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International Journal of Molecular... Oct 2022Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can...
Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.
Topics: Thermococcus; Inorganic Pyrophosphatase; Diphosphates; Archaeal Proteins; Pyrophosphatases; Uridine Diphosphate
PubMed: 36361526
DOI: 10.3390/ijms232112735 -
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 -
Current Topics in Medicinal Chemistry 2022Phosphodiesterases (PDEs) are a wide group of enzymes with multiple therapeutic actions, including vasorelaxation, cardiotonic, antidepressant, anti-inflammatory,... (Review)
Review
BACKGROUND
Phosphodiesterases (PDEs) are a wide group of enzymes with multiple therapeutic actions, including vasorelaxation, cardiotonic, antidepressant, anti-inflammatory, antithrombotic, anti-spasmolytic, memory-enhancing, and anti-asthmatic. PDEs with eleven subtypes from PDE-1 to PDE-11 typically catalyze the cleavage of the phosphodiester bond and, hence, degrades either cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).
OBJECTIVE
Several selective or non-selective inhibitors of the PDE subtypes are used clinically, i.e. sildenafil, rolipram, cysteine, etc. Recently, interest in plant-based pharmacologically bioactive compounds having potent PDEs inhibitory potential has increased. Purposely, extensive research has been carried out on natural products to explore new inhibitors of various PDEs. Therefore, this review summarizes the published data on natural PDEs inhibitors and their potential therapeutic applications.
METHODS
For this purpose, natural compounds with PDE inhibitory potential have been surveyed through several databases, including PubMed, Web of Sciences (WoS), Scopus, and Google Scholar.
RESULTS
According to a detailed literature survey, the most promising class of herbal compounds with PDE-inhibiting property has been found to belong to phenolics, including flavonoids (luteolin, kaempferol, icariin, etc.). Many other encouraging inhibitors from plants have also been identified, such as coumarins (23, 24) (licoarylcoumarin and glycocoumarin,), saponins (agapanthussaponins), lignans (31, 33) [(±)-schizandrin and kobusin], terpenes (28, 29, 31) (perianradulcin A, quinovic acid, and ursolic acid), anthraquinones (18, 19) (emodin and chrysophanol), and alkaloids (Sanjoinine-D) (36).
CONCLUSION
In this review, studies have revealed the PDE-inhibitory potential of natural plant extracts and their bioactive constituents in treating various diseases; however, further clinical studies comprising synergistic use of different therapies (synthetic & natural) to acquire multi-targeted results might also be a promising option.
Topics: Cyclic AMP; Cyclic GMP; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 34503407
DOI: 10.2174/1568026621666210909164118 -
Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies.Journal of Bone and Mineral Research :... Nov 2021Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial...
Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start-time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.
Topics: Familial Hypophosphatemic Rickets; Humans; Infant; Multidrug Resistance-Associated Proteins; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification
PubMed: 34355424
DOI: 10.1002/jbmr.4418 -
Spectrochimica Acta. Part A, Molecular... Jan 2023A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence "turn-off"...
A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence "turn-off" strategy. Carbon quantum dots@Cu(II)-based metal-organic framework nanotubes (CQDs@Cu-MOF) with length less than 300 nm and width less than 20 nm were synthesized. CQDs in the nanotubes exhibited weak fluorescence owing to static quenching. The coordination reaction between pyrophosphate ion (PPi) and Cu(II) decomposed CQDs@Cu-MOF and led to the release of CQDs, of which the fluorescence recovered. In the presence of PPase, the hydrolysis of PPi generated phosphate ion (Pi). CQDs@Cu-MOF remained their structural stability and the fluorescence turned off. The fluorescence intensity difference of the mixture of CQDs@Cu-MOF and PPi in the absence and presence of PPase (-ΔF) was proportional to the PPase concentration from 0.1 to 5 mU mL and that from 5 to 50 mU mL, and a limit of detection at 0.03 mU mL was obtained. PPase activity in human serum was analyzed using the proposed fluorescence sensor and the recovery values were found to vary from 95.0% to 104 %.
Topics: Carbon; Diphosphates; Fluorescence; Humans; Inorganic Pyrophosphatase; Metal-Organic Frameworks; Nanotubes, Carbon; Pyrophosphatases; Quantum Dots
PubMed: 36027790
DOI: 10.1016/j.saa.2022.121771 -
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 -
Veterinary Research Jan 2021Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The...
Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.
Topics: Animals; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Antibody Technique; Inorganic Pyrophosphatase; Larva; Mice; Mice, Inbred BALB C; Molting; Mutagenesis, Site-Directed; Phylogeny; Real-Time Polymerase Chain Reaction; Sequence Alignment; Trichinella spiralis; Trichinellosis
PubMed: 33413587
DOI: 10.1186/s13567-020-00877-8 -
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