-
Wiley Interdisciplinary Reviews. RNA 2023Inosine triphosphate pyrophosphatase (ITPase), encoded by the ITPA gene in humans, is an important enzyme that preserves the integrity of cellular nucleotide pools by... (Review)
Review
Inosine triphosphate pyrophosphatase (ITPase), encoded by the ITPA gene in humans, is an important enzyme that preserves the integrity of cellular nucleotide pools by hydrolyzing the noncanonical purine nucleotides (deoxy)inosine and (deoxy)xanthosine triphosphate into monophosphates and pyrophosphate. Variants in the ITPA gene can cause partial or complete ITPase deficiency. Partial ITPase deficiency is benign but clinically relevant as it is linked to altered drug responses. Complete ITPase deficiency causes a severe multisystem disorder characterized by seizures and encephalopathy that is frequently associated with fatal infantile dilated cardiomyopathy. In the absence of ITPase activity, its substrate noncanonical nucleotides have the potential to accumulate and become aberrantly incorporated into DNA and RNA. Hence, the pathophysiology of ITPase deficiency could arise from metabolic imbalance, altered DNA or RNA regulation, or from a combination of these factors. Here, we review the known functions of ITPase and highlight recent work aimed at determining the molecular basis for ITPA-associated pathogenesis which provides evidence for RNA dysfunction. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.
Topics: Humans; Nucleotides; RNA; Inosine; Inosine Triphosphate; Pyrophosphatases; DNA
PubMed: 37092460
DOI: 10.1002/wrna.1790 -
Biochemistry and Cell Biology =... Oct 2022Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription...
Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.
Topics: Diphosphates; Inorganic Pyrophosphatase; Magnesium; Pyrophosphatases; RNA
PubMed: 35926232
DOI: 10.1139/bcb-2022-0118 -
Journal of Zhejiang University.... Jan 2021Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a... (Review)
Review
Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.
Topics: BRCA1 Protein; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA-Binding Proteins; Endonucleases; Humans; Mad2 Proteins; Models, Biological; Poly ADP Ribosylation; Poly(ADP-ribose) Polymerase Inhibitors; Protein Interaction Domains and Motifs; Pyrophosphatases; RNA-Binding Proteins; Signal Transduction; Telomere-Binding Proteins; Tumor Suppressor p53-Binding Protein 1
PubMed: 33448186
DOI: 10.1631/jzus.B2000306 -
Biochemical and Biophysical Research... Dec 2020Inorganic pyrophosphatase (PPase) plays an essential role in energy conservation and provides energy for many biosynthetic pathways. Here, we present two...
Inorganic pyrophosphatase (PPase) plays an essential role in energy conservation and provides energy for many biosynthetic pathways. Here, we present two three-dimensional structures of PPase from Homo sapiens (Hu-PPase) at 2.38 Å and 3.40 Å in different crystallization conditions. One of the Hu-PPase structures complex of two magnesium metal ions was determined to be a monomer (Hu-PPase-mono) here, while the other one to be a dimer-dimer (Hu-PPase-dd). In each asymmetric unit of Hu-PPase-mono, there are four α-helices and ten β-strands and folds as a barrel structure, and the active site contains two magnesium ions. Like PPases from many species, we found that Hu-PPase was able to undergo self-assembly. To our surprise, disruption of the self-assembly of Hu-PPase did not influence its enzymatic activity or the ability to promote cell growth. Our work uncovered that different structure forms of Hu-PPase and found that the pyrophosphatase activity of Hu-PPase is independent of its self-assembly.
