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Cells coordinate their behaviors with the mechanical properties of the extracellular matrix (ECM). Tumor cells frequently harbor an enhanced nucleotide synthesis, presumably to meet the increased demands for rapid proliferation. Nevertheless, how ECM rigidity regulates nucleotide metabolism remains elusive. Here we show that shift from stiff to soft matrix blunts glycolysis-derived nucleotide synthesis in tumor cells. Soft ECM results in TNF receptor-associated factor 2 (TRAF2)-dependent K29 ubiquitination and degradation of phosphoribosyl pyrophosphate synthetase (PRPS)1/2. Recruitment of TRAF2 to PRPS1/2 requires phosphorylation of PRPS1 S285 or PRPS2 T285, which is mediated by low stiffness-activated large tumor suppressor (LATS)1/2 kinases. Further, non-phosphoryable or non-ubiquitinatable PRPS1/2 mutations maintain PRPS1/2 expression and nucleotide synthesis at low stiffness, and promote tumor growth and metastasis. Our findings demonstrate that PRPS1/2 stability and nucleotide metabolism is ECM rigidity-sensitive, and thereby highlight a regulatory cascade underlying mechanics-guided tumor metabolism reprogramming.

Phosphoribosyl pyrophosphate synthetases (EC 2.7.6.1) are key enzymes in the biological synthesis of phosphoribosyl pyrophosphate and are involved in diverse developmental processes. In our previous study, the

Neuroblastoma is an important problem in children. Long noncoding RNAs (lncRNAs) exhibit important roles in tumorigenicity of neuroblastoma. However, the role and mechanism of lncRNA small nucleolar RNA host gene 16 (SNHG16) in neuroblastoma tumorigenicity remain poorly understood. Forty-six neuroblastoma samples and 28 normal tissues were harvested. The levels of SNHG16, microRNA-15b-5p (miR-15b-5p), and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) were detected via quantitative reverse transcription PCR or western blot. Cell proliferation as well as cycle distribution were measured via 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide or flow cytometry. Cell metastasis was investigated via epithelial-mesenchymal transition or transwell assay. The target relationship of miR-15b-5p and SNHG16 or PRPS1 was explored via starBase and dual-luciferase reporter assay. The role of SNHG16 in neuroblastoma in vivo was analyzed using a xenograft model. We found SNHG16 and PRPS1 levels were increased in neuroblastoma tissues and cells. SNHG16 knockdown inhibited cell proliferation, increased the cell cycle distribution at G0/G1 phase, and decreased the cells at S phase. SNHG16 overexpression caused an opposite effect. SNHG16 silence suppressed neuroblastoma cell metastasis. PRPS1 knockdown constrained cell proliferation and metastasis and regulated cell cycle distribution. miR-15b-5p was sponged by SNHG16 and directly targeted PRPS1. miR-15b-5p knockdown or PRPS1 overexpression mitigated the influence of SNHG16 silence on cell cycle, proliferation, and metastasis. SNHG16 knockdown reduced xenograft tumor growth. In conclusion, SNHG16 downregulation suppressed neuroblastoma tumorigenicity by regulating cell cycle, proliferation, and metastasis via miR-15b-5p/PRPS1 axis.

Patients with relapsed/refractory Burkitt's lymphoma (BL) have a dismal prognosis. Current research efforts aim to increase cure rates by identifying high‐risk patients in need of more intensive or novel therapy. The 8q24 chromosomal translocation of the c‐Myc gene, a main molecular marker of BL, is related to the metabolism by regulating phosphoribosyl pyrophosphate synthetase 2 (PRPS2). In our study, BL showed significant resistance to thiopurines. PRPS2 homologous isoenzyme, PRPS1, was demonstrated to play the main role in thiopurine resistance. c‐Myc did not have direct effects on thiopurine resistance in BL for only driving PRPS2. PRPS1 wild type (WT) showed different resistance to 6‐mercaptopurine (6‐mp) in different metabolic cells because it could be inhibited by adenosine diphosphate or guanosine diphosphate negative feedback. PRPS1 A190T mutant could dramatically increase thiopurine resistance in BL. The interim analysis of the Treatment Regimen for Children or Adolescent with mature B cell non‐Hodgkin's lymphoma in China (CCCG‐B‐NHL‐2015 study) confirms the value of high‐dose methotrexate (MTX) and cytarabine (ARA‐C) in high‐risk paediatric patients with BL. However, there remains a subgroup of patients with lactate dehydrogenase higher than four times of the normal value (4N) for whom novel treatments are needed. Notably, we found that the combination of thiopurines and the phosphoribosylglycinamide formyltransferase (GART) inhibitor lometrexol could serve as a therapeutic strategy to overcome thiopurine resistance in BL.

Phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) is a rate-limiting enzyme and plays an important role in purine and pyrimidine nucleotide synthesis. Recent studies report that PRPS2 is involved in male infertility. However, the role of PRPS2 in hypospermatogenesis is unknown. In this study, the relationship of PRPS2 with hypospermatogenesis and spermatogenic cell apoptosis was investigated. The results showed that PRPS2 depletion increased the number of apoptotic spermatogenic cells in vitro. PRPS2 was downregulated in a mouse model of hypospermatogenesis. When PRPS2 expression was knocked down in mouse testes, hypospermatogenesis and accelerated apoptosis of spermatogenic cells were noted. E2F transcription factor 1 (E2F1) was confirmed as the target gene of PRPS2 and played a key role in cell apoptosis by regulating the P53/Bcl-xl/Bcl-2/Caspase 6/Caspase 9 apoptosis pathway. Therefore, these data indicate that PRPS2 depletion contributes to the apoptosis of spermatogenic cells and is associated with hypospermatogenesis, which may be helpful for the diagnosis of male infertility.

Pyrophosphate synthetase-1(PRS-1) is a crucial enzyme that catalyzes the synthesis of phosphoribosylpyrophosphate (PRPP) with substrate: adenosine triphosphate (ATP) and ribose-5-phophate(R5P) in the de novo pathways of purine and pyrimidine nucleotide synthesis. Mutation in PRPS1 can result in a series of diseases of purine metabolism, which includes PRS-1 superactivity. The common clinical phenotypes are hyperuricemia and hyperuricosuria. We identified a novel missense mutation in X-chromosomal gene PRPS1 in a young Chinese woman while her mother has heterogeneous genotype and phenotype. A 24-year-old Chinese female patient suffered hyperuricemia, gout, and recurrent hyperpyrexia for more than 6 years, and then was diagnosed with hyperandrogenism, insulin resistance (IR), and polycystic ovary syndrome (PCOS). A novel missense mutation, c.521(exon)G>T, p.(Gly174Val) was detected by next-generation sequencing (NGS) and confirmed by Sanger sequencing in the patient and her parents. Interestingly, her mother has the same heterozygous missense mutation but without uric acid overproduction which can be explained by the phenomenon of the skewed X-chromosome inactivation. The substituted amino acid Val for Gly174 is positioned in the pyrophosphate (PPi) binding loop, and this mutation impacts the binding rate of Mg2+-ATP complex to PRS-1, thus the assembling of homodimer is affected by changed Val174 leading to the instability of the allosteric site. Our report highlights the X-linked inheritance of gout in females caused by mutation in PRPS1 accompanied with severe metabolic disorders and recurrent hyperpyrexia.

Phosphoribosyl pyrophosphate synthetase-1 (PRPS-1; EC 2.7.6.1.) catalyzes the binding of phosphate-group to ribose 5-phosphate, forming the 5-phosphoribosyl-1-pyrophosphate, which is necessary for the salvage pathways of purine and pyrimidine, pyridine nucleotide cofactors - NAD and NADP, the amino acids histidine and tryptophan biosynthesis. We aimed to investigate the impact of the different effectors on the activity of PRPS-1, to check the activity of the enzyme in vitro in a wide range of pHs and investigate some structural essentials of the enzyme, isolated from brain and liver. Molecular docking analyses were used to delineate the essentials of PRPS-1 structure, to find out the existence of enzyme effectors. Previously created by us kit was used for determination of the activity of PRPS-1 based on the formation of the inorganic phosphates (λ = 700 nm, Cary 60, Agilent, USA). Effectors impact on the activity of PRPS-1 was evaluated. In silico results of the effectors were later proven by in vitro experiments. For the first time biochemical essentials, including the existence of the multiple pockets, involvement of the amino acids into the processes of interactions with the effectors, evolutional of the sequence conservation, tissue depended V

Nicotinamide mononucleotide (NMN), a precursor of NAD

In a recent issue of Science Advances, Srivastava et al. report a novel nucleotide biosynthesis regulatory role for UBR7 in NOTCH1-driven T-ALL. Here we will discuss their key findings and the implications for using cancer metabolism to understand the development and progression of T-ALL.

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways. The assembly of enzymes at the first branch point in de novo purine biosynthesis is coordinated, hierarchical, and based on their position within the pathway, while the enzymes at the second branch point are recruited to RNA stress granules. Consistent with distinct classes of structures being deployed at different control points in a pathway, we find that the first enzyme in the pathway, PRPP synthetase, forms evolutionarily conserved filaments that are sequestered in the nucleus in higher eukaryotes. These findings provide a roadmap for identifying additional conserved features of metabolic regulation by condensates/filaments.

