Structural Basis for the Specificity, Catalysis, and Regulation of Human Uridine-Cytidine Kinase

Suzuki, Nobuo; Koizumi, Katsuhisa; Fukushima, Masanori; Matsuda, Akira; Inagaki, Fuyuhiko

doi:10.1016/j.str.2004.02.038

Cited by 71 publications

(87 citation statements)

References 44 publications

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“…On the other hand, UCK-1 was not able to phosphorylate PSI-6130 or 2Ј-C-Me-C. These UCK-1 results are consistent with the findings of previous structural and biochemical studies, which showed that UCK has a high specificity for the 2Ј-hydroxyl group (28,29). Clearly, the presence of the 2Ј-methyl group also influences the affinities of these nucleosides analogs for UCK.…”

Section: Discussionsupporting

confidence: 90%

Mechanism of Activation of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Murakami¹,

Bao²,

Ramesh³

et al. 2007

Antimicrob Agents Chemother

102

101

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) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a K m of 81 M and a k cat of 0.007 s ؊1 , but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (K i ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 M. Similar results were obtained with 2-C-methyladenosine triphosphate (K i ‫؍‬ 1.5 M) and 2-C-methylcytidine triphosphate (K i ‫؍‬ 1.6 M). NS5B with the S282T mutation, which is known to confer resistance to 2-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.Hepatitis C virus (HCV) is an RNA virus which possesses a single-stranded positive-sense RNA as the viral genome. This viral RNA plays important roles during viral replication, as it serves as an mRNA for viral protein synthesis, a template for RNA replication, and a nascent RNA genome for a newly formed virus (17). HCV NS5B RNA-dependent RNA polymerase is a key enzyme in viral RNA replication. This enzyme, which does not require a primer for initiation of RNA synthesis, catalyzes de novo RNA synthesis (8, 11). Nucleoside analogs have been used to treat viral infections, such as herpes simplex virus, human immunodeficiency virus, and hepatitis B virus infections (5,6,21). These drugs are designed to inhibit viral polymerases by a process called chain termination, in which DNA synthesis is quenched by incorporating the triphosphate forms of these drugs, which lack the 3Ј-hydroxyl group on the sugar moiety. In order for nucleoside analogs to inhibit a viral polymerase, they must be transported into the cell and converted to the active 5Ј-triphosphate form by cellular kinases. 2Ј-C-Methylnucleosides have been investigated as anti-HCV agents targeting HCV NS5B RNA polymerase (2, 20). 2Ј-C-Methyladenosine (2Ј-C-Me-A) and 2Ј-C-methylguanosine (2Ј-C-Me-G) showed potent anti-HCV activities in a cell-based replicon assay, and their triphosphate forms inhibited replicase and NS5B RNA polymerase in vitro (20). In addition, 2Ј-C-Me-A exhibited significant activity against HCV in a cell culture system which involves complete HCV replication and which produces infectious HCV (16). A resistant replicon has been selected by passage of HCV in the...

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Section: Discussionsupporting

confidence: 90%

Mechanism of Activation of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Murakami¹,

Bao²,

Ramesh³

et al. 2007

Antimicrob Agents Chemother

102

101

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“…The first of these, choline kinase (CK), phosphorylates choline, to form phosphocholine. Uridine is phosphorylated by uridine-cytidine kinase (CDK) to form uridine triphosphate (UTP) [17], which is transformed in brain to cytidine triphosphate (CTP) by the enzyme CTP synthetase [18]. DHA is acylated by fatty acyl-CoA synthetase [19].…”

Section: Introductionmentioning

confidence: 99%

“…All of these enzymes have high Km's for their substrates relative to normal plasma and brain levels [18]. Uridine conversion to UTP and CTP is enhanced in PC12 cells [24] and rodent brain [14] when the saturation of uridine-cytidine kinase (UCK) [25,26,27,28,29,30] has been increased by pyrimidine administration [17]. Also, the concentration of CTP in brain is insufficient to saturate CTP: phosphocholine cytidylyltransferase (CT) [3,32].…”

Section: Introductionmentioning

confidence: 99%

Chronic administration of DHA and UMP improves the impaired memory of environmentally impoverished rats

