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BMC Pediatrics Jan 2024NUDT2 is an enzyme important for maintaining the intracellular level of the diadenosine tetraphosphate (Ap4A). Bi-allelic loss of function variants in NUDT2 has recently...
NUDT2 is an enzyme important for maintaining the intracellular level of the diadenosine tetraphosphate (Ap4A). Bi-allelic loss of function variants in NUDT2 has recently been reported as a rare cause of intellectual disability (ID). Herein, we describe a Chinese girl with ID, attention deficit hyperactivity disorder (ADHD), and motor delays with abnormal walking posture and difficulty climbing stairs, who bears compound heterozygous variants c.34 C > T (p.R12*) and c.194T > G (p.I65R) in NUDT2. Homozygous variants c.34 C > T (p.R12*) or c.186del (p.A63Qfs*3) in NUDT2 were previously reported to cause ID. This is the first patient with ID due to compound heterozygous variants in NUDT2 and p.I65R is a novel missense variant. This study enriched the genotype and phenotype of NUDT2-related ID and supported the critical developmental involvement of NUDT2.
Topics: Female; Humans; Intellectual Disability; Genotype; Phenotype; Mutation, Missense; Homozygote; Nudix Hydrolases; Phosphoric Monoester Hydrolases
PubMed: 38243213
DOI: 10.1186/s12887-024-04542-3 -
American Journal of Physiology. Cell... Mar 2024The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene...
The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization. We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.
Topics: Animals; Osteogenesis; Alkaline Phosphatase; Cell Differentiation; Phosphoric Diester Hydrolases; Calcinosis; Pyrophosphatases
PubMed: 38223929
DOI: 10.1152/ajpcell.00692.2023 -
EMBO Reports Feb 2024Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na- and/or H across membranes to the hydrolysis of...
Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na- and/or H across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na and H.
Topics: Pyrophosphatases; Membranes; Catalysis; Sodium
PubMed: 38182815
DOI: 10.1038/s44319-023-00037-x -
Virology Journal Jan 2024In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have...
BACKGROUND
In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues.
METHOD
Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated.
RESULTS
While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules.
CONCLUSION
Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.
Topics: Inosine Triphosphatase; Euphorbia; Phylogeny; Coinfection; RNA Viruses; Nucleotides; Potyviridae; Plant Viruses; Plants; Nucleic Acids; RNA, Viral; Genome, Viral
PubMed: 38178191
DOI: 10.1186/s12985-023-02257-y -
Heliyon Jan 2024In some patients especially those AMA negative, the diagnosis may be a challenge requiring liver biopsy. This study determined whether autotaxin, a secreted...
BACKGROUND
In some patients especially those AMA negative, the diagnosis may be a challenge requiring liver biopsy. This study determined whether autotaxin, a secreted lysophospholipase D encoded by the exonucleotide pyrophosphatase phosphodiesterase 2 gene, can be used as a serum biomarker for primary biliary cholangitis.
METHODS
Plasma samples were collected from 103 patients with PBC and 74 healthy controls. autotaxin levels were determined by Enzyme-linked immunosorbent assay, and its predictive value for diagnosing primary biliary cholangitis was analysed. The relationship between autotaxin and the clinical data was also evaluated.
RESULTS
Autotaxin levels in patients with primary biliary cholangitis were significantly higher than those in healthy control (median: 60.7 ng/ml vs. 32.6 ng/ml, P < 0.001). The cut-off value of autotaxin in patients with primary biliary cholangitis was 38.5 ng/ml, and the positivity rate was 33.9 %, calculated twice. The sensitivity, specificity, positive predictive value, and negative predictive value were 54.3 %, 93.1 %, 84.4 %, and 74.8 %, respectively, and the area under the curve was 0.73. Autotaxin level positively correlated with immunoglobulin M level (r = -0.22, P < 0.05) and Ludwig's classification (r = 0.76, P < 0.01) in patients with primary biliary cholangitis. The positivity rate of autotaxin (50.0 %) was higher than that of anti-sp100 (16.7 %) and anti-gp210 (11.1 %) antibodies in anti-mitochondrial antibody -negative patients with primary biliary cholangitis.
CONCLUSIONS
Autotaxin may be an effective noninvasive biomarker used in diagnosis, prognosis of primary biliary cholangitis, particularly in anti-mitochondrial antibody -negative patients.
PubMed: 38173498
DOI: 10.1016/j.heliyon.2023.e23438 -
Scientific Reports Jan 2024Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation...
Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (K ~ 4 nM) and myricetin (K ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.
Topics: Humans; Quercetin; Flavonoids; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 38167594
DOI: 10.1038/s41598-023-50590-7 -
Open Medicine (Warsaw, Poland) 2023This study aims to investigate the impact of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) on vascular calcification in rats. The rationale behind studying...
