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Biochimica Et Biophysica Acta. Proteins... May 2020Two groups of metabolically related enzymes, the Group III family of Fe-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates...
Two groups of metabolically related enzymes, the Group III family of Fe-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.
Topics: Bacterial Proteins; Catalytic Domain; Cupriavidus necator; Hydroxybutyrate Dehydrogenase; Kinetics; Mutation; NAD; Pyrophosphatases; Nudix Hydrolases
PubMed: 31981617
DOI: 10.1016/j.bbapap.2020.140376 -
Purinergic Signalling Sep 2021
Review
Topics: Biomarkers, Tumor; Genetic Variation; Humans; Mutation; Neoplasms; Oncogene Proteins; Protein Isoforms; Pyrophosphatases
PubMed: 34272651
DOI: 10.1007/s11302-021-09809-3 -
Cells Jan 2022Inosine triphosphate pyrophosphatase (ITPase) is an enzyme encoded by the gene and functions to prevent the incorporation of noncanonical purine nucleotides into DNA... (Review)
Review
Inosine triphosphate pyrophosphatase (ITPase) is an enzyme encoded by the gene and functions to prevent the incorporation of noncanonical purine nucleotides into DNA and RNA. Specifically, the ITPase catalyzed the hydrolysis of (deoxy) nucleoside triphosphates ((d) NTPs) into the corresponding nucleoside monophosphate with the concomitant release of pyrophosphate. Recently, thiopurine drug metabolites such as azathioprine have been included in the lists of ITPase substrates. Interestingly, inosine or xanthosine triphosphate (ITP/XTP) and their deoxy analogs, deoxy inosine or xanthosine triphosphate (dITP/dXTP), are products of important biological reactions such as deamination that take place within the cellular compartments. However, the incorporation of ITP/XTP, dITP/dXTP, or the genetic deficiency or polymorphism of the gene have been implicated in many human diseases, including infantile epileptic encephalopathy, early onset of tuberculosis, and the responsiveness of patients to cancer therapy. This review provides an up-to-date report on the ITPase enzyme, including information regarding its discovery, analysis, and cellular localization, its implication in human diseases including cancer, and its therapeutic potential, amongst others.
Topics: Humans; Inosine; Inosine Triphosphate; Mutation; Neoplasms; Nucleosides; Nucleotides; Pyrophosphatases; Inosine Triphosphatase
PubMed: 35159194
DOI: 10.3390/cells11030384 -
Nucleic Acids Research Sep 2022Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis...
Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.
Topics: Humans; Animals; Mice; Rats; Inosine Triphosphate; RNA; Pyrophosphatases; Inosine; Nucleotides
PubMed: 35979951
DOI: 10.1093/nar/gkac709 -
Cancer Discovery May 2022Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified...
ABSTRACT
Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.
SIGNIFICANCE
CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.
Topics: Breast Neoplasms; Female; Haptoglobins; Humans; Myeloid-Derived Suppressor Cells; Neoplasm Recurrence, Local; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 35191482
DOI: 10.1158/2159-8290.CD-21-0932 -
Cell Cycle (Georgetown, Tex.) Apr 2017
Topics: Animals; Endoplasmic Reticulum; Glycoproteins; Neoplasms; Pyrophosphatases; Tumor Suppressor Protein p53
PubMed: 28166433
DOI: 10.1080/15384101.2017.1291247 -
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 -
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 -
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 -
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