Nucleotide supply is essential for DNA replication in proliferating cells, including cancer cells. Ribose-phosphate diphosphokinase 1 (PRPS1) is a key enzyme to produce the consensus precursor of nucleotide synthesis. PRPS1 participates in the pentose phosphate pathway (PPP) by catalyzing the phosphoribosylation of D-ribose 5-phosphate (R-5P) to 5-phosphoribosyl-1-pyrophosphate. Therefore, PRPS1 not only controls purine biosynthesis and supplies precursors for DNA and RNA biosynthesis but also regulates PPP through a feedback loop of the PRPS1 substrate R-5P. However, it is still elusive whether PRPS1 enhances nucleotide synthesis during cell-cycle progression. In this study, we explore the role and activation mechanism of PRPS1 in cell-cycle progression of colorectal cancer, and observed a peak in its enzymatic activity during S phase. CDK1 contributes to upregulation of PRPS1 activity by phosphorylating PRPS1 at S103; loss of phosphorylation at S103 delayed the cell cycle and decreased cell proliferation. PRPS1 activity in colorectal cancer samples is higher than in adjacent tissue, and the use of an antibody that specifically detects PRPS1 phosphorylation at S103 showed consistent results in 184 colorectal cancer tissues. In conclusion, compared with upregulation of PRPS1 expression levels, increased PRPS1 activity, which is marked by S103 phosphorylation, is more important in promoting tumorigenesis and is a promising diagnostic indicator for colorectal cancer. Significance: These findings show that the enzymatic activity of PRPS1 is crucial for cell-cycle regulation and suggest PRPS1 phosphorylation at S103 as a direct therapeutic target and diagnostic biomarker for colorectal cancer.

Glioblastoma multiforme (GBM) is a refractory tumor with poor prognosis and requires more effective treatment regimens. It has been confirmed that long noncoding RNAs (lncRNAs) substantially regulate various human disease including GBM. However, the biological roles and its underlying molecular mechanisms still need to be further investigated. In this study, the biological function and potential molecular mechanism of lncHAS2-AS1 in GBM were explored. It was discovered that HAS2-AS1 was elevated in glioma tissues and correlated with the prognosis of patients with glioma. Reduction of HAS2-AS1 suppressed the migration and invasion in vitro and in vivo. The transcription factor STAT1 could raise HAS2-AS1 by binding to its promoter region. Besides, HAS2-AS1 could adjust PRPS1 via sponging miR-608 in a direct manner. On the whole, the results of this study evidence that HAS2-AS1 is an oncogene and a potential therapeutic target for GBM.

X-linked Charcot-Marie-Tooth disease type 5 (CMTX5) is an X-linked disorder characterized by early-onset sensorineural hearing impairment, peripheral neuropathy, and progressive optic atrophy. It is caused by a loss-of-function mutation in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes isoform I of phosphoribosyl pyrophosphate synthetase (PRS-I). A decreased activity leads to nonsyndromic sensorineural deafness (DFN2), CMTX5, and Arts syndrome depending upon residual PRS-I activity. Clinical and neurophysiological features of pediatric CMTX5 are poorly defined. We report two male siblings with peripheral neuropathy and prelingual sensorineural hearing loss who carried a novel c.319A>G (p.Ile107Val) PRPS1 missense mutation. They exhibited recurrent episodes of transient proximal muscle weakness, showing Gowers' sign and waddling gait after suffering from febrile illness. This transient weakness has not been previously reported in CMTX5. A patient with Arts syndrome was reported to have transient proximal weakness after febrile illness. The transient weakness presenting in both CMTX5 and Arts syndrome suggests an overlap of signs and a continuous spectrum of PRS-I hypoactivity disease. Children presenting with transient neurological signs should be evaluated for peripheral neuropathy and consider genetic analysis for PRPS1.

Phosphoribosyl pyrophosphate synthetase (PRS EC 2.7.6.1) is a rate-limiting enzyme that irreversibly catalyzes the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate (ATP). This key metabolite is required for the synthesis of purine and pyrimidine nucleotides, the two aromatic amino acids histidine and tryptophan, the cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), all of which are essential for various life processes. Despite its ubiquity and essential nature across the plant and animal kingdoms, PRPP synthetase displays species-specific characteristics regarding the number of gene copies and architecture permitting interaction with other areas of cellular metabolism. The impact of mutated PRS genes in the model eukaryote Saccharomyces cerevisiae on cell signalling and metabolism may be relevant to the human neuropathies associated with PRPS mutations. Human PRPS1 and PRPS2 gene products are implicated in drug resistance associated with recurrent acute lymphoblastic leukaemia and progression of colorectal cancer and hepatocellular carcinoma. The investigation of PRPP metabolism in accepted model organisms, e.g., yeast and zebrafish, has the potential to reveal novel drug targets for treating at least some of the diseases, often characterized by overlapping symptoms, such as Arts syndrome and respiratory infections, and uncover the significance and relevance of human PRPS in disease diagnosis, management, and treatment.

Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.

