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Identification and characterization of a human mitochondrial NAD kinase.

ABSTRACT: NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP(+) biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP(+) biosynthesis, which has pivotal roles in cells and organelles.

SUBMITTER: Ohashi K

PROVIDER: S-EPMC3535332 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Identification and characterization of a human mitochondrial NAD kinase.

Ohashi Kazuto K Kawai Shigeyuki S Murata Kousaku K

Nature communications 20120101

NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or ...[more]

PMID: 23212377

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Project description:BACKGROUND & AIMS:The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice. METHODS:We generated MNADK knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate, and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines. RESULTS:The KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of nonalcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein-hepatocyte specific and peroxisome proliferator-activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, an NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase. CONCLUSION:MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.

Project description:BackgroundInfectious coryza (IC) is an invasive upper respiratory disease caused by Avibacterium paragallinarum that affects birds, particularly chickens. The objective of this study is to isolate, characterize and molecularly identify the bacterium A. paragallinarum in poultry birds, as well as to determine its antibiotic sensitivity and resistance.MethodsA total of 10 chickens from four different Iranian farms with typical IC symptoms were used in this study. The nasal swabs were streaked onto chocolate agar plates and blood agar plates and incubated at 37°C in 5% CO2 for 24 to 48 h. As part of the identification of bacteria, bacteriological observations and polymerase chain reaction (PCR) testing are conducted. The antibiotic sensitivity tests were also performed using the disk diffusion method against A. paragallinarum and the prevalence in different farms was determined.ResultsBy using biochemical assays and PCR analyses, seven strains of A. paragallinarum were isolated from samples of four chicken farms with typical IC clinical signs. Most isolates (4/7) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. Three of the seven strains of A. paragallinarum were found to be novel NAD-independent under anaerobic conditions. There was one biochemical biovar identified in terms of carbohydrate fermentation patterns, although changes in maltose carbohydrate fermentation patterns were detected in the No. 5 strain. All isolates were sensitive to gentamicin and spectinomycin. Three novel NAD-independent strains (Nos.1, 5 and 7) were found to be multidrug-resistant (MDR) and resistant to at least three classes of antibiotics. There was greater antibiotic resistance in the three NAD-independent isolates than in normal NAD-dependent bacteria.ConclusionBy discovering NAD-independent forms of A. paragallinarum, these species have a greater range than previously believed. A clear, cautious approach should be taken in diagnosing and possibly controlling IC.

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Identification and characterization of a human mitochondrial NAD kinase.

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Identification and characterization of a human mitochondrial NAD kinase.

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