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Journal of Orthopaedic Surgery and... Jun 2023The deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) causes the phenotype similar to knee osteoarthritis (OA). However, the molecular mechanism...
OBJECTIVE AND BACKGROUND
The deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) causes the phenotype similar to knee osteoarthritis (OA). However, the molecular mechanism is poorly understood.
METHOD
The global deletion of Enpp1 (Enpp1) mice was created to analyze the role of Enpp1 in the progress of knee OA. The apoptosis, proliferation and chondrogenic differentiation ability of chondrocytes from wild-type (WT) and Enpp1 joints were compared. According to the results of high-throughput quantitative molecular measurements, the proteins of chondrocytes from WT and Enpp1 mice were used to explore the mechanism of Enpp1 deficiency-associated knee OA.
RESULT
In Enpp1 knee joints, we found significant chondrocyte apoptosis and proteomic results showed that abnormal expression of AMP-activated protein kinase (AMPK) signaling pathway may contribute to this phenotype. In primary chondrocyte cultures in vitro, Enpp1 deletion dramatically enhancing chondrocyte apoptosis. Meanwhile, we found Enpp1 deletion inhibits the phosphorylation of AMPK (P-AMPK). We also found that decreased level of P-AMPK and chondrocyte apoptosis, which are caused by Enpp1 deficiency, can be reversed by Acadesine (AICAR), the activator of AMPK.
CONCLUSION
Consequently, Enpp1 deficiency plays an essential role in knee OA by regulating AMPK signaling pathway.
Topics: Animals; Mice; AMP-Activated Protein Kinases; Apoptosis; Chondrocytes; Osteoarthritis, Knee; Phosphoric Diester Hydrolases; Proteomics; Pyrophosphatases; Signal Transduction
PubMed: 37370114
DOI: 10.1186/s13018-023-03923-1 -
The FEBS Journal Sep 2016Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as...
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two-repeat-containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three-fold structural symmetry.
Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Drosophila; Drosophila Proteins; Enzyme Stability; Evolution, Molecular; Gene Duplication; Genes, Insect; Models, Molecular; Phylogeny; Point Mutation; Protein Folding; Protein Structure, Quaternary; Pyrophosphatases; Sequence Homology, Amino Acid; Tandem Repeat Sequences
PubMed: 27380921
DOI: 10.1111/febs.13800 -
Journal of Bacteriology May 2017The genome of T4-type bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, and , would suffice for an NAD...
The genome of T4-type bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, and , would suffice for an NAD salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in , the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active , and a clone complements a mutant defective in both the bacterial and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD, and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected cells demonstrated and transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages. T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, and , share an evolutionary history in their respective phage-host group.
Topics: Bacteriophage T4; Escherichia coli; Genomics; Metabolic Networks and Pathways; NAD; Nicotinamide-Nucleotide Adenylyltransferase; Nucleotides; Pyridines; Pyrophosphatases; Vibrio parahaemolyticus
PubMed: 28167526
DOI: 10.1128/JB.00855-16 -
The Plant Cell May 2018Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we...
Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to PPa5) and vacuolar H-pyrophosphatase (H-PPase, AtVHP1/FUGU5) in In planta, five PPa isozymes tagged with GFP were detected in the cytosol and nuclei. Immunochemical analyses revealed a high abundance of PPa1 and the absence of PPa3 in vegetative tissue. In addition, the heterologous expression of each PPa restored growth in a soluble PPase-defective yeast strain. Although the quadruple knockout mutant plant showed no obvious phenotypes, H-PPase and PPa1 double mutants () exhibited significant phenotypes, including dwarfism, high PPi concentrations, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Altered cell arrangements and weakened cell walls in the root tip were particularly evident in and were more severe than in Our results indicate that H-PPase is essential for maintaining adequate PPi levels and that the cytosolic PPa isozymes, particularly PPa1, prevent increases in PPi concentrations to toxic levels. We discuss phenotypes in relation to metabolic reactions and PPi homeostasis.
Topics: Arabidopsis; Cytosol; Diphosphates; Inorganic Pyrophosphatase; Pyrophosphatases; Vacuoles
PubMed: 29691313
DOI: 10.1105/tpc.17.00911 -
Journal of Orthopaedic Surgery and... Oct 2022Apart from the current understanding of enzyme function, the mechanism of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) deficiency-associated osteoporosis...
BACKGROUND
Apart from the current understanding of enzyme function, the mechanism of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) deficiency-associated osteoporosis is unknown. We aimed to explore the changes in the expression of signaling pathways of bone tissues involved in Enpp1 deficiency.
METHODS
The body weights and morphology and histology of the bones of male Enpp1 knockout (KO) and wild-type (WT) mice were assessed. The humeri of WT and Enpp1 KO mice at 12 weeks of age were subjected to high-throughput quantitative molecular measurements, and bioinformatics analysis was performed. Proteins from humeri and calvarial pre-osteoblasts (Pobs) were used to verify the differentially expressed signaling pathways and to explain the mechanism of Enpp1 deficiency-associated osteoporosis.