Holguin

Huang

Liu

et al. 2008

Behavioural Brain Research

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Living in an enriched environment (EC) during development enhances memory function in adulthood; living in an impoverished environment (IC) impairs memory function. Compounds previously demonstrated to improve memory among IC rats include CDP-choline and uridine monophosphate (UMP). Brain phosphatidylcholine (PC) synthesis utilizes both the uridine formed from the metabolism of exogenous CDP-choline and UMP, and the choline formed from that of CDPcholine. It also uses the polyunsaturated fatty acid (PUFA) DHA, a precursor for the diacylglycerol incorporated into PC. DHA administration also improves cognition in young and aged rodents and humans; its effects on cognitively-impaired IC rats have not been characterized. We have thus examined the consequences of administering DHA (300 mg/kg ) by gavage, UMP (0.5% in the diet), or both compounds on hippocampal-and striatal-dependent forms of memory among rats exposed to EC or IC conditions for 1 month starting at weaning, and consuming a choline-containing diet. We observe that giving IC rats either dietary UMP or gavaged DHA improves performance on the hidden version of the Morris water maze (all P<0.05), a hippocampal-dependent task; coadministration of both phosphatide precursors further enhances the IC rats' performance on this task (P<0.001). Neither UMP, DHA, nor both compounds affects the performance of EC rats on the hidden version of the Morris water maze (P>0.05), nor the performance by IC or EC rats on the visible version of the Morris water maze (all P>0.05), a striatal-dependent task. We confirm that coadministration of UMP and DHA to rats increases brain levels of the phosphatides PC, PE, SM, PS, PI, and total brain phospholipid levels (all p<0.05), and show that rearing animals in an enriched environment also elevates brain PC, PS, and PI levels (all p<0.01) and total brain phospholipids (p<0.01) compared with their levels in animals reared in an IC environment. These findings suggest that giving DHA plus UMP can ameliorate memory deficits associated with rearing under impoverished conditions, and that this effect may be mediated in part through enhanced synthesis of brain membrane phosphatides.

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“…Brain PtdCho synthesis can be enhanced by its three circulating precursors (Kennedy and Weiss, 1956): choline, a pyrimidine nucleoside like uridine or cytidine, and a polyunsaturated fatty acid (PUFA) like docosahexaenoic acid (DHA) (Araki and Wurtman, 1998;Rapoport, 2001;Marszalek and Lodish, 2005); all three readily cross the blood-brain barrier (Li et al, 2001;Spector, 2001). Brain uridine can be phosphorylated by uridine kinase (Suzuki et al, 2004) to form uridine-5′-triphosphate (UTP), which can be aminated (by CTP synthetase) to cytidine-5′-triphosphate (CTP) (Genchev and Mandel, 1974). CTP subsequently forms cytidine-5′-diphosphocholine (CDP-choline) -PtdCho's immediate precursor -which combines with diacylglycerol (DAG) molecules, including those containing PUFA, to produce PtdCho (Weiss, 1995).…”

Section: Introductionmentioning

confidence: 99%

Dietary supplementation with uridine-5′-monophosphate (UMP), a membrane phosphatide precursor, increases acetylcholine level and release in striatum of aged rat

2007

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The biosynthesis of brain membrane phosphatides, e.g., phosphatidylcholine (PtdCho), may utilize three circulating compounds: choline, uridine (a precursor for UTP, CTP, and CDP-choline), and a PUFA (e.g., docosahexaenoic acid); moreover oral administration of the uridine source uridine-5′-monophosphate (UMP) can significantly increase levels of the phosphatides throughout the rodent brain. Since PtdCho can provide choline for acetylcholine (ACh) synthesis, we determined whether UMP administration also affects ACh levels in striatum and striatal extracellular fluid, in aged and young rats. Among aged animals consuming a UMP-containing diet (2.5%, w/w) for 1 or 6 weeks, baseline ACh levels in striatal dialysates rose from 73 fmol/min to 148 or 197 fmol/min (P<0.05). Consuming a lower dose (0.5%) for 1 week produced a smaller but still significant increase (from 75 to 92 fmol/min, P<0.05), and elevated striatal Ach content (by 16%; P<0.05). Dietary UMP (0.5%, 1 week) also amplified the increase in ACh caused by giving atropine (10 μM in the aCSF); atropine alone increased ACh concentrations from 81 to 386 fmol/min in control rats and from 137 to 680 fmol/min in those consuming UMP (P<0.05). Young rats eating the UMP-containing diet exhibited similar increases in basal ECF ACh (from 105 to 118 fmol/min) and in the increase produced by atropine (from 489 to 560 fmol/min; P<0.05). These data suggest that giving a uridine source may enhance some cholinergic functions, perhaps by increasing brain phosphatide levels. Scope: Section 3 (Neurophysiology, Neuropharmacology and other forms of Intercellular Communication)

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Structural Basis for the Specificity, Catalysis, and Regulation of Human Uridine-Cytidine Kinase

Cited by 71 publications

References 44 publications

Mechanism of Activation of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Mechanism of Activation of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

Chronic administration of DHA and UMP improves the impaired memory of environmentally impoverished rats

Dietary supplementation with uridine-5′-monophosphate (UMP), a membrane phosphatide precursor, increases acetylcholine level and release in striatum of aged rat

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