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Proceedings of the National Academy of... Apr 2020Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report...
Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP and 1,5-IP Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.
Topics: Diphosphates; Inositol Phosphates; Kinetics; Phosphorylation; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Saccharomyces cerevisiae; Signal Transduction
PubMed: 32303658
DOI: 10.1073/pnas.1908875117 -
Biomolecules Jan 2021Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) or Systemic Exertion Intolerance Disease (SEID) is a chronic multisystem illness of unconfirmed etiology.... (Review)
Review
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) or Systemic Exertion Intolerance Disease (SEID) is a chronic multisystem illness of unconfirmed etiology. There are currently no biomarkers and/or signatures available to assist in the diagnosis of the syndrome and while numerous mechanisms have been hypothesized to explain the pathology of ME/CFS, the triggers and/or drivers remain unknown. Initial studies suggested a potential role of the human herpesviruses especially Epstein-Barr virus (EBV) in the disease process but inconsistent and conflicting data led to the erroneous suggestion that these viruses had no role in the syndrome. New studies using more advanced approaches have now demonstrated that specific proteins encoded by EBV could contribute to the immune and neurological abnormalities exhibited by a subgroup of patients with ME/CFS. Elucidating the role of these herpesvirus proteins in ME/CFS may lead to the identification of specific biomarkers and the development of novel therapeutics.
Topics: Animals; Biomarkers; Disease Progression; Encephalomyelitis; Epstein-Barr Virus Infections; Fatigue Syndrome, Chronic; Herpesvirus 4, Human; Humans; Immune System; Inflammation; Pyrophosphatases; Virus Replication
PubMed: 33572802
DOI: 10.3390/biom11020185 -
RNA Biology Oct 2021Various kinds of cap structures, such as mG, triphosphate groups, NAD and dpCoA, protect the 5' terminus of RNA. The cap structures bond covalently to RNA and affect its...
Various kinds of cap structures, such as mG, triphosphate groups, NAD and dpCoA, protect the 5' terminus of RNA. The cap structures bond covalently to RNA and affect its stability, translation, and transport. The removal of the caps is mainly executed by Nudix hydrolase family proteins, including Dcp2, RppH and NudC. Numerous efforts have been made to elucidate the mechanism underlying the removal of mG, triphosphate group, and NAD caps. In contrast, few studies related to the cleavage of the RNA dpCoA cap have been conducted. Here, we report the hydrolytic activity of NudC towards dpCoA and dpCoA-capped RNA . We also determined the crystal structure of dimeric NudC in complex with dpCoA at 2.0 Å resolution. Structural analysis revealed that dpCoA is recognized and hydrolysed in a manner similar to NAD. In addition, NudC may also remove other dinucleotide derivative caps of RNA, which comprise the AMP moieties. NudC homologs in and exhibited similar dpCoA decapping (deCoAping) activity. These results together indicate a conserved mechanism underpinning the hydrolysis of dpCoA-capped RNA in both prokaryotes and eukaryotes.
Topics: Coenzyme A; Escherichia coli; Escherichia coli Proteins; Protein Conformation; Pyrophosphatases; RNA Caps; RNA, Bacterial; Nudix Hydrolases
PubMed: 34074215
DOI: 10.1080/15476286.2021.1936837 -
Journal of Medicinal Chemistry Nov 2023A lack of the T cell-inflamed tumor microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs). Activation of stimulator of interferon genes...
A lack of the T cell-inflamed tumor microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs). Activation of stimulator of interferon genes (STING)-mediated innate immunity has emerged as a novel therapeutic approach in cancer therapy. 2',3'-Cyclic GMP-AMP (cGAMP) is a natural STING agonist; however, cGAMP is subjected to endogenous degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). To improve the ICI response rate, we developed , a novel ENPP1 inhibitor with phthalazin-1(2)-one as the core scaffold. inhibited the cGAMP hydrolysis by ENPP1 in vitro (IC = 68 nM) and enhanced the STING-mediated type I interferon response in both immune and tumor cells. demonstrated excellent metabolic stability and bioavailability ( = 65%). Orally administered promoted tumor growth inhibition in a CT26 syngeneic model and increased the anti-PD-L1 response. Furthermore, -induced immunological memory prevented the tumor relapse against tumor rechallenge, suggesting the promising therapeutic potential of .
Topics: Humans; Phosphoric Diester Hydrolases; Neoplasms; Pyrophosphatases; Immunotherapy; Tumor Microenvironment
PubMed: 37963811
DOI: 10.1021/acs.jmedchem.3c01061 -
Pediatric Radiology Nov 2022Generalized arterial calcification of infancy (GACI), also known as idiopathic infantile arterial calcification, is a very uncommon genetic disorder characterized by... (Review)
Review
BACKGROUND
Generalized arterial calcification of infancy (GACI), also known as idiopathic infantile arterial calcification, is a very uncommon genetic disorder characterized by calcifications and stenoses of large- and medium-size arteries that can lead to end-organ damage.
OBJECTIVE
To describe changes in imaging findings in 10 children with GACI at a single institution from 2010 to 2021.
MATERIALS AND METHODS
In this retrospective study we reviewed initial and follow-up body imaging in children with genetic confirmation of GACI at our hospital. All initial images were analyzed for the presence and distribution of arterial calcifications, stenoses and wall thickening/irregularity within the chest, abdomen and pelvis. We compared available follow-up studies to the initial imaging findings. We extracted clinical information including prenatal and postnatal treatment from the children's medical records.
RESULTS
We evaluated 10 children (five boys) with a diagnosis of GACI. Median age at first body imaging was 8 days (range: 1 day to 5 years). Six children were identified prenatally and four postnatally. Postnatal presentation included cardiac failure, seizures and hypertension. Images in newborns (n = 8) most commonly showed diffuse arterial calcifications (6/8; 75%), while stenoses were less common (2/8; 25%) during this period. Two children were diagnosed after the neonatal period - one in infancy and one during childhood. In total, half the children (5/10; 50%) had arterial stenoses - three cases visualized at first imaging and two identified on follow-up images during infancy. Stenoses had completely resolved in one child (1/5; 20%) at last follow-up. Eight children received prenatal or postnatal treatment or both. All children who received both prenatal and postnatal treatment (n = 4) had completely resolved calcifications at last follow-up.
CONCLUSION
Children with GACI might have characteristic vascular calcifications at birth that raise the suspicion of this disease. Arterial calcifications decrease or disappear spontaneously or after treatment, but arterial stenoses usually persist. Calcifications and arterial stenoses can be easily identified and followed with non-contrast CT and CT angiography.
Topics: Male; Child; Humans; Infant, Newborn; Pyrophosphatases; Phosphoric Diester Hydrolases; Retrospective Studies; Constriction, Pathologic; Vascular Calcification
PubMed: 35438330
DOI: 10.1007/s00247-022-05364-0 -
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