Whereas nicotinamide metabolism is not conserved between vertebrates and fungi, presence of NRK genes suggests that the nicotinamide riboside kinase pathway is conserved more broadly in eukaryotes, though it is likely to be time and tissue-restricted in animals.
First reported in 1955, high doses of nicotinic acid are effective at reducing cholesterol levels (Altschul et al., 1955). Since the initial report, many controlled clinical studies have shown that nicotinic acid preparations, alone and in combination with HMG co-A reductase inhibitors, are effective in controlling low-density lipoprotein cholesterol, increasing high-density lipoprotein cholesterol, and reducing triglyceride and lipoprotein a levels in humans (Pasternak et al., 1996). Though nicotinic acid treatment effects all of the key lipids in the desirable direction and has been shown to reduce mortality in target populations (Pasternak et al., 1996), its use is limited because of a side effect of heat and redness termed “flushing,” which is significantly effected by the nature of formulation (Capuzzi et al., 2000). Thus, it will be important to test whether nicotinamide riboside supplementation is a preferred route to improve lipid profiles in humans. Additionally, study of the expression and regulation of NAD+ biosynthetic enzymes is expected to reveal approaches to sensitize tumors to compounds such as tiazofurin, to protect normal tissues from the toxicity of compounds such as tiazofurin adenine dinucleotide, and to stratify patients for the most judicious use of tiazofurin chemotherapy.
Experimental Procedures
S. cerevisiae Strains
Yeast diploid strain BY165, heterozygous for qns1 deletion and haploid BY165-1d carrying a chromosomal deletion of qns1 gene, transformed with plasmid pB175 containing QNS1 and URA3 were described previously (Bieganowski et al., 2003). Genetic deletions were introduced by direct transformation with PCR products (Brachmann et al., 1998) generated from primers listed in Supplemental Data available at
http://www.cell.com/cgi/content/full/117/4/495/DC1. Plating on media containing 5-fluoroorotic acid (Boeke et al., 1987) was after growth for 24 hr on complete media. The ado1 disruption cassette was constructed by PCR with primers 7041 and 7044 and plasmid pRS413 as a template. Yeast strain BY165 was transformed with this PCR product, and homologous recombination in histidine prototrophic transformants was confirmed by PCR with primers 7042 and 7043. This strain was transformed with plasmid pB175 and subjected to sporulation and tetrad dissection. One of the resulting haploids carrying qns1 and ado1 deletions and plasmid was selected for further experiments and named BY237. The urk1 deletion was introduced into strain BY237 by transformation with the product of the PCR amplification that used pRS415 as a template and primers 7051 and 7052. Disruption was confirmed by PCR with primers 7053 and 7054, and the resulting strain was named BY247. The rbk1 disruption cassette was constructed by PCR with primers 7063 and 7065 and plasmid pRS411 as a template. Disruption was introduced into strain BY242 by transformation with the product of this reaction and confirmed by PCR with primers 7062 and 7064. The resulting strain, carrying deletions of qns1, ado1, urk1, and rbk1 genes was named BY252. A qns1 yeast strain carrying disruption of the nrk1 locus was made by transformation of strain BY165-1d with an nrk1Δ::HIS3 cassette generated by PCR with primers 4750 and 4751 and plasmid pRS413 as template. Correct integration of the HIS3 marker into the nrk1 locus was confirmed by PCR with primers 4752 and 4753.
Nicotinamide Riboside and Whey Preparations
NAD+ (Sigma) concentration was determined by conversion to NADH with alcohol dehydrogenase using an absorption coefficient (340 nm) of 6200 cm−1 M−1. The concentration of NMN was determined by converting NAD+ to NMN plus AMP with rattlesnake venom NAD+ pyrophosphatase (E.C. 3.6.9.1, Sigma). Using 15,400 cm−1 M−1 as the absorption coefficient for AMP at 259 nm, we used relative peak areas to calculate the absorption coefficient (259 nm) of NMN to be 4740 cm−1 M−1. To prepare nicotinamide riboside, 120 μmol NMN (Sigma, concentration corrected by absorption) was treated with 1250 units of calf intestinal alkaline phosphatase (Sigma) for 1 hr at 37°C in 1 ml 100 mM NaCl, 20 mM Tris [pH 8.0], 5 mM MgCl2. After hydrolysis of NMN to nicotinamide riboside was verified by HPLC, phosphatase was removed by centrifuging the reaction through a 5000 Da filter (Millipore). A whey vitamin fraction of commercial nonfat cow's milk was prepared by adjusting the pH to 4 with HCl, stirring at 55°C for 10 min, removal of denatured casein by centrifugation, and passage through a 5000 Da filter. In yeast media, nicotinamide riboside was used at 10 μM and whey vitamin fraction at 50% by volume.
Yeast GST-ORF Library
Preparation of the fusion protein library was as described (Martzen et al. 1999, Phizicky et al. 2002) at a 500 ml culture scale for each of the 64 pools of 90–96 protein constructs. 10% of each pool preparation was assayed for Nrk activity in overnight incubations.
Nicotinamide Riboside Phosphorylation Assays
Reactions (0.2 ml), containing 100 mM NaCl, 20 mM Na HEPES [pH 7.2], 5 mM β-mercaptoethanol, 1 mM ATP, 5 mM MgCl2, and 500 μM nicotinamide riboside or alternate nucleoside were incubated at 30°C and terminated by addition of EDTA to 20 mM and heating for 2 min at 100°C. Specific activity assays, containing 50 ng to 6 μg enzyme depending on the enzyme and substrate, were incubated for 30 min at 30°C to maintain initial rate conditions. Reaction products were analyzed by HPLC on a strong anion exchange column with a 10 mM to 750 mM gradient of KPO4 [pH 2.6].
NRK Gene and cDNA Cloning and Enzyme Purification
The S. cerevisiae NRK1 gene was amplified from total yeast DNA with primers 7448 and 7449. The amplified DNA fragment was cloned in vector pSGA04 (Ghosh and Lowenstein, 1997) for E. coli expression using restriction sites for NdeI and XhoI included in primer sequences and the resulting plasmid was named pB446. Samples of cDNA made from human lymphocytes and spleen were used as a template for amplification of human NRK1 performed with primers 4754 and 4755. Plasmid pB449 was created by cloning of the product of this reaction between restriction sites NcoI and BamHI of vector pMR103 (Munson et al., 1994) for E. coli expression. Plasmid pB449 was used as a template for PCR reaction with primers 7769 and 7770. The product of this amplification was cloned between BamHI and XhoI sites of vector p425GAL1 (Mumberg et al., 1994) and the resulting plasmid carrying human NRK1 gene under GAL1 promoter control was named pB450. Human NRK2 cDNA was amplified with primers 7777 and 7776. The amplified fragment was digested with NdeI and XhoI enzymes and cloned in plasmid pSGA04 for E. coli expression (pB457) and into plasmid p425GAL1 for yeast expression (pB459). His-tagged enzymes were purified by immobilized cobalt affinity chromatography (Becton Dickenson).
Acknowledgements
This research was supported by National Cancer Institute grant CA77738.
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