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. 2014 Jun 13;289(24):16844-54.
doi: 10.1074/jbc.M114.551192. Epub 2014 Apr 28.

Identification of Yeast and Human 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters

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Identification of Yeast and Human 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters

Johanna Ceschin et al. J Biol Chem. .
Free PMC article

Abstract

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation.

Keywords: AMP-activated Kinase (AMPK); Nicotinamide; Nucleoside/Nucleotide Transport; Thiamine; Transporter.

Figures

FIGURE 1.
FIGURE 1.
AICAR accumulation and toxicity in yeast. A, addition of extracellular AICAr does not result in a massive intracellular AICAR accumulation in yeast. After an overnight preculture, wild-type cells (Y175) were diluted and kept in exponential phase for 24 h in SDcasaWAU medium. External AICAr (5 mm) was then added (red line) or not (blue line) for 20 min, and metabolites were extracted and separated by liquid chromatography (only the 60–65-min elution time region of the chromatogram is presented). AICAR intracellular concentration was determined by measuring yeast cell volume as described (1). The inset corresponds to a zoom of the indicated region. Internal AICAR values correspond to three independent extractions, and standard deviation is indicated. B, schematic representation of purine and histidine pathways in yeast. Ade, adenine; Hypox, hypoxanthine; PM, plasma membrane; PRPP, 5-phosphoribosyl-1-pyrophosphate. Only the enzymes mentioned in the text are shown (in blue). C, intracellular AICAR accumulation is strongly increased in the ade16 ade17 ade8 his1 mutant. The cells (Y6986) were grown in SDcasaWAU medium, treated (green line) or not (orange line) with AICAr (5 mm, 20 min), and the metabolites were extracted and separated as in Fig. 1A. Internal AICAR values correspond to three independent extractions, and standard deviation is indicated. N.D., not detectable. D, effect of external AICAr on growth of the ade8 his1 (Y2660) or ade16 ade17 ade8 his1 (Quadruple; Y6986) strains. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing external AICAr. Plates were imaged after an incubation of 2 days at the indicated temperature.
FIGURE 2.
FIGURE 2.
The S90P recessive mutation in the THI80 gene is responsible for AICAr hypersensitivity of the Y7506 mutant strain. A, the thi80 mutation is genetically linked to AICAr sensitivity. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing external AICAr. Parental strains ade16 ade17 ade8 his1 either mutated (Y7506) or not (control, Y7242) in the THI80 gene, and six derived spores are shown. The plates were imaged after 3 days at 37 °C. WT and mut, respectively, stand for wild-type (THI80) and mutated (thi80) versions of thiamine pyrophosphate kinase gene, as determined by sequencing. B, the thi80 (S90P) mutation leading to AICAr sensitivity is recessive. Diploid ade16 ade17 ade8 his1 strains mutated (thi80) or not (THI80) in thiamine pyrophosphate kinase gene were grown, diluted, and spotted on SDcasaWAU medium as in Fig. 2A. C, complementation by the wild-type THI80 gene. Haploid ade16 ade17 ade8 his1 strains (WT, Y7242; or thi80, Y7506) were transformed with a centromeric plasmid expressing (THI80) or not (vector) a wild-type copy of THI80. Transformants were grown, diluted, and spotted on SDcasaWA medium as in Fig. 2A. In A–C, the plates were imaged after 3 days at 37 °C. D, schematic representation of thiamine metabolism and resulting regulation of THI gene expression.
FIGURE 3.
FIGURE 3.
Mutations in THI3 and PDC2 lead to AICAr sensitivity. A and B, mutations in THI3 (A) and PDC2 (B) are linked to AICAr hypersensitivity. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing external AICAr. Strains correspond to ade16 ade17 ade8 his1 parental cells mutated (thi3, Y7321; or pdc2, Y7314) or not (control, Y6986) and to derived spores. The plates were imaged after 3 days at 30 °C. WT and mut, respectively, stand for wild-type and mutated versions of either THI3 or PDC2 gene as determined by sequencing of the corresponding gene. C and D, mutations in THI3 (C) and PDC2 (D) are haploinsufficient or semidominant. Diploid ade16 ade17 ade8 his1 strains each carrying the indicated combination of mutated (thi3 or pdc2) and wild-type (THI3 or PDC2) alleles were grown, diluted, and spotted on SDcasaWAU medium. The plates were imaged after 48 h at 30 °C.
FIGURE 4.
FIGURE 4.