Topics: Amino Acid Sequence; Binding Sites; Catalysis; Cell Proliferation; Crystallography, X-Ray; Dimerization; HEK293 Cells; HeLa Cells; Humans; Inorganic Pyrophosphatase; Magnesium; Models, Molecular; Protein Conformation; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Recombinant Proteins
PubMed: 33036755
DOI: 10.1016/j.bbrc.2020.09.139 -
Accounts of Chemical Research Apr 2021Bacterial infections caused by multi-drug-resistant Gram-negative pathogens pose a serious threat to public health. Gram-negative bacteria are characterized by the... (Review)
Review
Bacterial infections caused by multi-drug-resistant Gram-negative pathogens pose a serious threat to public health. Gram-negative bacteria are characterized by the enrichment of lipid A-anchored lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in the outer leaflet of their outer membrane. Constitutive biosynthesis of lipid A via the Raetz pathway is essential for bacterial viability and fitness in the human host. The inhibition of early-stage lipid A enzymes such as LpxC not only suppresses the growth of , , Enterobacter spp., and other clinically important Gram-negative pathogens but also sensitizes these bacteria to other antibiotics. The inhibition of late-stage lipid A enzymes such as LpxH is uniquely advantageous because it has an extra mechanism of bacterial killing through the accumulation of toxic lipid A intermediates, rendering LpxH inhibition additionally lethal to . Because essential enzymes of the Raetz pathway have never been exploited by commercial antibiotics, they are excellent targets for the development of novel antibiotics against multi-drug-resistant Gram-negative infections.This Account describes the ongoing research on characterizing the structure and inhibition of LpxC and LpxH, the second and fourth enzymes of the Raetz pathway of lipid A biosynthesis, in the laboratories of Dr. Pei Zhou and Dr. Jiyong Hong at Duke University. Our studies have elucidated the molecular basis of LpxC inhibition by the first broad-spectrum inhibitor, CHIR-090, as well as the mechanism underlying its spectrum of activity. Such an analysis has provided a molecular explanation for the broad-spectrum antibiotic activity of diacetylene-based LpxC inhibitors. Through the structural and biochemical investigation of LpxC inhibition by diacetylene LpxC inhibitors and the first nanomolar LpxC inhibitor, L-161,240, we have elucidated the intrinsic conformational and dynamics difference in individual LpxC enzymes near the active site. A similar approach has been taken to investigate LpxH inhibition, leading to the establishment of the pharmacophore model of LpxH inhibitors and subsequent structural elucidation of LpxH in complex with its first reported small-molecule inhibitor based on a sulfonyl piperazine scaffold.Intriguingly, although our crystallographic analysis of LpxC- and LpxH-inhibitor complexes detected only a single inhibitor conformation in the crystal lattice, solution NMR studies revealed the existence of multiple ligand conformations that together delineate a cryptic ligand envelope expanding the ligand-binding footprint beyond that observed in the crystal structure. By harnessing the ligand dynamics information and structural insights, we demonstrate the feasibility to design potent LpxC and LpxH inhibitors by merging multiple ligand conformations. Such an approach has enabled us to rationally design compounds with significantly enhanced potency in enzymatic assays and outstanding antibiotic activities and in animal models of bacterial infection. We anticipate that continued efforts with structure and ligand dynamics-based lead optimization will ultimately lead to the discovery of LpxC- and LpxH-targeting clinical antibiotics against a broad range of Gram-negative pathogens.
Topics: Amidohydrolases; Anti-Bacterial Agents; Drug Design; Enzyme Inhibitors; Gram-Negative Bacteria; Humans; Ligands; Molecular Dynamics Simulation; Molecular Structure; Pyrophosphatases
PubMed: 33720682
DOI: 10.1021/acs.accounts.0c00880 -
BMC Gastroenterology Jul 2023Thiopurines continue to play an important role in the treatment of inflammatory bowel disease (IBD). It is well known that thiopurines can cause several adverse...
BACKGROUND
Thiopurines continue to play an important role in the treatment of inflammatory bowel disease (IBD). It is well known that thiopurines can cause several adverse reactions. Especially, hematopoietic toxicity may lead to severe agranulocytosis. In a previous prospective study, we investigated the relationship between inosine triphosphate pyrophosphatase (ITPA) c.94c > a polymorphism, 6-thioguanine nucleotide (6-TGN) concentration and toxicity.
