RESUMEN
To explore the anti-hyperuricemia components in sunflower (Helianthus annuus L.) calathide extract (SCE), we identified abietic acid (AA) via liquid chromatography-mass spectrometry and found an excellent inhibitor of xanthine oxidase (IC50 = 10.60 µM, Ki = 193.65 nM) without cytotoxicity. Based on the transcriptomics analysis of the human embryonic kidney 293T cell model established using 1 mM uric acid, we evaluated that AA showed opposite modulation of purine metabolism to the UA group and markedly suppressed the intensity of purine nucleoside phosphorylase, ribose phosphate pyrophosphokinase 2, and ribose 5-phosphate isomerase A. Molecular docking also reveals the inhibition of purine nucleoside phosphorylase and ribose phosphate pyrophosphokinase 1. The SCE exhibits similar regulation of these genes, so we conclude that AA was a promising component in SCE against hyperuricemia. This present study provided a novel cell model for screening anti-hyperuricemia natural drugs in vitro and illustrated that AA, a natural diterpenoid, is a potential inhibitor of purine biosynthesis or metabolism.
Asunto(s)
Helianthus , Hiperuricemia , Humanos , Helianthus/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Simulación del Acoplamiento Molecular , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Células HEK293 , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/metabolismo , Riñón/metabolismo , Purinas/metabolismo , Xantina OxidasaRESUMEN
To gain a better insight of how Copper (Cu) ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (ΔcopBL ΔcopAZ; cop-). Exposure of the cop- strain to Cu(II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu(II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu(II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu(II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu(II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections.
Asunto(s)
Cobre , Staphylococcus aureus , Animales , Ratones , Staphylococcus aureus/metabolismo , Vía de Pentosa Fosfato , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Fosforribosil Pirofosfato/metabolismo , AdeninaRESUMEN
The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here we determine structures of human PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.
Asunto(s)
Ribosa-Fosfato Pirofosfoquinasa , Humanos , Sitio Alostérico , Mutación , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.
Asunto(s)
Mercaptopurina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Mercaptopurina/farmacología , Sistemas CRISPR-Cas/genética , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Recurrencia , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/uso terapéutico , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is the first enzyme in the de novo purine nucleotide synthesis pathway and is essential for cell development. However, the effect of PRPS1 on melanoma proliferation and metastasis remains unclear. This study aimed to investigate the regulatory mechanism of PRPS1 in the malignant progression of melanoma. Here, we found PRPS1 was upregulated in melanoma and melanoma cells. In addition, our data indicated that PRPS1 could promote the proliferation and migration and invasion of melanoma both in vitro and in vivo. PRPS1 also could inhibit melanoma cell apoptosis. Furthermore, we found NRF2 is an upstream transcription factor of PRPS1 that drive malignant progression of melanoma.
Asunto(s)
Melanoma , Ribosa-Fosfato Pirofosfoquinasa , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Nucleótidos de Purina , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Síndrome , Regulación hacia ArribaRESUMEN
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 Vmax differences were identified.
Asunto(s)
Fosforribosil Pirofosfato , Ribosa-Fosfato Pirofosfoquinasa , Difosfatos , Histidina , Simulación del Acoplamiento Molecular , NAD , NADP , Nucleótidos , Fosfatos , Fosforribosil Pirofosfato/química , Fosforribosil Pirofosfato/metabolismo , Purinas/metabolismo , Piridinas , Pirimidinas , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , TriptófanoRESUMEN
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.
Asunto(s)
Fosforribosil Pirofosfato , Pez Cebra , Adenosina Trifosfato/química , Animales , Humanos , NAD , Recurrencia Local de Neoplasia , Fosforribosil Pirofosfato/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Pez Cebra/metabolismoRESUMEN
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 PRPS1 gene was discovered as a key disease-resistance candidate gene in yellow drum, Nibea albiflora, in response to the infection of Vibrio harveyi, through genome-wide association analysis. This study mainly focused on the characteristics and its roles in immune responses of the PRPS1 gene in yellow drum. In the present study, the NaPRPS1 gene was cloned from yellow drum, encoding a protein of 320 amino acids. Bioinformatic analysis showed that NaPRPS1 was highly conserved during evolution. Quantitative RT-PCR demonstrated that NaPRPS1 was highly expressed in the head-kidney and brain, and its transcription and translation were significantly activated by V. harveyi infection examined by RT-qPCR and immunohistochemistry analysis, respectively. Subcellular localization revealed that NaPRPS1 was localized in cytoplasm. In addition, semi-in vivo pull-down assay coupled with mass spectrometry identified myeloid differentiation factor 88 (MyD88) as an NaPRPS1-interacting patterner, and their interaction was further supported by reciprocal pull-down assay and co-immunoprecipitation. The inducible expression of MyD88 by V. harveyi suggested that the linker molecule MyD88 in innate immune response may play together with NaPRPS1 to coordinate the immune signaling in yellow drum in response to the pathogenic infection. We provide new insights into important functions of PRPS1, especially PRPS1 in the innate immunity of teleost fishes, which will benefit the development of marine fish aquaculture.
Asunto(s)
Enfermedades de los Peces , Perciformes , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Vibrio , Animales , Enfermedades de los Peces/genética , Peces/genética , Estudio de Asociación del Genoma Completo , Inmunidad Innata/genética , Factor 88 de Diferenciación Mieloide/genética , Perciformes/genética , Fosforribosil Pirofosfato , Vibrio/fisiologíaRESUMEN
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.