Intracellular concentration of thiamine and AICAR are higher in the AICAr-hypersensitive mutants. A, metabolic analysis of AICAR and thiamine derivatives in the AICAr-sensitive mutants. After an overnight preculture, cells were diluted and kept in exponential phase for 24 h in SDcasaWAU medium containing AICAr (0.5 mm). Metabolites were then extracted and separated by liquid chromatography. TPP, thiamine pyrophosphate. The question mark corresponds to an unidentified peak. B, relative intracellular content of thiamine and AICAR derivatives. Quantifications were determined from at least three independent metabolite extractions and separations for each strain (control, Y6986; thi80, Y7506; thi3, Y7321; pdc2, Y7314), and error bars indicate variations to the mean. For each metabolite, content of the control strain (blue bar) was set at 1. C, chemical structure of adenosine, AICAr, NmR, and thiamine. D, AICAr uptake is strongly enhanced in the AICAr-hypersensitive mutants. the cells were exponentially grown for 24 h in SDcasaWAU medium, and [3H]AICAr uptake was determined from at least two independent kinetics, as described under “Experimental Procedures.” The error bars indicate variations to the mean. Control, Y6986; thi80, Y8845; thi3, Y7321; pdc2, Y7314.
FIGURE 5.
FIGURE 5.
AICAR accumulation and toxicity correlates with thiamine transporter expression and activity. A, THI7 expression is increased in AICAr-sensitive mutants. The ade16 ade17 ade8 his1 cells (control, Y7242; thi80, Y8845; thi3, Y8848; pdc2, Y8843) transformed with a THI7-LacZ fusion plasmid (p4884) were grown in SC-U-L medium to A600 nm = 1. Relative β-Gal activities were measured as described under experimental procedures and are given using the Y7242 strain as reference (set up at 1). B, overexpression of thiamine transporter genes leads to AICAr hypersensitivity. The ade16 ade17 ade8 his1 (Y6986) strain was transformed with a plasmid allowing or not (vector) the overexpression of either THI7 (p4678) or THI72 (p4680) gene. Transformants were diluted and spotted on SDcasaWA medium containing or not AICAr. The plates were imaged after 2 days at 30 °C. C, intracellular AICAR accumulation is enhanced by overexpression of thiamine carriers. Transformants from Fig. 5B were grown and treated with AICAr (5 mm) for 20 min, and metabolites were extracted and separated as in Fig. 1A. Internal AICAR values correspond to three independent determinations, and error bars indicate variations to the mean. AICAR content in the strain containing the empty vector was set up at 1. D, treatment with inhibitors of thiamine transporters severely affects AICAR accumulation. Intracellular AICAR content was determined on metabolites extracts from ade16 ade17 ade8 his1 cells (Y6986) grown in SDcasaWAU medium and incubated for 20 min with external AICAr (5 mm) as in Fig. 1A. Chloroquine (CQ, 2 mm) or amodiaquine (AQ, 100 μm) were added 1 min before AICAr addition. The results correspond to at least four independent metabolite extractions, and error bars indicate variations to the mean. AICAR content found in absence of inhibitor was set up at 1. E, chloroquine abolishes AICAr sensitivity of thi80 and pdc2 mutants. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing external AICAr (5 mm) and supplemented or not with chloroquine (2 mm). The plates were imaged after 2 days at 30 °C.
FIGURE 6.
FIGURE 6.
Nrt1 is the major AICAr transporter in yeast. A, deletion of NRT1 alleviates the growth defect of an ade16 ade17 ade8 his1 strain on AICAr. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing or not external AICAr (5 mm). All strains are in an ade16 ade17 ade8 his1 genetic background and carry the indicated additional knock-out (none, Y2950; thi7, Y8750; thi72, Y8755; nrt1, Y9116). The plates were imaged after 2 days at 37 °C. B, AICAR accumulation is severely decreased in the absence of NRT1. Relative intracellular AICAR contents were determined on metabolite extracts from strains grown in SD casaWAU medium and treated with external AICAr as in Fig. 1A. The amounts of AICAR found in the ade16 ade17 ade8 his1 strain (control, Y2950; content set up at 1) and isogenic mutant strains nrt1 (Y9116), thi7 (Y8750), thi72 (Y8755), and thi7 thi72 nrt1 (Y9188) are presented. C, AICAr uptake correlates with Nrt1 expression level. Triple mutant cells (thi7 thi72 nrt1; Y9188) were transformed with vectors allowing or not (−) expression of the NRT1 gene (CEN, centromeric plasmid: p4980; 2μ, multicopy plasmid: p4926). Transformants were exponentially grown for 24 h in SDcasaWA medium, and [3H]AICAr uptake was calculated from at least two independent kinetics. The error bars indicate variations to the mean. D, overexpression of NRT1 increases AICAr sensitivity. The ade16 ade17 ade8 his1 strain (Y2950) was transformed with a multicopy plasmid (p4926) allowing overexpression of NRT1. Transformants were grown overnight, serially diluted, and spotted on SDcasaWA medium containing external AICAr (5 mm) and in the presence or not of amodiaquine (AQ, 100 μm). The plates were imaged after 2 days at 37 °C. E, AICAr hypersensitivity of thi80 mutant is independent to the presence of NRT1 gene. