Encoding Phosphoribosylpyrophosphate Synthetase Research Articles

MicroRNA-124 Reduces the Pentose Phosphate Pathway and Proliferation by Targeting PRPS1 and RPIA mRNAs in Human Colorectal Cancer Cells

Cancer cells alter glucose metabolism to support their uncontrolled proliferation. Changes in microRNAs (miRNAs) have been associated with colorectal cancer (CRC) development and progression, but it is not clear whether they regulate metabolism in CRC cells. We aimed to identify miRNAs that alter glucose metabolism in CRC cells and to determine their effects on tumor development. CRC tissues and matched nontumor tissues were collected from 78 patients for messenger RNA (mRNA) analysis and from 112 patients for immunohistochemical analysis at the Fudan University Shanghai Cancer Center from 2005 through 2007. We integrated data on 100 miRNAs previously identified as potential regulators of glucose metabolism in a high-throughput screen with data on 66 miRNAs that often are deregulated in CRC cells. miRNAs with the potential to regulate glucose metabolism in CRC cells were blocked with mimics, and effects on lactate production were measured in CRC cell lines. miRNAs and their targets were overexpressed from lentivirals in CRC cell lines (LoVo and HCT-116) or knocked down with small interfering RNAs. The cells were analyzed in proliferation and colony formation assays and for growth as xenograft tumors in mice. We identified 3 miRNAs that significantly inhibited lactate production in 3 CRC cell lines; miR124-3p (miR124) had the strongest effect. By using complementary DNA microarray analyses, we identified 67 mRNAs that were reduced in CRC cell lines that overexpressed miR124; the mRNAs encoding phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and ribose-5-phosphate isomerase-A (RPIA) were found to be direct targets of miR124. Knockdown of PRPS1 and RPIA, as well as overexpression of miR124, each reduced glucose consumption and adenosine triphosphate in level CRC cells. Conversely, overexpression of PRPS1 or RPIA restored glycometabolism to these cells. RPIA and PRPS1 contribute to nucleotide metabolism and supply precursors for DNA and RNA biosynthesis. CRC cells that overexpressed miR124 or with knockdown of RPIA or PRPS1 had reduced DNA synthesis and proliferation, whereas cells incubated with an inhibitor of miR124 had significantly increased DNA synthesis and proliferation and formed more colonies. LoVo cells that overexpressed miR124 formed smaller xenograft tumors that controlled cells in mice, and had lower levels of PRPS1 and RPIAmRNA and protein. Compared with normal colorectal tissues, levels of miR124 were reduced significantly in CRC tissues from patients, whereas levels of PRPS1 and RPIA increased, which was associated with reduced patient survival times. miR124 inhibits DNA synthesis and proliferation by reducing levels of pentose phosphate pathway enzymes in CRC cells. Expression of miR124 and its targets correlate with survival times and might be used in prognosis.

Exome Sequencing Reveals a NovelPRPS1Mutation in a Family with CMTX5 without Optic Atrophy

BackgroundX-linked Charcot-Marie-Tooth disease type 5 (CMTX5) is caused by mutations in the gene encoding phosphoribosyl pyrophosphate synthetase I (PRPS1). There has been only one case report of CMTX5 patients. The aim of this study was to identify the causative gene in a family with CMTX with peripheral neuropathy and deafness.Case ReportA Korean family with X-linked recessive CMT was enrolled. The age at the onset of hearing loss of the male proband was 5 months, and that of steppage gait was 6 years; he underwent cochlear surgery at the age of 12 years. In contrast to what was reported for the first patients with CMTX5, this patient did not exhibit optic atrophy. Furthermore, there was no cognitive impairment, respiratory dysfunction, or visual disturbance. Assessment of his family history revealed two male relatives with very similar clinical manifestations. Electrophysiological evaluations disclosed sensorineural hearing loss and peripheral neuropathy. Whole-exome sequencing identified a novel p.Ala121Gly (c.362C>G) PRPS1 mutation as the underlying genetic cause of the clinical phenotype.ConclusionsA novel mutation of PRPS1 was identified in a CMTX5 family in which the proband had a phenotype of peripheral neuropathy with early-onset hearing loss, but no optic atrophy. The findings of this study will expand the clinical spectrum of X-linked recessive CMT and will be useful for the molecular diagnosis of clinically heterogeneous peripheral neuropathies.

