Role of PRPS2 as a prognostic and therapeutic target in osteosarcoma.

AIMS: Osteosarcoma (OS) is the most common primary malignant tumour of the bone. However, further improvement in survival has not been achieved due to a lack of well-validated prognostic markers and more effective therapeutic agents. Recently, the c-Myc-phosphoribosyl pyrophosphate synthetase 2 (PRPS2) pathway has been shown to promote nucleic acid metabolism and cancer cell proliferation in malignant melanoma; phosphorylated mammalian target of rapamycin (p-mTOR) has been upregulated and an effective therapeutic target in OS. However, the p-mTOR-PRPS2 pathway has not been evaluated in OS. METHODS: In this study, the expression level of PRPS2, p-mTOR and marker of proliferation (MKI-67) was observed in a cohort of specimens (including 236 OS cases and 56 control samples) using immunohistochemistry, and the association between expression level and clinicopathological characteristics of patients with OS was analysed. RESULTS: PRPS2 protein level, which is related to tumour proliferation, was higher in OS cells (p=0.003) than in fibrous dysplasia, and the higher PRPS2 protein level was associated with a higher tumour recurrence (p=0.001). In addition, our statistical analysis confirmed that PRPS2 is a novel, independent prognostic indicator of OS. Finally, we found that the expression of p-mTOR was associated with the poor prognosis of patients with OS (p<0.05). CONCLUSIONS: PRPS2 is an independent prognostic marker and a potential therapeutic target for OS.

Arts syndrome is caused by loss-of-function mutations in PRPS1.

Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy. Linkage analysis in a Dutch family and an Australian family suggested that the candidate gene maps to Xq22.1-q24. Oligonucleotide microarray expression profiling of fibroblasts from two probands of the Dutch family revealed reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1). Subsequent sequencing of PRPS1 led to the identification of two different missense mutations, c.455T-->C (p.L152P) in the Dutch family and c.398A-->C (p.Q133P) in the Australian family. Both mutations result in a loss of phosphoribosyl pyrophosphate synthetase 1 activity, as was shown in silico by molecular modeling and was shown in vitro by phosphoribosyl pyrophosphate synthetase activity assays in erythrocytes and fibroblasts from patients. This is in contrast to the gain-of-function mutations in PRPS1 that were identified previously in PRPS-related gout. The loss-of-function mutations of PRPS1 likely result in impaired purine biosynthesis, which is supported by the undetectable hypoxanthine in urine and the reduced uric acid levels in serum from patients. To replenish low levels of purines, treatment with S-adenosylmethionine theoretically could have therapeutic efficacy, and a clinical trial involving the two affected Australian brothers is currently underway.

Mutations in PRPS1, which encodes the phosphoribosyl pyrophosphate synthetase enzyme critical for nucleotide biosynthesis, cause hereditary peripheral neuropathy with hearing loss and optic neuropathy (cmtx5).

We have identified missense mutations at conserved amino acids in the PRPS1 gene on Xq22.3 in two families with a syndromic form of inherited peripheral neuropathy, one of Asian and one of European descent. The disease is inherited in an X-linked recessive manner, and the affected male patients invariably develop sensorineural hearing loss of prelingual type followed by gating disturbance and visual loss. The family of European descent was reported in 1967 as having Rosenberg-Chutorian syndrome, and recently a Korean family with the same symptom triad was identified with a novel disease locus CMTX5 on the chromosome band Xq21.32-q24. PRPS1 (phosphoribosyl pyrophosphate synthetase 1) is an isoform of the PRPS gene family and is ubiquitously expressed in human tissues, including cochlea. The enzyme mediates the biochemical step critical for purine metabolism and nucleotide biosynthesis. The mutations identified were E43D, in patients with Rosenberg-Chutorian syndrome, and M115T, in the Korean patients with CMTX5. We also showed decreased enzyme activity in patients with M115T. PRPS1 is the first CMT gene that encodes a metabolic enzyme, shedding a new light on the understanding of peripheral nerve-specific metabolism and also suggesting the potential of PRPS1 as a target for drugs in prevention and treatment of peripheral neuropathy by antimetabolite therapy.

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine.

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.

Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit.

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.

