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International Journal of Molecular... Feb 2022Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial...
Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-β1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5'-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-β1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.
Topics: Calcium Pyrophosphate; Chondrocalcinosis; Chondrocytes; Histone Deacetylase Inhibitors; Humans; Pyrophosphatases
PubMed: 35269745
DOI: 10.3390/ijms23052604 -
Biochemical and Biophysical Research... Oct 2014There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins.... (Review)
Review
There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.
Topics: Animals; Biological Transport, Active; Cytosol; Dolichols; Endoplasmic Reticulum; Glycosylation; Humans; Models, Biological; Oligosaccharides; Polysaccharides; Protein Folding; Pyrophosphatases
PubMed: 24866240
DOI: 10.1016/j.bbrc.2014.05.075 -
Human Mutation Dec 2022Loss-of-function variants in the ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) cause ENPP1 Deficiency, a rare disorder characterized by... (Review)
Review
Loss-of-function variants in the ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) cause ENPP1 Deficiency, a rare disorder characterized by pathological calcification, neointimal proliferation, and impaired bone mineralization. The consequence of ENPP1 Deficiency is a broad range of age dependent symptoms and morbidities including cardiovascular complications and 50% mortality in infants, autosomal recessive hypophosphatemic rickets type 2 (ARHR2) in children, and joint pain, osteomalacia and enthesopathies in adults. Recent research continues to add to the growing clinical presentation profile as well as expanding the role of ENPP1 itself. Here we review the current knowledge on the spectrum of clinical and genetic findings of ENPP1 Deficiency reported in patients diagnosed with GACI or ARHR2 phenotypes using a comprehensive database of known ENPP1 variants with associated clinical data. A total of 108 genotypes were identified from 154 patients. Of the 109 ENPP1 variants reviewed, 72.5% were demonstrably disease-causing, a threefold increase in pathogenic/likely pathogenic variants over other databases. There is substantial heterogeneity in disease severity, even among patients with the same variant. The approach to creating a continuously curated database of ENPP1 variants accessible to clinicians is necessary to increase the diagnostic yield of clinical genetic testing and accelerate diagnosis of ENPP1 Deficiency.
Topics: Humans; Familial Hypophosphatemic Rickets; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 36150100
DOI: 10.1002/humu.24477 -
MBio Jun 2022Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase...
Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP derepress the regulon while mutations that ablate IP synthesis are hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1-385). Reaction of Asp1 kinase with IP and ATP resulted in both IP phosphorylation to 1-IP and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP is 22-fold faster than with IP and is strongly biased in favor of IP synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of expression correlated with Asp1's kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP or 5-IP. We report that inorganic phosphate is a concentration-dependent enabler of net IP synthesis by full-length Asp1 , by virtue of its antagonism of IP turnover. Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP. IP dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP is 22-fold faster than with IP and is strongly biased in favor of IP synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP turnover.
Topics: Acid Phosphatase; Adenosine Triphosphate; Diphosphates; Gene Expression; Inositol Phosphates; Multifunctional Enzymes; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 35536002
DOI: 10.1128/mbio.01034-22 -
The New Phytologist Mar 2023In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain...
In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.
Topics: Adenosine Triphosphate; DNA; Inosine Triphosphate; Nucleic Acids; Purine Nucleotides; Pyrophosphatases; RNA
PubMed: 36464781
DOI: 10.1111/nph.18656 -
Biochemistry. Biokhimiia Aug 2020Proteins of the NUDIX hydrolase (NUDT) superfamily that cleave organic pyrophosphates are found in all classes of organisms, from archaea and bacteria to higher... (Review)
Review
Proteins of the NUDIX hydrolase (NUDT) superfamily that cleave organic pyrophosphates are found in all classes of organisms, from archaea and bacteria to higher eukaryotes. In mammals, NUDTs exhibit a wide range of functions and are characterized by different substrate specificity and intracellular localization. They control the concentration of various metabolites in the cell, including key regulatory molecules such as nicotinamide adenine dinucleotide (NAD), ADP-ribose, and their derivatives. In this review, we discuss the role of NUDT proteins in the metabolism of NAD and ADP-ribose in human and animal cells.
Topics: Adenosine Diphosphate Ribose; Animals; Escherichia coli; Humans; Intracellular Space; Mammals; NAD; Oxidation-Reduction; Pyrophosphatases; Saccharomyces cerevisiae; Substrate Specificity; Nudix Hydrolases
PubMed: 33045949
DOI: 10.1134/S0006297920080040 -
DNA Repair Aug 2016Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important... (Review)
Review
Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions.
Topics: DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; Genome, Human; Genomic Instability; Humans; Nucleic Acid Precursors; Nucleotides; Oxidation-Reduction; Oxidative Stress; Phosphoric Monoester Hydrolases; Pyrophosphatases; Nudix Hydrolases
PubMed: 27311542
DOI: 10.1016/j.dnarep.2016.05.032 -
Nature Chemical Biology Oct 2020The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites,...
The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
Topics: Binding Sites; Cell Line; Drug Design; Drug Development; Escherichia coli; Humans; Inorganic Pyrophosphatase; Models, Molecular; Protein Binding; Protein Conformation; Pyrophosphatases; Structure-Activity Relationship
PubMed: 32690945
DOI: 10.1038/s41589-020-0592-z -
Journal of Lipid Research Jan 2016The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX... (Review)
Review
The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX is expressed in multiple tissues and participates in numerous key physiologic and pathologic processes, including neural development, obesity, inflammation, and oncogenesis, through the generation of the bioactive lipid, lysophosphatidic acid. Overwhelming evidence indicates that altered ATX activity leads to oncogenesis and cancer progression through the modulation of multiple hallmarks of cancer pathobiology. Here, we review the structural and catalytic characteristics of the ectoenzyme, how its expression and maturation processes are regulated, and how the systemic integration of its pleomorphic effects on cells and tissues may contribute to cancer initiation, progression, and therapy. Additionally, the up-to-date spectrum of the most frequent ATX genomic alterations from The Cancer Genome Atlas project is reported for a subset of cancers.
Topics: Animals; Carcinogenesis; Cell Transformation, Neoplastic; Disease Progression; Humans; Neoplasms; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 25977291
DOI: 10.1194/jlr.R060020 -
Bioscience, Biotechnology, and... 2015Nudix hydrolases are widely distributed in all kingdoms of life and have the potential to hydrolyze a wide range of organic pyrophosphates, including nucleoside di- and... (Review)
Review
Nudix hydrolases are widely distributed in all kingdoms of life and have the potential to hydrolyze a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, nucleotide coenzymes, nucleotide sugars, and RNA caps. However, except for E. coli MutT and its orthologs in other organisms that sanitize oxidized nucleotides to prevent DNA and RNA mutations, the functions of Nudix hydrolases had largely remained unclear until recently, because many members of this enzyme family exhibited broad substrate specificities. There is now increasing evidence to show that their functions extend into many aspects of the regulation of cellular responses. This review summarizes current knowledge on the molecular and enzymatic properties as well as physiological functions of Arabidopsis Nudix hydrolases. The information presented here may provide novel insights into the physiological roles of these enzymes in not only plant species, but also other organisms.
Topics: Amino Acid Sequence; Arabidopsis; Germination; Molecular Sequence Data; Pyrophosphatases; Stress, Physiological; Nudix Hydrolases
PubMed: 25483172
DOI: 10.1080/09168451.2014.987207