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735mg cytidine/l using glucose as substrate, which was approx. 128% higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2%) slightly and increased glucose uptake (72%). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.

The yeast PRS3 gene is required for cell integrity, cell cycle arrest upon nutrient deprivation, ion homeostasis and the proper organization of the actin cytoskeleton.

PRS3 is one of a family of five genes encoding phosphoribosylpyrophosphate synthetase, an enzyme which catalyses the first step in a variety of biosynthetic pathways, including purine and pyrimidine biosynthesis. We report here that prs3Delta mutants have a number of phenotypes that suggest an unexpected role for PRS3 in linking nutrient availability to cell cycle progression, cell integrity and the actin cytoskeleton. Upon nutrient limitation, prs3Delta mutants fail to arrest in G(1)-cells remain budded and a significant fraction have a G(2) DNA content. Furthermore, in such conditions, prs3Delta mutants have a disorganized actin cytoskeleton: actin accumulates in one or two intensely staining clumps per cell. Prs3Delta mutants also show defects in ion homeostasis and cell integrity. They fail to grow on medium containing 1.0 M NaCl, 5 mM caffeine or when incubated at 37 degrees C. The caffeine and temperature sensitivity are rescued by supplementing the growth medium with 1.0 M sorbitol. These phenotypes resemble those of whi2Delta mutations and indeed, a prs3 allele was recovered in a colony-sectoring screen for mutations that are co-lethal with whi2Delta. However, further investigation showed that the prs3Delta whi2Delta double mutant was viable, with no obvious growth defect compared to either single mutant. In the same colony-sectoring assay, an mpk1 allele was also recovered. Multicopy PRS3 rescued the caffeine sensitivity of this mpk1 allele.

Characterisation of the gene encoding phosphoribosylpyrophosphate synthetase ( PRS) of Giardia intestinalis

Characterisation of the gene encoding phosphoribosylpyrophosphate synthetase ( PRS) of Giardia intestinalis

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell.

Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression.

The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs. Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms

The 5′ regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes ( PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5′ flanking regions cloned contribute to the cell-differential expression of these two genes.

Structure of the rat PRPS1 gene encoding phosphoribosylpyrophosphate synthetase subunit I.

Phosphoribosylpyrophosphate (PP-Rib-P) synthetase (EC 2.7.6.1) subunit I gene (PRPS1) is constitutively expressed in various tissues (Taira, M., Iizasa, T., Yamada, K., Shimada, H., and Tatibana, M. (1989) Biochim. Biophys. Acta 1007, 203-208). We report here the exon-intron organization and the transcription promoter sequence of rat PRPS1 gene. This gene has 22 kilobases and is split into 7 exons ranging in size from 99 to 251 base pairs (bp), except for exon 7 (1008 bp). A putative PP-Rib-P binding site is encoded in exon 5. The exon-intron boundaries are similar to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays with the use of RNA from rat Yoshida ascites sarcoma cells led to the identification of four possible transcription start points closely spaced between 126 and 129 bp from the ATG initiation codon. In the upstream region from the transcriptional start sites, we observed a TATA-like sequence (TAATTTAAT) at nucleotides -28, a CCAAT element (AGCCAATC) at nucleotides -80, and three GC boxes (putative Sp1-binding sites) at nucleotides -103, -43, and -10. A comparison of the promoter region for PRPS1 with those of other housekeeping genes revealed a homology resembling that of the beta-actin gene.

Phosphoribosylpyrophosphate (PRPP)‐less mutants of Escherichia coli

A DNA fragment encoding kanamycin resistance was inserted in vitro into a plasmid-borne prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. The resulting plasmids were subsequently transferred to the chromosome by homologous recombination and the haploid strains prs-3::KanR and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show that phosphoribosylpyrophosphate synthetase is dispensable for E. coli.

Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli.

The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage lambda D69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.

Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli

A mutant of Escherichia coli with a partially defective phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase) has been characterized genetically. The genetic lesion causing the altered phosphoribosylpyrophosphate synthetase, prs, was mapped at 26 min on the linkage map by conjugation. Transductional analysis of the prs region established the gene order as purB-fadR-dadR-tre-pth-prs-hemA-trp. Two additional mutations were identified in the mutant: one in gsk, the gene encoding guanosine kinase, and one in lon, conferring a mucoid colony morphology. The contribution of each mutation to the phenotype of the mutant has been evaluated.