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing or not external AICAr (2 mm). All strains are in an ade16 ade17 ade8 his1 genetic background and carry the indicated additional knock-out (thi80, Y7506; nrt1, Y9116; thi80 nrt1, Y9437). The plates were imaged after 2 days at 37 °C.
FIGURE 7.
FIGURE 7.
AICAr uptake occurs through adenosine transport systems in human cells. A, AICAR accumulation in various human cell lines. The cells grown in complete medium were treated with external AICAr (250 μm) for 12 h, and metabolites were extracted and separated by liquid chromatography as described under “Experimental Procedures.” The tissular origin of the different human cell lines is as follows: SF188, pediatric glioblastoma; U87 and SF126, adult glioblastomas; NHA/TS, immortalized astrocytes; A549, lung carcinoma; Huh7, hepatocellular carcinoma. B, SF188 cells accumulate AICAR when incubated with AICAr. The cells were treated (red line) or not (blue line) with external AICAr (500 μm) for 20 min, and metabolites were extracted and separated by liquid chromatography. The inset corresponds to a zoom of the indicated region. AICAR content in the absence of external AICAr was set up at 1. C, effect of various compounds on intracellular AICAR accumulation. Relative AICAR content was determined in SF188 cells grown and treated with external AICAr as in Fig. 7B and in the presence or the absence (control, set up at 1) of potential AICAr uptake competitors (100 μm each) added 1 min before AICAr treatment (500 μm, 20 min). D, dipyridamole alleviates AICAr accumulation in SF188 cells. Relative AICAR content was determined after metabolite extractions of cells grown and treated with AICAr (500 μm, 20 min) as in Fig. 7B, but in the absence (control, set up at 1) or presence of increasing concentrations of the nucleoside transporter inhibitors dipyridamole (DP) added 1 min before AICAr. E, dipyridamole protects SF188 cells against AICAr toxicity. Cell proliferation was determined by using the WST-1 colorimetric assay on SF188 cells grown for 3 days in the absence (blue triangles) or in the presence of 0.3 mm (orange circles) or 1 mm (red circles) of external AICAr and in the presence or not of dipyridamole. WST-1 signal obtained in the absence of both AICAR and inhibitors was set up at 1. F, expression of the human nucleoside transporters hENT1, hENT2, and hCNT3 enhances AICAr uptake in yeast. Yeast cells (KY114 (38) expressing or not (control) the various human nucleoside transporters (hENT1, SLC29A1; hENT2, SLC29A2; hCNT2, SLC28A2; hCNT3, SLC28A3)) were exponentially grown for 24 h in SDcasaWA medium prior to [3H]AICAr uptake measurements determined as in Fig. 4D. A–F, in all panels, values correspond to at least three independent determinations, and error bars indicate variations to the mean.
FIGURE 8.
FIGURE 8.
AICAr uptake and AICAR toxicity are AMPK-independent in mouse fibroblasts. A, the AMPK catalytic subunits α1 and α2 are not required for AICAR accumulation in MEFs. Relative AICAR content was determined in MEF double knock-out (AMPKα KO stands for AMPKα1/α2 KO) or not (AMPKα WT) in AMPKα genes. The cells were grown and treated with external AICAr as in Fig. 7B and in the presence (+DP) or the absence (−DP) of dipyridamole (2.5 μm) added 1 min before AICAr treatment (500 μm, 20 min). AICAR content was set up at 1 for WT MEF in the absence of DP. B, AICAR effect on cell proliferation is not altered in the absence of AMPK-α1 and -α2 catalytic subunits. Cell proliferation was measured using WT cells (red curves) or AMPKα1/α2 KO cells (green curves) MEF grown for 3 days in the presence (squares) or the absence (triangles) of AICAr (1 mm). C and D, dipyridamole protects MEF against AICAr toxicity in an AMPK-independent manner. Cell proliferation was determined using the WST-1 test on wild-type and AMPKα KO MEFs grown for 3 days in the absence (blue triangles) or in the presence of either 0.3 mm (orange circles) or 1 mm (red circles) of external AICAr and in the presence or not of dipyridamole. A–D, in all panels, values correspond to at least three independent determinations, and errors bars indicate variations to the mean.

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