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Journal of Visualized Experiments : JoVE Nov 2019Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into...
Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.
Topics: Anti-Bacterial Agents; Drug Evaluation, Preclinical; Enzyme Inhibitors; Membranes; Molybdenum; Pyrophosphatases; Thermotoga maritima
PubMed: 31814619
DOI: 10.3791/60619 -
Journal of Enzyme Inhibition and... Dec 2022In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of...
In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found possessed high potency (IC = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN- and IP-10 were induced by in a concentration dependent manner. Finally, we discovered that causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.
Topics: Animals; Mice; Phosphoric Diester Hydrolases; Pyrimidines; Pyrophosphatases; Pyrroles; Structure-Activity Relationship
PubMed: 36069240
DOI: 10.1080/14756366.2022.2119566 -
Human Mutation Mar 2022Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate...
Clinico-radiological features, molecular spectrum, and identification of prognostic factors in developmental and epileptic encephalopathy due to inosine triphosphate pyrophosphatase (ITPase) deficiency.
Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate pyrophosphatase, an essential enzyme in purine metabolism. We delineate the genotypic and phenotypic spectrum of DEE 35, analyzing possible predictors for adverse clinical outcomes. We investigated a cohort of 28 new patients and reviewed previously described cases, providing a comprehensive characterization of 40 subjects. Exome sequencing was performed to identify underlying ITPA pathogenic variants. Brain MRI (magnetic resonance imaging) scans were systematically analyzed to delineate the neuroradiological spectrum. Survival curves according to the Kaplan-Meier method and log-rank test were used to investigate outcome predictors in different subgroups of patients. We identified 18 distinct ITPA pathogenic variants, including 14 novel variants, and two deletions. All subjects showed profound developmental delay, microcephaly, and refractory epilepsy followed by neurodevelopmental regression. Brain MRI revision revealed a recurrent pattern of delayed myelination and restricted diffusion of early myelinating structures. Congenital microcephaly and cardiac involvement were statistically significant novel clinical predictors of adverse outcomes. We refined the molecular, clinical, and neuroradiological characterization of ITPase deficiency, and identified new clinical predictors which may have a potentially important impact on diagnosis, counseling, and follow-up of affected individuals.
Topics: Epilepsy, Generalized; Humans; Inosine; Inosine Triphosphate; Microcephaly; Mutation; Prognosis; Pyrophosphatases; Inosine Triphosphatase
PubMed: 34989426
DOI: 10.1002/humu.24326 -
The Protein Journal Feb 2024Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in...
Recent clinical data have identified infant patients with lethal ITPA deficiencies. ITPA is known to modulate ITP concentrations in cells and has a critical function in neural development which is not understood. Polymorphism of the ITPA gene affects outcomes for both ribavirin and thiopurine based therapies and nearly one third of the human population is thought to harbor ITPA polymorphism. In a previous site-directed mutagenesis alanine screen of the ITPA substrate selectivity pocket, we identified the ITPA mutant, E22A, as a gain-of function mutant with enhanced ITP hydrolysis activity. Here we report a rational enzyme engineering experiment to investigate the biochemical properties of position 22 ITPA mutants and find that the E22D ITPA has two- and four-fold improved substrate selectivity for ITP over the canonical purine triphosphates ATP and GTP, respectively, while maintaining biological activity. The novel E22D ITPA should be considered as a platform for further development of ITPA therapies.
Topics: Humans; Pyrophosphatases; Inosine Triphosphatase; Polymorphism, Genetic; Genotype
PubMed: 38066288
DOI: 10.1007/s10930-023-10162-0 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jan 2018Aphids are major agricultural pests that cause significant yield losses of crops each year. (E)-β-farnesene (EβF), as the main component of the aphid alarm pheromones,... (Review)
Review
Aphids are major agricultural pests that cause significant yield losses of crops each year. (E)-β-farnesene (EβF), as the main component of the aphid alarm pheromones, can interrupt aphid feeding and cause other conspecies in the vicinity to become agitated or disperse from their host plant. Furthermore, EβF can function as a kairomone in attracting aphid predators. EβF synthase genes, which encode enzymes that convert farnesyl diphosphate (FPP) to the acyclic sesquiterpene EβF, have been isolated and characterized from peppermint (Mentha × piperita and Mentha asiatica), Yuzu (Citrus junos), Douglas fir (Pseudotsuga menziesii), sweet wormwood (Artemisia annua) and chamomile (Matricaria recutita), respectively. Transgenic plant overexpressing EβF synthase genes has been one of the most efficient strategies for aphid management. In this review, the current statuses of transgenic plants engineered for aphid resistance were summarized. The plant-derived EβF synthase genes with their potential roles in aphid management via genetic-modified (GM) approaches were reviewed. The existing problem in GM plants with EβF synthase gene, such as low EβF emission was usually detected in the transgenic plant, was discussed and the development direction in this area was proposed.