RESULTS
Enpp1 KO mice had significantly lower body weight and trabecular bone mass in the hindlimbs than WT mice. Proteomics and immunoblotting showed that Enpp1 deletion downregulated the expression of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in bones. Lysophosphatidic acid (LPA) was involved in activating the MKK3/p38 MAPK/PCNA pathway and proliferating Pobs in Enpp1 KO mice, whereas a p38 MAPK inhibitor suppressed the LPA-induced pro-proliferation phenotype (p < 0.05).
CONCLUSION
The inhibition of MKK3/p38 MAPK/PCNA pathway plays an important role in the development of osteoporosis caused by Enpp1 deficiency, and LPA partially rescued the proliferation of pre-osteoblasts via the MKK3/p38 MAPK/PCNA pathway.
Topics: Animals; Male; Mice; MAP Kinase Kinase 3; Mice, Knockout; Osteoporosis; p38 Mitogen-Activated Protein Kinases; Phosphoric Diester Hydrolases; Proliferating Cell Nuclear Antigen; Pyrophosphatases; Signal Transduction
PubMed: 36243801
DOI: 10.1186/s13018-022-03349-1 -
Nature Communications Jul 2021Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of...
Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.
Topics: Acyclovir; Adolescent; Adult; Aged; Animals; Antibiotic Prophylaxis; Antiviral Agents; Biological Variation, Population; Cell Line; Child; Child, Preschool; Crystallography, X-Ray; Cytomegalovirus; Cytomegalovirus Infections; DNA, Viral; Disease Models, Animal; Drug Resistance, Viral; Female; Ganciclovir; Hematopoietic Stem Cell Transplantation; Host Microbial Interactions; Humans; Infant; Infant, Newborn; Male; Middle Aged; Muromegalovirus; Pharmacogenomic Variants; Polymorphism, Single Nucleotide; Pyrophosphatases; Treatment Outcome; Young Adult
PubMed: 34234136
DOI: 10.1038/s41467-021-24509-7 -
Microbiology Spectrum Feb 2023Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper...
Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI's repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. and analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life. Stl, the Staphylococcus aureus pathogenicity island (SaPI) master repressor, targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. This fascinating phage-SaPI arms race is exemplified by the Stl from SaPIbov1 which targets phage dimeric and trimeric dUTPases (Duts), structurally unrelated proteins with identical functions in the phages. By solving the structure of the Stl in complex with a prokaryotic (M. tuberculosis) and a eukaryotic (human) trimeric Dut, we showed that Stl has developed a sophisticated substrate mimicry strategy to target trimeric Duts. Since all these Duts present identical catalytic mechanisms, Stl is able to interact with Duts from different kingdoms. In addition, modeling with monomeric Dut supports the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor.
Topics: Humans; Bacteriophages; Pyrophosphatases; Genomic Islands; Repressor Proteins
PubMed: 36622213
DOI: 10.1128/spectrum.03232-22 -
Cellular and Molecular Gastroenterology... 2021
Review
Topics: Animals; Female; Genotype; Immunosuppressive Agents; Mice; Pregnancy; Purines; Pyrophosphatases
PubMed: 33766784
DOI: 10.1016/j.jcmgh.2021.03.006 -
Plant Disease Nov 2022Next generation sequencing has been used to identify and characterize the full genome sequence of a cassava-infecting torradovirus, revealing the presence of a Maf/HAM1...
Next generation sequencing has been used to identify and characterize the full genome sequence of a cassava-infecting torradovirus, revealing the presence of a Maf/HAM1 domain downstream of the RNA-dependent RNA-polymerase (RdRp) domain in RNA1 in all isolates sequenced. A similar domain is also found in unrelated potyvirids infecting Euphorbiaceae hosts in the Americas and cassava in Africa. Even though cassava torrado-like virus (CsTLV) could not be mechanically transmitted to a series of herbaceous hosts, it can be efficiently transmitted by bud graft-inoculation to different cassava landraces. Our bioassays show that CsTLV has a narrow host range. Crystal-like structures of isometric virus-like particles were observed in cells of plants with single infection by CsTLV, and consistently induced chlorotic leaf spots and affected root yields significantly. Moreover, CsTLV infection induces changes in the accumulation of total sugars in storage roots. Field surveys indicated the presence of CsTLV in the main cassava growing regions of Colombia, and the occurrence of two different cassava-infecting torradovirus species. Profiles of small RNAs of 21 to 24 nucleotides in length, derived from CsTLV RNAs targeted by cassava RNA silencing defense mechanisms, are also reported.
Topics: Manihot; Pyrophosphatases; Plant Diseases; RNA; Colombia
PubMed: 35471077
DOI: 10.1094/PDIS-11-21-2520-RE -
American Journal of Hematology Jul 2019
Review
Topics: Female; Genotype; Humans; Leukemia; Male; Mercaptopurine; Methyltransferases; Pyrophosphatases; Thioguanine
PubMed: 30945335
DOI: 10.1002/ajh.25485