoA. Under these conditions, the specific activity per pmol of Nudt19 was lower than that of Nudt7, but the enzyme readily hydrolyzed short and medium chain acyl-CoAs, malonyl- and succinyl-CoA, in addition to free CoA (Fig. 1A). Exceptions were acetyl-CoA and the synthetic monobromobimane (mBB)-CoA, which were excellent substrates for Nudt7, but not for Nudt19. Nudt19 also had no detectable hydrolytic activity against ATP, ADP, NAD+ NADH, NADP+, and NADPH (data not shown), confirming the specificity of this enzyme for CoA (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar). The mRNA levels of Nudt19 are highly abundant in mouse kidneys (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar, 30Snider L.D. King D. Lingrel J.B. Androgen regulation of MAK mRNAs in mouse kidney.J. Biol. Chem. 1985; 260 (2410417): 9884-9893Abstract Full Text PDF PubMed Google Scholar). To examine the distribution of this protein in mouse tissues, we generated a polyclonal antibody against the full-length mouse Nudt19. Consistent with the mRNA expression pattern, Nudt19 protein showed the highest abundance in the kidneys, with lower but still detectable levels in skeletal muscle and brain (Fig. 1C). This expression pattern was strikingly different from Nudt7, which was expressed at the highest levels in the liver and, to a significantly lower extent, in white and brown adipose tissue (Fig. 1C). The high expression level of Nudt19, combined with the minimally detectable Nudt7 protein in the kidneys (Fig. 1C), suggested that Nudt19 was the major of the two isoforms in these organs. To determine whether Nudt19 regulated CoA levels in vivo, we obtained Nudt19+/− mice from the KOMP repository and bred them to generate Nudt19−/− mice and wildtype littermate controls. The Nudt19−/− mice were viable, fertile, and outwardly normal. Successful deletion of the gene was verified by PCR analysis of tail biopsies (Fig. 2A), whereas Western blotting analysis confirmed the lack of Nudt19 protein expression (Fig. 2B). Additionally, enzymatic assays confirmed the lack of any residual formation of 3′,5′-ADP in kidney homogenates obtained from Nudt19−/− mice (Fig. 2C). The concentration of CoA was measured in the kidneys of Nudt19−/− and control mice in the fed state and following an overnight fast. In the fed state, the kidneys of the Nudt19−/− mice exhibited a significant 20% increase in the concentration of CoA compared with the wildtype mice (Fig. 2D). Fasting induced an increase in kidney CoA levels in both wildtype and Nudt19−/− mice. Under these conditions, the concentration of CoA in the Nudt19−/− kidneys tended to be higher than control mice, but the difference did not reach statistical significance. No compensatory increase in Nudt7 transcript levels was detected in the kidneys of the Nudt19−/− mice in either the fed or fasted states (Fig. 2E). Combined, these data showed that Nudt19 was the major CoA diphosphohydrolase in the kidneys and that this enzyme contributed to the regulation of kidney CoA levels in vivo. Mouse Nudt7 and other CoA diphosphohydrolases from Saccharomyces cerevisiae and Caenorhabditis elegans localize to the peroxisomes (17Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357 (11415433): 33-3810.1042/0264-6021:357003310.1042/bj3570033Crossref PubMed Scopus (79) Google Scholar, 22Cartwright J.L. Gasmi L. Spiller D.G. McLennan A.G. The Saccharomyces cerevisiae PCD1 gene encodes a peroxisomal nudix hydrolase active toward coenzyme A and its derivatives.J. Biol. Chem. 2000; 275 (10922370): 32925-3293010.1074/jbc.M005015200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 31AbdelRaheim S.R. McLennan A.G. The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase.BMC Biochem. 2002; 3 (11943069): 5Crossref PubMed Scopus (20) Google Scholar), which suggests that these enzymes might specifically regulate the peroxisomal pool of CoA (32Hunt M.C. Tillander V. Alexson S.E. Regulation of peroxisomal lipid metabolism: the role of acyl-CoA and coenzyme A metabolizing enzymes.Biochimie. 