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Proceedings of the National Academy of... Dec 2023Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the...
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.
Topics: Female; Humans; Breast Neoplasms; Immunity, Innate; Membrane Proteins; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 38117852
DOI: 10.1073/pnas.2313693120 -
The Protein Journal Feb 2024Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in...
Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.
Topics: Humans; Pyrophosphatases; Inosine Triphosphatase; Polymorphism, Genetic; Genotype
PubMed: 38066288
DOI: 10.1007/s10930-023-10162-0 -
Scientific Reports Dec 2023Rheumatic joints have an altered cartilage turnover. Cartilage intermediate layer protein 1 (CILP-1) is secreted from articular chondrocytes and deposited into the...
Rheumatic joints have an altered cartilage turnover. Cartilage intermediate layer protein 1 (CILP-1) is secreted from articular chondrocytes and deposited into the cartilage extracellular matrix. We developed an immunoassay targeting a Matrix Metalloproteinase (MMP)-generated neo-epitope of CILP-1, named CILP-M. Human articular cartilage was cleaved with proteolytic enzymes and CILP-M levels were measured. We also quantified CILP-M in two studies from patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS) and osteoarthritis (OA) and explored the monitoring and prognostic potential of CILP-M in TNF-α inhibitory treatment and modified Stoke AS Spine Score (mSASSS) progression. CILP-M was generated by MMP-1, -8 and -12. In the discovery study, CILP-M was significantly higher in patients with RA, AS and OA than healthy donors (p < 0.01, p < 0.001, p < 0.05) with an area under the curve (AUC) between the diseased groups and healthy donors > 0.95 (p < 0.001). In the validation study, patients with RA and AS had significantly higher CILP-M levels than healthy controls (p < 0.001) and AUC > 0.90 (p < 0.001). Patients with AS treated with TNF- α inhibitory treatment in the validation study had significantly lower CILP-M levels after treatment (p = 0.004). CILP-M may provide useful insights into cartilage degradation processes in rheumatic diseases.
Topics: Humans; Arthritis, Rheumatoid; Biomarkers; Cartilage, Articular; Matrix Metalloproteinase 1; Osteoarthritis; Spondylitis, Ankylosing; Tumor Necrosis Factor-alpha; Pyrophosphatases; Extracellular Matrix Proteins
PubMed: 38066013
DOI: 10.1038/s41598-023-48787-x -
PLoS Pathogens Dec 2023During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its...
During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.
Topics: Animals; Humans; Plasmodium falciparum; Proton Pumps; Trophozoites; Diphosphates; Protons; Erythrocytes; Pyrophosphatases; Malaria, Falciparum; Antimalarials
PubMed: 38048362
DOI: 10.1371/journal.ppat.1011818 -
The Journal of Biological Chemistry Jan 2024Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage...
Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.
Topics: Animals; Humans; Mice; Aging; Cartilage; Luciferases; Mice, Knockout; Osteoporosis; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 38042486
DOI: 10.1016/j.jbc.2023.105512 -
International Journal of Molecular... Nov 2023Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular...
Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (β-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, β-HIVS treatment decreased dUTPase expression. β-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. β-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.
Topics: Humans; Epithelial Cell Adhesion Molecule; Carcinoma, Hepatocellular; Liver Neoplasms; Cell Line, Tumor; Neoplastic Stem Cells; Fluorouracil
PubMed: 38003473
DOI: 10.3390/ijms242216283 -
Metabolites Nov 2023Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known...
Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known disease-causing enzymes involved in galactose metabolism. Using whole-exome sequencing, we identified a homozygous missense variant in encoding the cytosolic pyrophosphatase 1 (PPA1), c.557C>T (p.Thr186Ile). The enzyme activity of PPA1 was determined using a colorimetric assay, and the protein content was visualized via western blotting in skin fibroblasts from one of the affected individuals. The galactolytic activity of the affected fibroblasts was determined by measuring extracellular acidification with a Seahorse XFe96 analyzer. PPA1 activity decreased to 22% of that of controls in the cytosolic fraction of homogenates from patient fibroblasts. PPA1 protein content decreased by 50% according to western blot analysis, indicating a reduced stability of the variant protein. The extracellular acidification rate was reduced in patient fibroblasts when galactose was used as a substrate. Untargeted metabolomics of blood samples revealed an elevation of other metabolites related to pyrophosphate metabolism. Besides hyperbilirubinemia in the neonatal period in one child, both children were clinically unremarkable at the ages of 3 and 14 years, respectively. We hypothesize that the observed metabolic derangement is a possible mild manifestation of PPA1 deficiency. Unresolved abnormalities in galactosemia screening might result in the identification of more individuals with PPA1 deficiency, a newly discovered inborn metabolic disorder (IMD).
PubMed: 37999237
DOI: 10.3390/metabo13111141 -
Theranostics 2023One of the hallmarks of osteoarthritis (OA), the most common degenerative joint disease, is increased numbers of senescent chondrocytes. Targeting senescent...