To investigate the expression of phosphoribosyl pyrophosphate synthase 2 (PRPS2) in the human testis and its clinical significance.Using quantitative real-time PCR (qRT-PCR) and immunohistochemistry, we detected the expression of PRPS2 mRNA in the testis tissue of the men with normal spermatogenesis or mile, moderate or severe hypospermatogenesis (HS) and that of the PRPS2 protein in the testicular biopsy tissue of 67 adult males. Then, we analyzed the relationship of the PRPS2 expressions with the testicular histological types and clinical parameters of the subjects.The expression of PRPS2 mRNA in the testis tissue was significantly higher in the normal spermatogenesis group than in the moderate and severe HS groups (P0.01). The positive expression of the PRPS2 protein was 70.0% in the normal spermatogenesis group, 66.7% in the mild HS group, 50.0% in the moderate HS group and 23.8% in the severe HS group, significantly higher in the normal spermatogenesis and mild HS groups than in the moderate and severe HS groups (P0.01). No significant correlation, however, was observed between the PRPS2 expression and clinical parameters of the subjects (P0.05).PRPS2 is lowly expressed in the testis tissue of the men with hypospermatogenesis and its expression level may help the diagnosis of male infertility and the prediction of the spermatogenic function of the testis.

We describe two sporadic and two familial cases with loss-of-function variants in PRPS1, which is located on the X chromosome and encodes phosphoribosyl pyrophosphate synthetase 1 (PRS-1). We illustrate the clinical variability associated with decreased PRS-1 activity, ranging from mild isolated hearing loss to severe encephalopathy. One of the variants we identified has already been reported with a phenotype similar to our patient's, whereas the other three were unknown. The clinical and biochemical information we provide will hopefully contribute to gain insight into the correlation between genotype and phenotype of this rare condition, both in females and in males. Moreover, our observation of a new family in which hemizygous males display hearing loss without any neurological or ophthalmological symptoms prompts us to suggest analysing PRPS1 in cases of isolated hearing loss. Eventually, PRPS1 variants should be considered as a differential diagnosis of mitochondrial disorders.

Metabolic reprogramming to fulfill the biosynthetic and bioenergetic demands of cancer cells has aroused great interest in recent years. However, metabolic reprogramming for cancer metastasis has not been well elucidated. Here, we screened a subpopulation of breast cancer cells with highly metastatic capacity to the lung in mice and investigated the metabolic alternations by analyzing the metabolome and the transcriptome, which were confirmed in breast cancer cells, mouse models, and patients’ tissues. The effects and the mechanisms of nucleotide de novo synthesis in cancer metastasis were further evaluated in vitro and in vivo. In our study, we report an increased nucleotide de novo synthesis as a key metabolic hallmark in metastatic breast cancer cells and revealed that enforced nucleotide de novo synthesis was enough to drive the metastasis of breast cancer cells. An increased key metabolite of de novo synthesis, guanosine-5'-triphosphate (GTP), is able to generate more cyclic guanosine monophosphate (cGMP) to activate cGMP-dependent protein kinases PKG and downstream MAPK pathway, resulting in the increased tumor cell stemness and metastasis. Blocking de novo synthesis by silencing phosphoribosylpyrophosphate synthetase 2 (PRPS2) can effectively decrease the stemness of breast cancer cells and reduce the lung metastasis. More interestingly, in breast cancer patients, the level of plasma uric acid (UA), a downstream metabolite of purine, is tightly correlated with patient’s survival. Our study uncovered that increased de novo synthesis is a metabolic hallmark of metastatic breast cancer cells and its metabolites can regulate the signaling pathway to promote the stemness and metastasis of breast cancer., Metabolic reprogramming to fulfil the biosynthetic and bioenergetic demands of cancer cells is well known, but a role in metastasis is less clear. This study shows that nucleotide metabolic reprogramming leads to an enhancement of the cGMP-PKG-MAPK axis, enabling breast cancer cells to effectively colonize and grow in the lung.

Background and study aims The metabolism of dietary fructose by ketohexokinase (KHK) is an important step in glucose metabolism in various tumour types. However, the expression, function and underlying mechanisms of KHK in oesophageal squamous cell carcinoma (ESCC) remain largely unclear. The objective of this study was to investigate the effects of KHK-A, a peripheral isoform of KHK, on the proliferation of ESCC cell lines. Material and methods The function and mechanism of KHK-A in ESCC cells were investigated by constructing stable KHK-A-knockdown and -overexpressing ESCC cell lines (KYSE410 and KYSE150, respectively). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and colony formation assays were used to analyse the effects of KHK-A on cell proliferation , cell cycle and colony formation, respectively. KHK-A and phosphoribosyl pyrophosphate synthetase isoform 1 (PRPS1) mRNA and protein expressions in several ESCC cell lines were determined using routine reverse transcription-polymerase chain reaction and immunoblotting, respectively. KHK and PRPS1 expressions in ESCC tumour tissues and corresponding adjacent non-tumour tissues were evaluated according to the gene expression omnibus (GEO) database (GSE20347). Results In vitro experiments showed that KHK-A significantly promoted cell proliferation by modulating the G1/S phase transition in the cell cycle, which was probably regulated by PRPS1 expression. GEO database-based analysis showed that KHK levels were significantly higher in the ESCC tissues than in the corresponding adjacent non-tumour tissues. Pearson’s correlation coefficient analysis showed that KHK expression in ESCC cell lines and tissues was significantly positively associated with the up-regulation of PRPS1, suggesting that KHK-A levels regulate PRPS1 expression in ESCC. Conclusion KHK-A may serve as a driving gene in ESCC for the activation of PRPS1, resulting in the up-regulation of PRPS1. This could lead to enhanced nucleic acid synthesis for tumourigenesis. Our study showed that KHK-A is a potential target for ESCC diagnosis and therapy.

