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Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS–MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.

Abstract Uridine phosphorylases are known as key targets for the development of new anticancer and antiparasitic agents. Crystals of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae were grown in microgravity by the capillary counter-diffusion method on board of the International Space Station. The three-dimensional structure of this enzyme was determined at atomic (1.04 Å) resolution (RCSB PDB ID: 6Z9Z). Alternative conformations of long fragments (β-strands and adjacent loops) of the protein molecule were found for the first time in the three-dimensional structure of uridine phosphorylase in the absence of specific bound ligands. Apparently, these alternative conformations are related to the enzyme function. Conformational analysis with Markov state models demonstrated that conformational rearrangements can occur in the ligand-free state of the enzyme.

Crystals of mutants of uridine phosphorylase from Shewanella oneidensis MR-1 at the active-site threonine residue were obtained, and the three-dimensional structures of the mutants were determined. It was shown that the loop 161–179 responsible for the nucleoside recognition is involved in stabilization of the hexameric structure of the protein and its disorder significantly facilitates the access of nucleoside to the enzyme active site. The role of conformational changes in the enzyme function is discussed.

Colorectal cancer (CRC) is commonly associated with aberrant transcription regulation, but characteristics of the dysregulated transcription factors in CRC pathogenesis remain to be elucidated. In the present study, core-binding factor β (CBFβ) is found to be significantly upregulated in human CRC tissues and correlates with poor survival rate of CRC patients. Mechanistically, CBFβ is found to promote CRC cell proliferation, migration, invasion, and inhibit cell apoptosis in a RUNX2-dependent way. Transcriptome studies reveal that CBFβ and RUNX2 form a transcriptional complex that activates gene expression of OPN, FAM129A, and UPP1. Furthermore, CBFβ significantly promotes CRC tumor growth and live metastasis in a mouse xenograft model and a mouse liver metastasis model. In addition, tumor-suppressive miR-143/145 are found to inhibit CBFβ expression by specifically targeting its 3'-UTR region. Consistently, an inverse correlation between miR-143/miR-145 and CBFβ expression levels is present in CRC patients. Taken together, this study uncovers a novel regulatory role of CBFβ-RUNX2 complex in the transcriptional activation of OPN, FAM129A, and UPP1 during CRC development, and may provide important insights into CRC pathogenesis.

A library of strains producing recombinant nucleoside phosphorylases (NPs) and their mutant and hybrid forms from various mesophilic and extremophilic microorganisms was constructed based on Escherichia coli cells. Substrates were shown to stabilize the NP structure upon thermal exposure, with the inorganic phosphate ion playing a decisive role in the process. Bioinformatics analyses made it possible to assume that the N‑terminal structure of NPs is largely responsible for their thermal stability. A hybrid thymidine phosphorylase (TPP) was constructed via the replacement of the N-terminal fragment (amino acid residues 1–62) of E. coli TPP with the corresponding TPP fragment from the thermophilic bacterium Geobacillus stearothermophilus. Higher thermal stability was observed for the hybrid TPP. The primary structure of E. coli uridine phosphorylase (UDP) was found to have a sequence, 25-Pro-Gly-Asp-Pro-30 (amino acid residues are numbered as in E. coli UDP), that is highly conserved among UDPs of mesophilic microorganisms. The E. coli UDP (Asp27Gly) mutant was constructed and similarly showed a higher thermal stability than the original form. The architecture of the phosphate-binding site and features of its function were assumed to be crucial for the thermal stability of the enzyme.

Metabolic pathways in cancer cells typically become reprogrammed to support unconstrained proliferation. These abnormal metabolic states are often accompanied by accumulation of high concentrations of ATP in the cytosol, a phenomenon known as the Warburg Effect. However, how high concentrations of ATP relate to the functional state of proteins is poorly understood. Here, we comprehensively studied the influence of ATP levels on the functional state of the human enzyme, uridine phosphorylase I (hUP1), which is responsible for activating the chemotherapeutic pro‐drug, 5‐fluorouracil. We found that elevated levels of ATP decrease the stability of hUP1, leading to the loss of its proper folding and function. We further showed that the concentration of hUP1 exerts a critical influence on this ATP‐induced destabilizing effect. In addition, we found that ATP interacts with hUP1 through a partially unfolded state and accelerates the rate of hUP1 unfolding. Interestingly, some structurally similar metabolites showed similar destabilization effects on hUP1. Our findings suggest that metabolites can alter the folding and function of a human protein, hUP1, through protein destabilization. This phenomenon may be relevant in studying the functions of proteins that exist in the specific metabolic environment of a cancer cell.

Uridine phosphorylase 1 (UPP1) has been reported as an oncogene in several malignancies. In glioma, the role of UPP1 remains unclear. This study was performed to explore its role in glioma at transcriptional level. Totally, 998 glioma patients with clinical data were enrolled, including 301 mRNA microarray data from Chinese Glioma Genome Atlas (CGGA) dataset and 697 RNAseq data from The Cancer Genome Atlas (TCGA) dataset. Statistical analysis was performed with R language. UPP1 expression level was positively correlated with WHO grade of glioma. UPP1 was significantly upregulated in mesenchymal subtype and could serve as a potential biomarker for this subtype. Based on most correlated genes of UPP1, Gene ontology analysis revealed that UPP1 was profoundly associated with immune and inflammatory response. Gene Sets Variation Analysis was further performed and showed that UPP1 was particularly correlated with MHC‐II and LCK, which were mainly associated with activities of antigen‐presenting cells and T cells. Moreover, UPP1 was found to be synergistic with various immune checkpoint members, especially with PD1 pathway and B7‐H3. Finally, Kaplan‐Meier curves revealed that higher UPP1 indicated significantly shorter survival for glioma patients. Taken together, UPP1 played an oncogenic role in glioma via suppressing tumor‐related immune response., UPP1 is positively associated with WHO grade of glioma. UPP1 is mainly involved in tumor‐induced immune response, particularly associated with antigen‐presenting cell‐related immune response and T‐cell activation. High expression level of UPP1 predicts poor prognosis in glioma.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