One of the hallmarks of osteoarthritis (OA), the most common degenerative joint disease, is increased numbers of senescent chondrocytes. Targeting senescent chondrocytes or signaling mechanisms leading to senescence could be a promising new therapeutic approach for OA treatment. However, understanding the key targets and links between chondrocyte senescence and OA remains unclear. Senescent chondrocytes were identified from , double-knockout mice lacking and (dKO) and applied to microarray. The presence of forkhead transcription factor M1 (FOXM1) was detected in aged, dKO, and destabilization of the medial meniscus (DMM) cartilages and articular chondrocytes, and the effect of overexpression and acetyl-CoA treatment on cartilage homeostasis was examined using immunohistochemistry, quantitative real-time PCR (qRT-PCR), cell apoptosis and proliferation assay, and safranin O staining. Delivery of Rho@PAA-MnO (MnO nanosheet) or heparin-ACBP/COS-GA- (ACBP-) nanoparticles into DMM cartilage was performed Here, we propose the specific capture of acetyl-CoA with the delivery of ( siRNA () to prevent cartilage degradation by inhibiting the axis of chondrocyte senescence. dKO stimulate chondrocyte senescence via the upregulation of and contribute to severe cartilage breakdown. We found that the accumulation of acetyl-CoA in the dKO mice may be responsible for the upregulation of during OA pathogenesis. Moreover, scavenging reactive oxygen species (ROS) induced by chondrocyte senescence via the implantation of MnO nanosheets or delivery of functionalized with acetyl-CoA binding protein (ACBP) to capture acetyl-CoA using an injectable bioactive nanoparticle (-ACBP-NP) significantly suppressed DMM-induced cartilage destruction. We found that the loss of and stimulates chondrocyte senescence via the upregulation of and accumulation of acetyl-CoA, and the application of -ACBP-NP is a potential therapeutic strategy for OA treatment.
Topics: Animals; Mice; Acetyl Coenzyme A; Cellular Senescence; Chondrocytes; Manganese Compounds; Mice, Knockout; Osteoarthritis; Oxides; Nudix Hydrolases
PubMed: 37908734
DOI: 10.7150/thno.89033 -
Medicine Oct 2023To investigate associations between inosine triphosphatase (ITPA) gene polymorphisms and long-term outcomes among chronic hepatitis C (CHC) patients in Northeast China...
To investigate associations between inosine triphosphatase (ITPA) gene polymorphisms and long-term outcomes among chronic hepatitis C (CHC) patients in Northeast China treated with Peg-interferon (IFN)/ribavirin (RBV). CHC patients who received Peg-IFN-2a/RBV treatment during between 2011 and 2013 at 5 hepatitis centers in Northeast China were enrolled. ITPA single nucleotide polymorphisms rs1127354 and rs7270101 from all patients were detected and their associations with 5-year outcomes were analyzed. A total of 635 patients, including 421 infected with hepatitis C virus (HCV) genotype 1 and 214 infected with non-genotype 1 were included. No significant differences were observed in the distribution frequencies of ITPA rs1127354 variants and ITPase activity between patients with HCV genotype 1 and non-genotype 1. In patients who received more than 80% of the planned RBV dose, the 5-year virological response rate and the improvement in liver fibrosis were higher in those with ITPA rs1127354 non-CC with ITPase activity <25% compared with these outcomes in patients with ITPA rs1127354 CC with 100% ITPase activity. Multiple regression analysis revealed that HCV genotype non-1, low baseline HCV ribose nucleic acid (RNA) levels (≤4 × 105 IU/mL), interleukin-28B rs12979860 CC genotype, low baseline liver fibrosis (Fibroscan 0-2), and ITPA rs1127354 non-CC genotype were independent predictors for a high long-term virological response rate, whereas interleukin-28B rs12979860 CC genotype, ITPA rs1127354 non-CC genotype, and low baseline liver fibrosis were independent predictors for improvement of liver fibrosis. ITPA rs1127354 polymorphisms is predictors of long-term outcomes in CHC patients treated with Peg-IFN/RBV.
Topics: Humans; Ribavirin; Hepatitis C, Chronic; Antiviral Agents; Follow-Up Studies; Treatment Outcome; Pyrophosphatases; Interferons; Polymorphism, Single Nucleotide; Genotype; Hepacivirus; Liver Cirrhosis; Interleukins; Inosine Triphosphatase
PubMed: 37904484
DOI: 10.1097/MD.0000000000035508 -
Plant Biotechnology Journal Mar 2024Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing a significant loss in crop yield and ornamental value....
Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing a significant loss in crop yield and ornamental value. Biotechnology approaches manipulating specific genes prove to be effective strategies in crop trait modification. The Arabidopsis vacuolar pyrophosphatase gene AVP1, the rice SUMO E3 ligase gene OsSIZ1 and the cyanobacterium flavodoxin gene Fld have previously been implicated in regulating plant stress responses and conferring enhanced tolerance to different abiotic stresses when individually overexpressed in various plant species. We have explored the feasibility of combining multiple favourable traits brought by individual genes to acquire superior plant performance. To this end, we have simultaneously introduced AVP1, OsSIZ1 and Fld in creeping bentgrass. Transgenic (TG) plants overexpressing these three genes performed significantly better than wild type controls and the TGs expressing individual genes under both normal and various abiotic stress conditions, exhibited significantly enhanced plant growth and tolerance to drought, salinity and heat stresses as well as nitrogen and phosphate starvation, which were associated with altered physiological and biochemical characteristics and delicately fine-tuned expression of genes involved in plant stress responses. Our results suggest that AVP1, OsSIZ1 and Fld function synergistically to regulate plant development and plant stress response, leading to superior overall performance under both normal and adverse environments. The information obtained provides new insights into gene stacking as an effective approach for plant genetic engineering. A similar strategy can be extended for the use of other beneficial genes in various crop species for trait modifications, enhancing agricultural production.
Topics: Stress, Physiological; Plants, Genetically Modified; Arabidopsis; Arabidopsis Proteins; Plant Development; Gene Expression Regulation, Plant; Droughts; Plant Proteins
PubMed: 37902192
DOI: 10.1111/pbi.14216