Cloned pigs generated by the somatic cell transfer nuclear (SCNT) technique are highly valuable for agriculture, biomedicine, and life sciences. However, the neonatal mortality rate of cloned pigs is very high. The reasons causing the massive loss of cloned pigs during their neonatal ages are unclear. In the present study, we found that the neonatal death of cloned pigs was associated with aberrant purine metabolism, impaired renal morphology and function, and decreased hepatic Hprt1 expression. The downregulation of Hprt1, a key purine metabolism regulation gene, in the liver was responsible for the elevation of an important purine metabolite, uric acid, in the serum, causing abnormalities in kidney morphology and function and leading to death of neonatal cloned pigs. This study provided insights into the pathophysiological mechanisms underlying the neonatal death of clone pigs, and results will help improve their survival rate.

Pluripotent stem cells (PSCs) have a unique energetic and biosynthetic metabolism compared with typically differentiated cells. However, the metabolism profiling of PSCs and its underlying mechanism are still unclear. Here, we report PSCs metabolism profiling and identify the purine synthesis enzymes, phosphoribosyl pyrophosphate synthetase 1/2 (PRPS1/2), are critical for PSCs stemness and survival. Ultra-high performance liquid chromatography/mass spectroscopy (UHPLC-MS) analysis revealed that purine synthesis intermediate metabolite levels in PSCs are higher than that in somatic cells. Ectopic expression of PRPS1/2 did not improve purine biosynthesis, drug resistance, or stemness in PSCs. However, knockout of PRPS1 caused PSCs DNA damage and apoptosis. Depletion of PRPS2 attenuated PSCs stemness and assisted PSCs differentiation. Our finding demonstrates that PRPS1/2-mediated purine biosynthesis is critical for pluripotent stem cell stemness and survival.

AimsOsteosarcoma (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.MethodsIn 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.ResultsPRPS2 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 (pConclusionsPRPS2 is an independent prognostic marker and a potential therapeutic target for OS.

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4Â g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes., This study was supported by the Portuguese Foundation for Science and Technology (FCT) by the strategic funding of UID/BIO/ 04469/2013 unit, MIT-Portugal Program (PhD grant PD/BD/128247/ 2016 to Joana T. Cunha), COMPETE 2020 (POCI-01-0145-FEDER006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and MultiBiorefinery project (POCI-01-0145-FEDER-016403). Funding received by Institute for Bioengineering and Biosciences (IBB) from FCT (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project No. 007317) is acknowledged. BJ was supported through the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI)., info:eu-repo/semantics/publishedVersion

Objectives Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods Whole exome sequencing was performed in affected females and their fathers. Results Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) μmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.

Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strainThermus thermophilusHB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Å resolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits inT. thermophilusPRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic β-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.

Arts syndrome is characterized by early-onset hypotonia, ataxia, intellectual disability, sensorineural hearing impairment, progressive optic atrophy, and a tendency to develop infections. Arts syndrome is an X-linked disorder caused by a loss-of-function mutation in the PRPS1 gene, which encodes phosphoribosylpyrophosphate synthetase 1. Only three families have been reported. Here, we report another family with Arts syndrome. The initial symptoms of the 1-year-old proband were hypotonia and ataxia, worsening recurrent infection-triggered muscle weakness, motor and intellectual developmental delay, and hearing loss. Both central nervous system involvement and peripheral neuropathy were demonstrated. His three maternal uncles had died before the age of 3years. A genetic analysis of PRPS1 revealed a novel missense mutation, c.367C>G (p.His123Asp). PRPS enzymatic activity was markedly reduced in the patient. His mother was supposed to be an asymptomatic carrier. Arts syndrome should be included in the differential diagnosis of infantile hypotonia and weakness aggravated by recurrent infection with a family history of X-linked inheritance.