The high-resolution three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complex with the competitive inhibitor 2,2'-anhydrouridine was determined by X-ray diffraction (RCSBPDB ID: 6RCA). The three-dimensional structure of this complex is compared with the previously determined structures of V. cholerae uridine phosphorylase in complex with the substrate (uridine) and S. typhimurium uridine phosphorylase in complex with 2,2'-anhydrouridine. The protein–inhibitor and protein–substrate binding free energies were calculated by the free-energy perturbation method. The number of stable hydrogen bonds between the 2,2'-anhydrouridine molecule and the active site of the enzyme is smaller and these bonds are longer compared to the natural substrate of the enzyme (uridine). However, calculations taking into account solvation energy of the molecule and the entropy effects showed that the binding of the inhibitor (2,2'-anhydrouridine) at the active site of the protein is energetically more favorable than the binding of the native substrate (uridine). These results may be useful in the design of new inhibitors with a higher selectivity for the binding sites of uridine phosphorylases.

Objective Protein tyrosine kinases (PTKs) regulate osteoarthritis (OA) progression by activating a series of signal transduction pathways. However, the roles of protein tyrosine phosphatases (PTPs) in OA remain obscure. Methods The expression of 107 PTP genes in human OA cartilage was analyzed based on a single-cell sequencing dataset. The enzyme activity of the PTP SHP2 was detected in primary chondrocytes after interleukin (IL)-1β treatment and in human OA cartilage. Destabilized medial meniscus (DMM) model and IL-1β-stimulated primary mouse chondrocytes were treated with an SHP2 inhibitor and celecoxib (a clinical drug for the treatment of OA). The function of SHP2 in OA pathogenesis was further verified in Aggrecan-CreERT ; SHP2 flox/flox mice. The downstream protein expression profile and dephosphorylated substrate of SHP2 were examined by tandem mass tag (TMT) labeling-based global proteomic and stable isotope labeling using amino acids in cell culture (SILAC)-labeled tyrosine phosphoproteomic analysis, respectively. Results SHP2 enzyme activity significantly increased in human OA samples with serious articular cartilage injury and in IL-1β-stimulated chondrocytes. Pharmacological inhibition or genetic deletion of SHP2 ameliorated OA progression. SHP2 inhibitors dramatically reduced the expression of cartilage degradation-related genes and simultaneously promoted the expression of cartilage synthesis-related genes. Mechanistically, SHP2 inhibition suppressed the dephosphorylation of DOK1 and subsequently reduced the expression of uridine phosphorylase 1 and increased uridine level, thereby contributing to the homeostasis of cartilage metabolism. Conclusions SHP2 is a novel accelerator of the imbalance in the cartilage homeostasis. Specific inhibition of SHP2 may ameliorate OA by maintaining the anabolic and catabolic balance.

Thyroid cancer incidence has been continuity increasing worldwide. Uridine phosphorylase 1 (UPP1) is a protein‐coding gene and has been detected that UPP1 was the higher expression in many solid malignancies, just as head and neck cancers, breast cancer, compared with paired normal tissue. But the act of UPP1 in thyroid cancer is not explicit. In this article, we investigate the function of UPP1 expression in thyroid cancer. The Cancer Genome Atlas (TCGA) unpaired thyroid cancer and normal RNA‐seq data were downloaded, and our paired thyroid cancer and normal samples were analysed by a polymerase chain reaction. The expression of UPP1 was regulated by transfected small interfering RNA, and the function of UPP1 was determined via migration, invasion and cell proliferation assays. Western blot assay was achieved to determine the UPP1 expression correlates with the function of 5‐FU regulate epithelial‐mesenchymal transition. The significant upregulation of UPP1 in thyroid cancer tissues compared with normal thyroid tissues was revealed by our data and TCGA data. UPP1 overexpression was significantly correlated with lymph node metastasis, tumour stage and tumour size. In the cell, experiments showed that UPP1 low expression significantly suppressed the migration, invasion and proliferation. Western blot assay proves the effect of UPP1 expression on 5‐FU regulates epithelial‐mesenchymal transition pathway. UPP1 plays a crucial oncogene in thyroid cancer. Our findings indicate that UPP1 might be a biomarker of thyroid cancer and may act by regulating epithelial‐mesenchymal transition (EMT).

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai‐1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS‐PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT‐2 cells, which are an IL‐2‐dependent T cell line, nor did it modify IL‐2 production by Con A‐stimulated mouse spleen cells. The N‐terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai‐1 was cloned into expression vector pQE‐60 in Escherichia coli XL‐1 Blue. Recombinant UPase (rUPase) tagged with His at the C‐terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A‐stimulated mouse spleen cells and may be a virulence factor.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general., The conversion of simple reagents into the 1st antiviral for COVID-19 was realized through the use of a multienzyme cascade made possible by the development of an efficient phosphate catalytic cycle.