Topics: Animals; Aphids; Metabolic Engineering; Plants, Genetically Modified; Pyrophosphatases; Sesquiterpenes
PubMed: 29380567
DOI: 10.13345/j.cjb.170093 -
International Journal of Developmental... Feb 2018There is growing evidence that over consumption of high-fat foods and insulin resistance may alter hippocampal-dependent cognitive function. To study the individual... (Review)
Review
There is growing evidence that over consumption of high-fat foods and insulin resistance may alter hippocampal-dependent cognitive function. To study the individual contributions of diet and peripheral insulin resistance to learning and memory, we used a transgenic mouse line that overexpresses ecto-nucleotide pyrophosphatase phosphodiesterase-1 in adipocytes, which inhibits the insulin receptor. Here, we demonstrate that a model of peripheral insulin resistance exacerbates high-fat diet induced deficits in performance on the Morris Water Maze task. This finding was then reviewed in the context of the greater literature to explore potential mechanisms including triglyceride storage, adiponectin, lipid composition, insulin signaling, oxidative stress, and hippocampal signaling. Together, these findings further our understanding of the complex relationship among peripheral insulin resistance, diet and memory.
Topics: Animals; Brain; Cognition Disorders; Diet, High-Fat; Insulin Resistance; Mice; Mice, Transgenic; Oxidative Stress; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 28373023
DOI: 10.1016/j.ijdevneu.2017.03.011 -
Archives of Biochemistry and Biophysics Oct 2020A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG...
A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200-250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Desulfitobacterium; Inorganic Pyrophosphatase; Ligands; Mutagenesis; Sequence Deletion
PubMed: 32810477
DOI: 10.1016/j.abb.2020.108537 -
Pharmacogenetics and Genomics May 2017Thiopurines have a narrow therapeutic range because of frequent toxicity (i.e. marrow suppression), which is only partly explained by TPMT genetic polymorphisms,...
Thiopurines have a narrow therapeutic range because of frequent toxicity (i.e. marrow suppression), which is only partly explained by TPMT genetic polymorphisms, especially within Asian populations. Recent studies have identified NUDT15 variation as another important factor affecting thiopurine metabolism. In this study, a total of four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile, and p.Val18_Val19insGlyVal) were genotyped in 920 Korean individuals using direct sequencing of NUDT15 for the first time in a Korean population. The allele frequencies were 86.7% for NUDT15*1, and 4.4, 6.9, 0.4, 1.1, and 0.50% for *2, *3, *4, *5, and *6, respectively. The NUDT15 phenotypes based on diplotypes included normal activity (n=692), intermediate activity (n=209), and low activity (n=19), occurring in 75.2, 22.7, and 2.1% of the population, respectively. This study was the first to report NUDT15 variants other than NUDT15*3 in the Korean population and more individuals who were categorized as having intermediate or low NUDT15 activity in our study than in previously reported studies in the Korean population (24.8 vs. 19.4%, P<0.05). This study is useful for future clinical studies on thiopurine pharmacogenetics and dosage adjustment in the Korean population.
Topics: Adolescent; Adult; Asian People; Child; Female; Genotype; Humans; Male; Middle Aged; Pharmacogenomic Variants; Pyrophosphatases; Republic of Korea; Young Adult
PubMed: 28277331
DOI: 10.1097/FPC.0000000000000274 -
The FEBS Journal Sep 2016ADP-ribosylation is a conserved post-translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription,...
ADP-ribosylation is a conserved post-translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP-ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP-ribosylation can be reversed by the macrodomain-containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP-ribose as well as consecutive ADP-ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP-ribose) to mono(ADP-ribose). Recently, proteins containing the NUDIX domain - namely human NUDT16 and bacterial RppH - have been shown to process in vitro protein ADP-ribosylation through an alternative mechanism, converting it into protein-conjugated ribose-5'-phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP-ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families - Nudix and ENPP/NPP - are able to metabolize protein-conjugated ADP-ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein-conjugated mono(ADP-ribose) and poly(ADP-ribose) into mass spectrometry-friendly pR tags, thus facilitating the identification of ADP-ribosylation sites.
Topics: ADP Ribose Transferases; Adenosine Diphosphate Ribose; Amino Acid Sequence; Animals; Binding Sites; Humans; In Vitro Techniques; Mice; Phosphoric Diester Hydrolases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Domains; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Pyrophosphatases; Recombinant Proteins; Sequence Homology, Amino Acid; Tandem Mass Spectrometry
PubMed: 27406238
DOI: 10.1111/febs.13811 -
Protein Science : a Publication of the... Oct 2020The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major...
The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown. To further our understanding of NTPDase4 function, we determined its crystal structure. The enzyme adopts a wide open, inactive conformation. Differences in the nucleotide-binding site relative to its homologs could account for its substrate selectivity. The putative membrane-interacting loop of cell-surface NTPDases is drastically altered in NTPDase4, potentially affecting its interdomain dynamics at the Golgi membrane.
Topics: Animals; Crystallography, X-Ray; Humans; Protein Domains; Protein Structure, Secondary; Pyrophosphatases; Sf9 Cells; Spodoptera
PubMed: 32767432
DOI: 10.1002/pro.3926