This study aims to investigate the impact of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) on vascular calcification in rats. The rationale behind studying ENPP1's role in vascular calcification lies in its potential to modulate calcification processes. Understanding this relationship can offer insights into novel therapeutic avenues for addressing vascular calcification-related disorders. In this experiment, vascular smooth muscle cell (VSMC) calcification was induced using β-glycerophosphoric acid. Subsequently, recombinant AAV9-carrying ENPP1 was introduced into VSMCs to achieve both and overexpression of ENPP1. The findings indicate that ENPP1 overexpression significantly reduces calcium and phosphorus content in the aorta ( < 0.05). Alizarin red and von Kossa staining reveal notable reductions in calcium salt deposits in VSMCs and aorta, respectively. Notably, the expression levels of BMP-2, PINP, OC, and BALP were substantially decreased in VSMCs ( < 0.05), underscoring ENPP1's role in impeding osteoblast-like transdifferentiation of VSMCs. Additionally, ENPP1 overexpression led to a significant increase in pyrophosphate (PPi) levels compared to control rats ( < 0.05). In conclusion, this study suggests that ENPP1 contributes to alleviating vascular calcification by elevating PPi levels and inhibiting the phenotypic transformation of VSMCs. These findings shed light on the potential therapeutic role of ENPP1 in mitigating vascular calcification-related complications.
PubMed: 38152331
DOI: 10.1515/med-2023-0861 -
International Journal of Molecular... Dec 2023Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating...
Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.
Topics: Pyrophosphatases; Cystathionine beta-Synthase; Catalytic Domain; Bacterial Proteins; Nucleotides
PubMed: 38138989
DOI: 10.3390/ijms242417160 -
Biomolecules Dec 2023The role of altered brain mitochondrial regulation in psychiatric pathologies, including Major Depressive Disorder (MDD), has attracted increasing attention. Aberrant...
The role of altered brain mitochondrial regulation in psychiatric pathologies, including Major Depressive Disorder (MDD), has attracted increasing attention. Aberrant mitochondrial functions were suggested to underlie distinct inter-individual vulnerability to stress-related MDD syndrome. In this context, insulin receptor sensitizers (IRSs) that regulate brain metabolism have become a focus of recent research, as their use in pre-clinical studies can help to elucidate the role of mitochondrial dynamics in this disorder and contribute to the development of new antidepressant treatment. Here, following 2-week chronic mild stress (CMS) using predation, social defeat, and restraint, MDD-related behaviour and brain molecular markers have been investigated along with the hippocampus-dependent performance and emotionality in mice that received the IRS dicholine succinate (DS). In a sucrose test, mice were studied for the key feature of MDD, a decreased sensitivity to reward, called anhedonia. Based on this test, animals were assigned to anhedonic and resilient-to-stress-induced-anhedonia groups, using a previously established criterion of a decrease in sucrose preference below 65%. Such assignment was based on the fact that none of control, non-stressed animals displayed sucrose preference that would be smaller than this value. DS-treated stressed mice displayed ameliorated behaviours in a battery of assays: sucrose preference, coat state, the Y-maze, the marble test, tail suspension, and nest building. CMS-vulnerable mice exhibited overexpression of the inflammatory markers , , and , as well as and -R, in various brain regions. The alterations in hippocampal gene expression were the closest to clinical findings and were studied further. DS-treated, stressed mice showed normalised hippocampal expression of the plasticity markers , , , , , , and . DS-treated and non-treated stressed mice who were resilient or vulnerable to anhedonia were compared for hippocampal mitochondrial pathway regulation using Illumina profiling. Resilient mice revealed overexpression of the mitochondrial complexes NADH dehydrogenase, succinate dehydrogenase, cytochrome 1, cytochrome oxidase, F-type and V-type ATPases, and inorganic pyrophosphatase, which were decreased in anhedonic mice. DS partially normalised the expression of both ATPases. We conclude that hippocampal reduction in ATP synthesis is associated with anhedonia and pro-inflammatory brain changes that are ameliorated by DS.
Topics: Mice; Animals; Depression; Anhedonia; Depressive Disorder, Major; Resilience, Psychological; Mitochondrial Dynamics; N-Methylaspartate; Hippocampus; Mice, Inbred Strains; Sucrose; Adenosine Triphosphatases; Gene Expression
PubMed: 38136653
DOI: 10.3390/biom13121782 -
MBio Feb 2024The inositol pyrophosphate signaling molecule 1,5-IP is an agonist of RNA 3-processing and transcription termination in fission yeast that regulates the expression of...
Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.
The inositol pyrophosphate signaling molecule 1,5-IP is an agonist of RNA 3-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes , , and . IP is synthesized from 5-IP by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter that abolishes toxicity in YES medium. This result, and the fact that mRNA expression is increased by >40-fold in cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to cells in a Tgp1-dependent manner, and (ii) induced overexpression of in cells also elicits toxicity dependent on GPC. suppressor screens yielded a suite of single missense mutations in the essential IP kinase Kcs1 that generates 5-IP, the immediate precursor to IP. Transcription profiling of the mutants in an background revealed the downregulation of the same phosphate acquisition genes that were upregulated in cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP governs repression of fission yeast phosphate homeostasis genes , , and by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP levels elicit precocious lncRNA termination, leading to derepression of the genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP agonism of transcription termination. It was assumed that IP toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP and the essential Kcs1 kinase that converts IP to 5-IP, the immediate precursor of IP.
Topics: Schizosaccharomyces; Inositol; Diphosphates; Schizosaccharomyces pombe Proteins; RNA, Long Noncoding; Membrane Transport Proteins; Pyrophosphatases; Inositol Phosphates; Peptide Fragments; Thyroglobulin; Phosphotransferases (Phosphate Group Acceptor)
PubMed: 38133430
DOI: 10.1128/mbio.03062-23