2014; 98 (24389458): 45-5510.1016/j.biochi.2013.12.018Crossref PubMed Scopus (61) Google Scholar, 33Antonenkov V.D. Hiltunen J.K. Peroxisomal membrane permeability and solute transfer.Biochim. Biophys. Acta. 2006; 1763 (17045662): 1697-170610.1016/j.bbamcr.2006.08.044Crossref PubMed Scopus (55) Google Scholar). Nudt19 contains a C-terminal peroxisome targeting signal type 1 (PTS1), Ala-His-Leu, and was originally identified as a peroxisomal enzyme by the proteomic analysis of kidney peroxisomes (16Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393 (16185196): 537-54310.1042/BJ20050893Crossref PubMed Scopus (70) Google Scholar). Recently, proteomic studies conducted on isolated mitochondria have also led to the annotation of Nudt19 as a mitochondrial enzyme in the Mitocarta2.0 inventory (29Calvo S.E. Clauser K.R. Mootha V.K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins.Nucleic Acids Res. 2016; 44 (26450961): D1251-D125710.1093/nar/gkv1003Crossref PubMed Scopus (854) Google Scholar). Given the common contamination of isolated peroxisomes and mitochondria with variable amounts of other organelles, we used immunofluorescence and confocal microscopy to determine the intracellular localization of Nudt19 in whole HEK 293 cells (Fig. 3). HEK 293 cells were transiently transfected with constructs expressing Nudt19 with N- or C-terminal GFP tags. Nuclei were stained with DAPI, mitochondria were stained with MitoTracker® Orange CMTMRos, and peroxisomes were visualized with an antibody against the endogenous peroxisomal protein PMP70. When expressed with an N-terminal GFP tag that left the PTS1 exposed, Nudt19 colocalized with PMP70, as shown by the yellow pixels resulting from the overlap of the green and red contributions (Fig. 3, A–C). Masking the PTS1 of Nudt19 with a C-terminal GFP tag prevented the peroxisomal localization (Fig. 3, G–I) but did not direct the protein to the mitochondria (Fig. 3, J–L), leading instead to a diffuse cytoplasmic localization. Overall, these results supported the conclusion that Nudt19 was a peroxisomal enzyme. As such, the 20% increase measured in the whole kidney homogenates from fed Nudt19−/− mice (Fig. 2D) could be an underestimation of the local accumulation of CoA in the kidney peroxisomes of these animals. Deletion of Nudt19 led to a significant increase in kidney CoA levels in fed but not fasted mice (Fig. 2D). This suggested that, in a wildtype mouse, the activity of Nudt19 might be higher in the fed state compared with the fasted state. We analyzed Nudt19 mRNA and protein expression levels in the kidneys of mice fed ad libitum and mice fasted for up to 48 h, but we did not detect any difference (Fig. 4, A–C). We then focused our attention on metabolite regulation. We screened a panel of 30 compounds, including a large number of substrates and products of peroxisomal metabolism, for their effect on the activity of recombinant Nudt19 (Table 1). None of the compounds tested significantly stimulated Nudt19 activity. Instead, we found that a select group of bile acids inhibited the enzyme by >50%. More specifically, chenodeoxycholic acid (CDCA) and its conjugated derivatives, taurochenodeoxycholic acid and glycochenodeoxycholic acid, decreased Nudt19 activity by 60–75% with respect to vehicle control (Table 1). More hydrophobic bile acids, such as lithocholic acid and ursocholanic acid, were also potent inhibitors of Nudt19, and α-muricholic acid, which derives from CDCA by the addition of a 6β-hydroxyl group, still retained significant potency against the enzyme. In contrast, cholic acid and its glycine and taurine conjugates exhibited decreased potency against Nudt19 compared with CDCA. Furthermore, other steroid compounds, such as progesterone, pregnenolone, and corticosterone, had no significant effect on the activity of the enzyme, confirming the specificity of the inhibitory effect of CDCA and derived bile acids on Nudt19 (Table 1).Table 1Effect of selected metabolites on the activity of Nudt19 and Nudt7MetaboliteActivity relative to vehicleNudt19Nudt7%Hydrogen peroxide (10 μm)+16−11Hydrogen peroxide (100 μm)+3+5ATP+6+2NAD+−5+1NADH−13+20NADP+−16+4NADPH−5+7NMN−8−1Pyru" @default.
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• W2787361799 date "2018-03-01" @default.
• W2787361799 modified "2023-10-18" @default.
• W2787361799 title "Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform" @default.
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