Effects of uridine diphosphoglucose (UDPG) infusion on 5-phosphoribosyl pyrophosphate (PRPP) levels of mouse tissues.

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.

Human phosphoribosylpyrophosphate synthetase: radioimmunochemical quantitation in erythrocytes and fibroblasts.

PRPP synthetase catalyzes the synthesis of PRPP, a regulatory substrate in the pathway of purine nucleotide synthesis de novo. We have developed a specific assay for quantitative determination of PRPP synthetase immunologically cross-reactive material in human erythrocyte and fibroblast extracts. The sensitivity of the radioimmunoassay (0.3% and 0.08% of normal mean cross-reactive material in erythrocytes and fibroblasts, respectively) was equivalent to that of the enzymatic activity assay, but enzyme protein initially present in relatively inactive monomeric and smaller aggregated forms was radioimmunochemically measurable. The radioimmunoassay was utilized in conjunction with the enzymatic assay to study normal PRPP synthetase and PRPP synthetases from five affected male patients (in four families) in whom inherited enzyme superactivity was associated with increased rates of PRPP and purine nucleotide synthesis and gout with excessive uric acid excretion. Despite increased enzymatic activities in patients' cell extracts, values for cross-reactive material were within the ranges measured in the respective normal cell extracts. Thus, calculated absolute specific activities (nmol/hr/mg cross-reactive material) of patients' PRPP synthetases were substantially greater than those of normal PRPP synthetase. Moreover, absolute specific activities in hemolysates from both patients and normal individuals were in close agreement with the enzyme-specific activities measured in preparations of erythrocyte PRPP synthetase purified to homogeneity from the corresponding patient or normal source. These findings provided evidence for the accuracy and specificity of the radioimmunoassay and supported previous evidence for increased maximal reaction velocity as the basis of superactivity of the patients' enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Additional data from two kindreds with genetically induced deficiencies of erythrocyte pyrimidine nucleotidase.

Two subjects, not previously reported in detail, had severe inherited deficiencies of erythrocyte pyrimidine nucleotidase. This was manifested hematologically by moderate hemolytic anemia with splenomegaly, morphologically by punctate basophilic stippling of Wright's stained erythrocytes, and biochemically by intraerythrocytic accumulation of pyrimidine nucleotides, elevated concentrations of reduced glutathione, and partial deficiencies of ribosephosphate pyrophosphokinase. All 5 of their children were asymptomatic and phenotypically normal except for intermediate reductions in activities of pyrimidine nucleotidase consistent with heterozygosity for an autosomal recessive defect.

Mutant feedback-resistant phosphoribosylpyrophosphate synthetase associated with purine overproduction and gout. Phosphoribosylpyrophosphate and purine metabolism in cultured fibroblasts.

We have reported previously two siblings with gout and uric acid lithiasis associated with excessive purine production. In the erythrocytes of these patients, phosphoribosylpyrophosphate (PRPP) synthetase exhibited resistance to feedback-inhibition by normal cell constituents such as guanosine-5'-diphosphate (GDP) and adenosine-5'-diphosphate (ADP), resulting in superactivity of the mutant enzyme and consequently in increased PRPP content and availability for nucleotide synthesis. Erythrocyte PRPP content and availability were normal in the propositus' parents, his healthy brother and three sons, and they all had normal serum level and urinary excretion of uric acid, except for the mother who was hyperuricosuric. To further characterize this mutation we studied PRPP and purine metabolism in cultured fibroblasts of the affected family. PRPP synthetase in dialyzed lysates of fibroblasts from the propositus and his mother exhibited increased specific activity, more markedly at low inorganic phosphate concentration, and decreased sensitivity to inhibition by ADP and GDP, PRPP content and availability and the rate of de novo purine nucleotide synthesis were markedly increased in the fibroblasts of the propositus and to a lesser extent in the fibroblasts of his mother but were normal in the fibroblasts of the other family members investigated. The fibroblast studies demonstrate the following sequence of abnormalities: feedback-resistance of PRPP synthetase; superactivity of this enzyme in normal physiological milieu; increased availability of PRPP; and increased de novo synthesis of purine nucleotides. The pattern of inheritance of this disorder is compatible with both an X-linked recessive and autosomal dominant traits.