Asunto(s)
Fosforribosil Pirofosfato , Ribosa-Fosfato Pirofosfoquinasa , Ligasas/metabolismo , Nucleótidos/metabolismo , Fosforilación , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
Nicotinamide mononucleotide (NMN), a precursor of NAD+, can be synthesized by the conversion of nicotinamide with the help of nicotinamide phosphoribosyl transferase (NAMPT) via the salvage pathway. NMN has recently gained great attention as an excellent therapeutic option due to its long-term effective pharmacological activities. In this study, we constructed a recombinant strain of Escherichia coli by inserting NAMPT and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and PRPS2 (from Homo sapiens) genes to investigate the effect of PRPS1 and PRPS2 on NMN synthesis. The metabolically engineered strain of E. coli BL21 (DE3) exhibited 1.57 mM NMN production in the presence of Mg2+ and phosphates in batch fermentation studies. For further improvement in NMN production levels, effects of different variables were studied using a response surface methodology approach. A significant increment was achieved with a maximum of 2.31 mM NMN production when supplemented with 1% ribose, 1 mM Mg2+ and phosphate, and 0.5% nicotinamide in the presence of a lactose (1%) inducer. Additionally, insertion of the PRPS1 and PRPS2 genes in the phosphoribosyl pyrophosphate synthesis pathway and individual gene expression studies facilitated a higher NMN production at the intracellular level than the reported studies. The strain exhibited intracellular production of NMN from cheap substrates such as glucose, lactose, and nicotinamide. Hence, the overall optimized process can be further scaled up for the economical production of NMN using a recombinant strain of E. coli BL21 (DE3), which is the future perspective of the current study.
Asunto(s)
Escherichia coli/metabolismo , Mononucleótido de Nicotinamida/biosíntesis , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Vías Biosintéticas/fisiología , Glucosa/metabolismo , Ingeniería Metabólica/métodos , NAD/metabolismo , Niacinamida/metabolismo , Nucleótidos/metabolismoRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , Purinas/biosíntesis , Ribosa-Fosfato Pirofosfoquinasa/genética , Apoptosis/genética , Línea Celular Tumoral , Autorrenovación de las Células/genética , Supervivencia Celular/genética , Cromatografía Liquida , Daño del ADN/genética , Resistencia a Antineoplásicos/genética , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Espectrometría de Masas , Metaboloma , Nucleótidos de Purina , Purinas/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/metabolismo , Células Madre Neoplásicas/metabolismo , Nucleótidos/metabolismo , Adulto , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , China , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Metabolómica/métodos , Ratones , Ratones Endogámicos BALB C , Nucleótidos/biosíntesis , Purinas , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Carcinogénesis/metabolismo , MicroARNs/metabolismo , Neuroblastoma/metabolismo , ARN Largo no Codificante/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Niño , Técnicas de Silenciamiento del Gen/métodos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Neuroblastoma/genética , Neuroblastoma/patología , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
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.
Asunto(s)
Clonación de Organismos/efectos adversos , Hipoxantina Fosforribosiltransferasa/metabolismo , Riñón/fisiopatología , Hígado/fisiopatología , Mortalidad/tendencias , Técnicas de Transferencia Nuclear/efectos adversos , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Animales , Femenino , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Ribosa-Fosfato Pirofosfoquinasa/genética , PorcinosRESUMEN
BACKGROUND: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown. RESULTS: By generating novel alleles in the zebrafish PRPS paralogs, prps1a and prps1b, we gained new insight into the role of PRPS filaments during eye development. We found that mutations in prps1a alone are sufficient to generate abnormally small eyes along with defects in head size, pigmentation, and swim bladder inflation. Furthermore, a loss-of-function mutation that truncates the Prps1a protein resulted in the failure of PRPS filament assembly. Lastly, in mutants that fail to assemble PRPS filaments, we observed disorganization of the actin network in the lens fibers. CONCLUSIONS: The truncation of Prps1a blocked PRPS filament formation and resulted in a disorganized lens fiber actin network. Altogether, these findings highlight a potential role for PRPS filaments during lens fiber organization in zebrafish.
Asunto(s)
Cristalino/embriología , Cristalino/crecimiento & desarrollo , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Actinas/metabolismo , Sacos Aéreos/embriología , Alelos , Animales , Ojo/embriología , Ojo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Microscopía Fluorescente , Mutación , Pigmentación , Polimerizacion , Retina/embriología , Epitelio Pigmentado de la Retina/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Apoptosis/genética , Factor de Transcripción E2F1/metabolismo , Oligospermia/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Animales , Caspasa 6/metabolismo , Caspasa 9/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Factor de Transcripción E2F1/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN/metabolismo , Distribución Aleatoria , Transducción de Señal , Espermatocitos/fisiología , Testículo/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Proteína bcl-X/metabolismoRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , MicroARNs/genética , ARN Largo no Codificante/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Pronóstico , Ribosa-Fosfato Pirofosfoquinasa/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Autofagia/genética , Lisosomas/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Pleiotropía Genética/genética , Lisosomas/metabolismo , Mutación , Mutación Missense , Proteostasis/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Redes y Vías Metabólicas , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Proteínas Mitocondriales/genética , Ribosa-Fosfato Pirofosfoquinasa/genética , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.