Background The symptoms of phosphoribosyl pyrophosphate synthetase 1 (PRPS1) deficiency diseases have been reported to be alleviated by medication. In the present study, we report biochemical data that favor PRPS1 deficiency-related hearing loss as a potential target for pharmaceutical treatment. Methods We recruited 42 probands from subjects aged less than 15 years with a moderate degree of nonsyndromic autosomal-recessive or sporadic sensorineural hearing loss (SNHL) in at least one side. Molecular genetic testing, including targeted exome sequencing (TES) of 129 genes for deafness, and in silico prediction were performed. Results A strong candidate variant (p.A82P) of PRPS1 is co-segregated with SNHL in X-linked recessive inheritance from one Korean multiplex SNHL family. Subsequent measurement of in vitro enzymatic activities of PRPS1 from erythrocytes of affected and unaffected family members, as well as unrelated normal controls, confirmed a pathogenic role of this variant. In detail, compared to normal hearing controls (0.23-0.26 nmol/ml/h), the proband, the affected sibling and their normal hearing mother demonstrated a significantly decreased PRPS1 enzymatic activity (0.07, 0.03 and 0.11 nmol/ml/h, respectively). This novel loss-of-function mutation of PRPS1 (p.A82P) is the ninth and sixth most reported mutation in the world and in Asia, respectively. Conclusions DFNX1 was found to account for approximately 2.4% (1/42) of moderate SNHL in a Korean pediatric population. Confirmation of PRPS1 activity deficiency and an audiologic phenotype that initially begins in a milder form of SNHL, as in our family, should indicate the need for rigorous genetic screening as early as possible.

Background CMTX5 is characterized by peripheral neuropathy, early‐onset sensorineural hearing impairment, and optic neuropathy. Only seven variants have been reported and no genotype‐phenotype correlations have yet been established. PRPS1 has a crystallographic structure, as it is composed of three dimers that constitute a hexamer. Methods Next‐generation sequencing (NGS) was performed using a custom 92‐gene panel designed for the diagnosis of Charcot‐Marie‐Tooth (CMT) and associated neuropathies. Results We report the case of a 35‐year‐old male, who had presented CMT and hearing loss since childhood associated to bilateral optic neuropathy without any sign of retinitis pigmentosa. A new hemizygous variant on chromosomic position X:106,882,604, in the PRPS1 gene, c.202A > T, p.(Met68Leu) was found. This change is predicted to lead to an altered affinity between the different subunits in the dimer, thereby may prevent the hexamer formation. Conclusion CMTX5 is probably under‐diagnosed, as an overlap among the different features due to PRPS1 exists. Patients who developed polyneuropathy associated to sensorineural deafness and optic atrophy during childhood should be assessed for PRPS1.

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

Kinases are among the most important families of enzymes involved in cell signaling. In this study, we employed a recently developed parallel-reaction monitoring (PRM)-based targeted proteomic method to examine the reprogramming of the human kinome during colorectal cancer (CRC) metastasis. We were able to quantify the relative expression of 299 kinase proteins in a pair of matched primary/metastatic CRC cell lines. We also found that, among the differentially expressed kinases, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) promotes the migration and invasion of cultured CRC cells through regulating the activity of matrix metalloproteinase 9 (MMP-9) and the expression of E-cadherin. Moreover, we found that the up-regulation of PRPS2 in metastatic CRC cells could be induced by the MYC proto-oncogene. Together, our unbiased kinome profiling approach led to the identification, for the first time, of PRPS2 as a promoter for CRC metastasis.

Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders., Author summary Phosphoribosyl pyrophosphate synthetase (PRPS) is an important enzyme in nucleotide synthesis: the building blocks of DNA and RNA and other important metabolites. Importantly, while PRPS is mutated in neurological disorders such as Arts syndrome, Charcot-Marie-Tooth disease, and nonsyndromic sensorineural deafness, it is currently unclear why PRPS dysfunction leads to neurological disorders. In this study, we engineered a Drosophila model of ARTS syndrome and discovered that PRPS mutations result in defects in lysosome-mediated and autophagy processes, which are known to be important for neuronal homeostasis. Our study provides crucial insights into the way that PRPS mutations contribute to neurological disorders.

X-linked Charcot-Marie-Tooth disease-5 (CMTX5) is a rare hereditary disorder caused by mutations in the gene for phosphoribosyl pyrophosphate synthetase-1 (PRPS1). We investigated a boy with a novel PRPS1 mutation (c.334G>C, p.V112L) via genetic, neuropathological and enzymatic tests. The proband was a 13-year-old boy with congenital non-syndromic sensorineural deafness. At 3 year old, he developed progressive distal weakness of all limbs with muscle atrophy of both hands and shanks. Nerve conduction study revealed the loss of sensory nerve action potentials, and slowing down of motor nerve conduction velocities with a decrease of amplitudes of compound motor action potentials. Visual evoked potentials and brainstem auditory evoked potentials were not bilaterally evocable. Sural biopsy proved the loss of myelinated nerve fibers, with axonal degeneration, regenerating clusters and onion bulbs. Enzymatically, PRPS1 activity was close to zero in the proband and mildly reduced in his mother, compared with controls. To our knowledge, this is the first report of CMTX5 in a Chinese population. The genetic finding has expanded the genotypic spectrum of PRPS1 mutations.