METHODS
To clarify the cause of thiopurine toxicity, we analysed nucleoside disphosphate-linked moiety X-type motif 15 (NUDT15) gene polymorphisms, i.e., R139C, V18I, and V19_V19insGV, and measured 6-mercaptopurines and 6-methylmercaptopurines (6-MMP) using the archived blood samples collected from 49 IBD patients for our previous study.
RESULTS
The ITPA c.94c > a polymorphism was detected in 19 patients (38.7%, all heterozygous). The R139C polymorphism was found in 10 patients (20.4%, 1 homozygous, 9 heterozygous), V18_V19insGV in 7 patients (14.3%, all heterozygous), and V18I in 2 patients (4.08%, all heterozygous). Although R139C was more strongly associated with leukopenia than c.94c > a, there were no significant correlations with 6-TGN and 6-MMP levels, as for c.94c > a. The leukopenia incidence rates for each gene polymorphism were 0% in those with all wild-type genes, 21.4% for c.94c > a only, 42.9% for NUDT15 polymorphism (s) only, and 80.0% for both polymorphisms.
CONCLUSIONS
All cases of leukopenia were associated with ITPA c.94c > a and/or polymorphism of NUDT15 and the risk of developing leukopenia was synergistically increased by ITPA and NUDT15 gene polymorphism. However, there was no association between the level of azathioprine metabolites and these polymorphisms.
Topics: Humans; Azathioprine; East Asian People; Inflammatory Bowel Diseases; Leukopenia; Mercaptopurine; Pyrophosphatases
PubMed: 37454061
DOI: 10.1186/s12876-023-02881-6 -
Experimental Dermatology Apr 2022Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes...
Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PP ) concentration in the circulation. Although PP is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na P O is absorbed in humans. Here, we report that gelatin-encapsulated Na H P O has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K H P O form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6 mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PP in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.
Topics: Animals; Dietary Supplements; Diphosphates; Humans; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification
PubMed: 34758173
DOI: 10.1111/exd.14498 -
Expert Opinion on Therapeutic Patents Jul 2022Ectobucleotidases are a broad class of extracellular nucleotide and nucleoside hydrolyzing enzymes. Since they play a crucial role in mediating purinergic cell... (Review)
Review
INTRODUCTION
Ectobucleotidases are a broad class of extracellular nucleotide and nucleoside hydrolyzing enzymes. Since they play a crucial role in mediating purinergic cell signalling, they are promising therapeutic targets for treatment of a range of disorders, including fibrosis, tumor metastasis, inflammation, multiple sclerosis, and autoimmune diseases. Hence selective inhibtors of ectonulceotidases are of great interest for therapeutic intervention.
AREA COVERED
Many compounds have demonstrated promising inhibitory potential against ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs). The chemistry and clinical applications of NPP inhibitors patented between 2015 and 2020 are discussed in this review.
EXPERT OPINION
In recent years, there has been a lot of effort towards finding effective and selective inhibitors of NPPs. Even though a number of inhibitors are known, only a few in vivo investigations have been published. In addition to IOA-289, which has passed Phase Ia clinical trials, potent NPP2/ATX inhibitor compounds such as BLD-0409, IPF and BBT-877 have been placed in phase I clinical studies. Some of the most promising NPP2/ATX inhibitors in recent years are closely related analogs of previously known inhibitors, such as PF-8380. Knowledge of the structure activity relationship of such promising inhibitors can potentially translate into the discovery of more potent and effective inhibitors of NPP.
Topics: Humans; Patents as Topic; Phosphoric Diester Hydrolases; Pyrophosphatases; Structure-Activity Relationship
PubMed: 35333684
DOI: 10.1080/13543776.2022.2058874 -
Journal of Cellular Physiology Apr 2021Arterial medial calcification (AMC), the deposition of hydroxyapatite in the medial layer of the arteries, is a known risk factor for cardiovascular events. Oxidative...