Background Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) is an enzyme that catalyzes the ATP-dependent conversion of ribose-5-phosphate to phosphoribosyl pyrophosphate. The reaction product is a key precursor for the biosynthesis of purine and pyrimidine nucleotides. Results We report the 2.2 Å crystal structure of the E. coli ribose-phosphate pyrophosphobinase (EcKPRS). The protein has two type I phosphoribosyltransferase folds, related by 2-fold pseudosymmetry. The propeller-shaped homohexameric structure of KPRS is composed of a trimer of dimers, with the C-terminal domains forming the dimeric blades of the propeller and the N-terminal domains forming the hexameric core. The key, conserved active site residues are well-defined in the structure and positioned appropriately to bind substrates, adenosine monophosphate and ribose-5-phosphate. The allosteric site is also relatively well conserved but, in the EcKPRS structure, several residues from a flexible loop occupy the site where the allosteric modulator, adenosine diphosphate, is predicted to bind. The presence of the loop in the allosteric site may be an additional level of regulation, whereby low affinity molecules are precluded from binding. Conclusions Overall, this study details key structural features of an enzyme that catalyzes a critical step in nucleotide metabolism. This work provides a framework for future studies of this important protein and, as nucleotides are critical for viability, may serve as a foundation for the development of novel anti-bacterial drugs.

The five-membered PRS gene family of Saccharomyces cerevisiae is an example of gene duplication allowing the acquisition of novel functions. Each of the five Prs polypeptides is theoretically capable of synthesising PRPP but at least one of the following heterodimers is required for survival: Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5. Prs3 contains a pentameric motif (284)KKCPK(288) found only in nuclear proteins. Deletion of (284)KKCPK(288) destabilises the Prs1/Prs3 complex resulting in a cascade of events, including reduction in PRPP synthetase activity and altered cell wall integrity (CWI) as measured by caffeine sensitivity and Rlm1 expression. Prs3 also interacts with the kinetochore-associated protein, Nuf2. Following the possibility of (284)KKCPK(288)-mediated transport of the Prs1/Prs3 complex to the nucleus, it may interact with Nuf2 and phosphorylated Slt2 permitting activation of Rlm1. This scenario explains the breakdown of CWI encountered in mutants lacking PRS3 or deleted for (284)KKCPK(288). However, removal of NHR1–1 from Prs1 does not disrupt the Prs1/Prs3 interaction as shown by increased PRPP synthetase activity. This is evidence for the separation of the two metabolic functions of the PRPP-synthesising machinery: provision of PRPP and maintenance of CWI and is an example of evolutionary development when multiple copies of a gene were present in the ancestral organism.

Allopurinol is a commonly used medication to treat hyperuricemia and its complications. Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to enhanced hypouricemic effect of allopurinol based on uric acid metabolism enzyme XOD. In this study, we evaluated whether Pallidifloside D (5mg/kg) enhanced hypouricemic effect of allopurinol (5mg/kg) related to others uric acid metabolism enzymes such as PRPS, HGPRT and PRPPAT. We found that, compared with allopurinol alone, the combination of allopurinol and Pallidifloside D significantly up-regulated HGPRT mRNA expression and down-regulated the mRNA expression of PRPS and PRPPAT in PC12 cells (all P

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

Relapse is the main cause of mortality in patients with glioblastoma multiforme (GBM). Treatment options involve surgical resection followed by a combination of radiotheraphy and chemotherapy with temozolomide. Several genes and genetic pathways have been identified to contribute to therapeutic resistance, giving rise to recurrence of the malignancy. In the last decades, glioma stem cells (GSCs) with the capacity of self-renewal have been demonstrated to maintain tumor propagation and treatment resistance. Here, we isolated CD133-positive (CD133+) and CD133-negative (CD133-) cells from glioblastoma U98G and U87MG cell lines. The role of phosphoribosylpyrophosphate synthetase 1 (PRPS1), which catalyzes the first step of the synthesis of nucleotide, in proliferation and apoptosis was investigated. We found that PRPS1 had a remarkable effect on cell proliferation and sphere formation in both CD133+ and CD133- cells. Compared to CD133- cells, CD133+ cells exhibited more significant results in cell apoptosis assay. CD133+ T98G and U87MG cells were used in xenograft mouse model of tumor formation. Interestingly, the mice implanted with PRPS1 knockdown T98G or U87MG stem cells exhibited prolonged survival time and reduced tumor volume. By immunostaining caspase-3 in tumor tissues of these mice, we demonstrated that the apoptotic activities in tumor cells were positively correlated to the survival time but negatively correlated to PRPS1 expression. Our results indicate that PRPS1 plays an important role in proliferation and apoptosis in GSCs and provide new clues for potential PRPS1-targeted therapy in GBM treatment.

Relapse‐specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate‐limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5‐Fluorouracil (5‐FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5‐FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5‐FU to FUMP and FUTP in Reh cells, to promote 5‐FU‐induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re‐targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse‐specific mutations of PRPS1 might benefit from 5‐FU‐based chemotherapy in clinical settings.