Arterial medial calcification (AMC), the deposition of hydroxyapatite in the medial layer of the arteries, is a known risk factor for cardiovascular events. Oxidative stress is a known inducer of AMC and endogenous antioxidants, such as glutathione (GSH), may prevent calcification. GSH synthesis, however, can be limited by cysteine levels. Therefore, we assessed the effects of the cysteine prodrug 2-oxothiazolidine-4-carboxylic acid (OTC), on vascular smooth muscle cell (VSMC) calcification to ascertain its therapeutic potential. Human aortic VSMCs were cultured in basal or mineralising medium (1 mM calcium chloride/sodium phosphate) and treated with OTC (1-5 mM) for 7 days. Cell-based assays and western blot analysis were performed to assess cell differentiation and function. OTC inhibited calcification ≤90%, which was associated with increased ectonucleotide pyrophosphatase/phosphodiesterase activity, and reduced apoptosis. In calcifying cells, OTC downregulated protein expression of osteoblast markers (Runt-related transcription factor 2 and osteopontin), while maintaining expression of VSMC markers (smooth muscle protein 22α and α-smooth muscle actin). GSH levels were significantly reduced by 90% in VSMCs cultured in calcifying conditions, which was associated with declines in expression of gamma-glutamylcysteine synthetase and GSH synthetase. Treatment of calcifying cells with OTC blocked the reduction in expression of both enzymes and prevented the decline in GSH. This study shows OTC to be a potent and effective inhibitor of in vitro VSMC calcification. It appears to maintain GSH synthesis which may, in turn, prevent apoptosis and VSMCs gaining osteoblast-like characteristics. These findings may be of clinical relevance and raise the possibility that treatment with OTC could benefit patients susceptible to AMC.
Topics: Alkaline Phosphatase; Apoptosis; Cell Differentiation; Cells, Cultured; Glutamate-Cysteine Ligase; Glutathione; Glutathione Synthase; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteoblasts; Phosphoric Diester Hydrolases; Prodrugs; Pyrophosphatases; Pyrrolidonecarboxylic Acid; Thiazolidines; Vascular Calcification
PubMed: 32918744
DOI: 10.1002/jcp.30036 -
Journal of Orthopaedic Surgery and... Jul 2021Intervertebral disk degeneration (IDD) is caused by nucleus pulposus (NP) degeneration and extracellular matrix (ECM) remodeling and cartilage intermediate layer protein...
BACKGROUND
Intervertebral disk degeneration (IDD) is caused by nucleus pulposus (NP) degeneration and extracellular matrix (ECM) remodeling and cartilage intermediate layer protein (CILP) expression has been confirmed to be increased in IDD. This study is mainly conducted to clarify the mechanism of CILP in the NP cell degeneration and ECM remodeling in IDD.
METHODS
CILP expression in the degenerated NP tissues and cells is quantified by quantitative real-time PCR and western blot. CILP function is assessed by cell cycle assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry, β-galactosidase staining, and the detection of ECM-related molecules aggrecan, collagen type I, collagen type II, MMP-3, and MMP-9 expression is accomplished by qRT-PCR. The potential mechanism is authenticated by dual-luciferase reporter gene assay.
RESULTS
CILP was increased in the degenerated NP tissues and cells, and the knockdown of CILP promoted the NP cell cycle, increased cell activity, and repressed cell apoptosis and repressed cell senescence and ECM production. Moreover, miR-330-5p targeted the CILP 3'-untranslated region, and miR-330-5p negatively regulated CILP expression. Moreover, the overexpression of miR-330-5p repressed NP cell degeneration and ECM remodeling to relieve IDD by downregulating CILP.
CONCLUSION
MiR-330-5p represses NP cell degeneration and ECM remodeling to ameliorate IDD by downregulating CILP.
Topics: Down-Regulation; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Intervertebral Disc Degeneration; MicroRNAs; Nucleus Pulposus; Pyrophosphatases; Up-Regulation
PubMed: 34233701
DOI: 10.1186/s13018-021-02582-4