Hereditary deafness is often a neurosensory disorder and affects the quality of life of humans. Only three X-linked genes (POU class 3 homeobox 4 (POU3F4), phosphoribosyl pyrophosphate synthetase 1 (PRPS1), and small muscle protein X-linked (SMPX)) are known to be involved in nonsyndromic hearing loss. Four PRPS1 missense mutations have been found to associate with X-linked nonsyndromic sensorineural deafness (DFNX1/DFN2) in humans. However, a causative relationship between PRPS1 mutations and hearing loss in humans has not been well studied in any animal model. Phosphoribosyl pyrophosphate synthetase 1 (PRS-I) is highly conserved in vertebrate taxa. In this study, we used the zebrafish as a model to investigate the auditory role of zebrafish orthologs (prps1a and prps1b) of the human PRPS1 gene with whole mount in situ hybridization, reverse transcription polymerase chain reaction, phenotypic screening, confocal imaging, and electrophysiological methods. We found that both prps1a and prps1b genes were expressed in the inner ear of zebrafish. Splice-blocking antisense morpholino oligonucleotides (MO1 and MO2) caused exon-2 skip and intron-2 retention of prps1a and exon-2 skip and intron-1 retention of prps1b to knock down functions of the genes, respectively. MO1 and MO2 morphants had smaller otic vesicles and otoliths, fewer inner ear hair cells, and lower microphonic response amplitude and sensitivity than control zebrafish. Therefore, knockdown of either prps1a or prps1b resulted in significant sensorineural hearing loss in zebrafish. We conclude that the prps1 genes are essential for hearing in zebrafish, which has the potential to help us understand the biology of human deafness DFNX1/DFN2. Anat Rec, 303:544-555, 2020. © 2019 American Association for Anatomy.

Bovine spongiform encephalopathy (BSE) is caused by different prion strains that are discriminated by the molecular characteristics of the pathological prion protein. In 2011, Switzerland reported two presumptive cases of BSE in cattle with a prion protein phenotype different from previously described strains, and it was unclear whether these findings were related to a transmissible disease and have implications on animal and public health. In this study, brain tissues of these cases were inoculated into transgenic mice expressing the bovine prion protein (BoPrP-Tg110) and into cattle. Clinical and pathological investigations as well as molecular testing did not provide evidence for the presence of BSE in the Swiss cases after two passages in BoPrP-Tg110 mice and a challenge period of 3.5 years in cattle. This lack of disease transmission suggests that the Swiss 2011 cases were not affected by a prion disease and were unrelated to the feed-born BSE epidemic.

Understanding the evolution of a new metabolic capability in full mechanistic detail is challenging, as causative mutations may be masked by non-essential "hitchhiking" mutations accumulated during the evolutionary trajectory. We have previously used adaptive laboratory evolution of a rationally engineered ancestor to generate an Escherichia coli strain able to utilize CO2 fixation for sugar synthesis. Here, we reveal the genetic basis underlying this metabolic transition. Five mutations are sufficient to enable robust growth when a non-native Calvin–Benson–Bassham cycle provides all the sugar-derived metabolic building blocks. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO2 fixation cycle toward biomass (prs, serA, and pgi), or in key regulators of carbon metabolism (crp and ppsR). Using suppressor analysis, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the enzymatic constraints that are essential to the metabolic stability of autocatalytic cycles and are relevant to future efforts in constructing non-native carbon fixation pathways., An E. coli strain able to use CO2 fixation for sugar synthesis was previously generated by experimental evolution of an engineered strain. Here, Herz et al. show that specific mutations in five genes, encoding carbon metabolism enzymes or key regulators, are sufficient to enable robust growth of the strain.

PRPS1 encodes isoform I of phosphoribosylpyrophosphate synthetase (PRS-I), a key enzyme in nucleotide biosynthesis. Different missense mutations in PRPS1 cause a variety of disorders that include PRS-I superactivity, nonsyndromic sensorineural hearing impairment, Charcot-Marie-Tooth disease, and Arts syndrome. It has been proposed that each mutation would result in a specific phenotype, depending on its effects on the structure and function of the enzyme.Thirteen Spanish unrelated families segregating X-linked hearing impairment were screened for PRPS1 mutations by Sanger sequencing. In two positive pedigrees, segregation of mutations was studied, and clinical data from affected subjects were compared.We report two novel missense mutations in PRPS1, p.Ile275Thr and p.Gly306Glu, which were found in the propositi of two unrelated Spanish families, both subjects presenting with nonsyndromic hearing impairment. Further investigation revealed syndromic features in other hemizygous carriers from one of the pedigrees. Sequencing of genes that are functionally related to PRPS1 did not reveal any candidate variant that might act as a phenotype modifier.This case of intrafamilial phenotypic variation associated with a single PRPS1 mutation complicates the genotype-phenotype correlations, which makes genetic counseling of mutation carriers difficult because of the wide spectrum of severity of the associated disorders.