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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 28-32, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922591

RESUMO

OBJECTIVE: To explore the genetic basis of an infant featuring congenital cataract, developmental delay and proteinuria. METHODS: Clinical data and peripheral blood samples of the family were collected. Potential variants were screened by using targeted capture and high-throughput sequencing on a NextSeq 500 platform. Suspected variant was verified by quantitative PCR. Pathogenicity of the candidate variant was predicted based on clinical presentation and laboratory tests. RESULTS: The infant's phenotypes included brain development retardation and proteinuria. Cranial MRI indicated widening of cerebral fissure, bilateral frontal and temporal subarachnoid cavities, and dysplasia of white matter myelination in posterior angular of ventricle. A novel duplication of exons 5 to 16 of the OCRL gene was found in the patient. His mother has carried the same duplication variant. CONCLUSION: The duplication variant of the OCRL gene probably underlies the oculo-cerebro-renal syndrome in the infant. Due to the heterogeneity of its clinical manifestation, pertinent genetic detection is essential for acurrate diagnosis of patients who have the related phenotypes.


Assuntos
Síndrome Oculocerebrorrenal , Monoéster Fosfórico Hidrolases , Éxons/genética , Testes Genéticos , Humanos , Lactente , Síndrome Oculocerebrorrenal/genética , Fenótipo , Monoéster Fosfórico Hidrolases/genética
2.
Plant Mol Biol ; 102(3): 323-337, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900819

RESUMO

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ferro/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Difosfato de Adenosina/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clorofila , Cloroplastos/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Homeostase , Mitocôndrias/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genética , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo
3.
Insect Mol Biol ; 29(1): 48-55, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31294881

RESUMO

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.


Assuntos
Bombyx/enzimologia , Monoéster Fosfórico Hidrolases/química , Serina/biossíntese , Sequência de Aminoácidos , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Seda
4.
J Agric Food Chem ; 67(49): 13518-13525, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31757125

RESUMO

Coordinating unsaturated metal sites (CUS) on the surface of metal-organic frameworks (MOFs) could be used to adsorb His-tagged proteins. The specific adsorption between CUS and His-tagged proteins could reduce preparation steps, shorten preparation time, and could also avoid the binding between the metal ion of metalloenzyme active center and the chelating agent to ensure the enzyme activity. In this study, MIL-88A was synthesized by hydrothermal method and used to purify and immobilize His-tagged organophosphohydrolase (OpdA) in one step for organophosphate bioremediation. Under optimized conditions, OpdA@MIL-88A had a maximal activity of 1554 U/gprotein, which was nearly 5 times higher than free OpdA. Compared with free OpdA, OpdA@MIL-88A exhibited improved organic solvent tolerance, SDS tolerance, thermal stability, and storage stability. OpdA@MIL-88A was used to degrade organophosphorus pesticides on grapes and cucumbers. After reuse 6 times, OpdA@MIL-88A retained more than 66% and 61% of the initial activity, respectively. Therefore, this proposed strategy provided a facile and effective method for degradation of organophosphorus pesticides.


Assuntos
Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Estruturas Metalorgânicas/química , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Monoéster Fosfórico Hidrolases/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Meios de Cultura/química , Meios de Cultura/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Compostos Organofosforados/química , Praguicidas/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
5.
J Ind Microbiol Biotechnol ; 46(12): 1725-1731, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31501960

RESUMO

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.


Assuntos
Arabinose/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiose , Etanol/metabolismo , Fermentação , Heptoses/metabolismo , Via de Pentose Fosfato , Monoéster Fosfórico Hidrolases/genética , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência
6.
Nat Commun ; 10(1): 4424, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562313

RESUMO

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5'-3' RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5'-3' exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Mutagênese , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Estabilidade de RNA
7.
Cell Biochem Biophys ; 77(4): 357-366, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31562588

RESUMO

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.


Assuntos
Corrente Citoplasmática/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Physarum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
8.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546755

RESUMO

Fungal diseases are a serious health burden worldwide with drug resistance compromising efficacy of the limited arsenal of antifungals available. New drugs with novel mechanisms of action are desperately needed to overcome current challenges. The screening of the Aspergillus fumigatus genome identified 35 phosphatases, four of which were previously reported as essential for viability. In addition, we validated another three essential phosphatases. Phosphatases control critical events in fungi from cell wall integrity to cell cycle, thus they are attractive targets for drug development. We used VSpipe v1.0, a virtual screening pipeline, to evaluate the druggability of the seven essential phosphatases and identify starting points for drug discovery. Targeted virtual screening and evaluation of the ligand efficiency plots created by VSpipe, enabled us to define the most favourable chemical space for drug development and suggested different modes of inhibition for each phosphatase. Interestingly, the identified ligand binding sites match with functional sites (active site and protein interaction sites) reported for other yeast and human homologues. Thus, the VSpipe virtual screening approach identified both druggable and functional sites in these essential phosphatases for further experimental validation and antifungal drug development.


Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Genoma Fúngico , Monoéster Fosfórico Hidrolases/genética , Análise de Sequência de DNA , Software , Aspergillus fumigatus/genética , Ciclo Celular/genética
9.
BMC Plant Biol ; 19(1): 381, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477017

RESUMO

BACKGROUND: Trehalose-6-phosphate phosphatases (TPPs), which are encoded by members of the TPP gene family, can improve the drought tolerance of plants. However, the molecular mechanisms underlying the dynamic regulation of TPP genes during drought stress remain unclear. In this study, we explored the function of an Arabidopsis TPP gene by conducting comparative analyses of a loss-of-function mutant and overexpression lines. RESULTS: The loss-of-function mutation of Arabidopsis thaliana TPPF, a member of the TPP gene family, resulted in a drought-sensitive phenotype, while a line overexpressing TPPF showed significantly increased drought tolerance and trehalose accumulation. Compared with wild-type plants, tppf1 mutants accumulated more H2O2 under drought, while AtTPPF-overexpressing plants accumulated less H2O2 under drought. Overexpression of AtTPPF led to increased contents of trehalose, sucrose, and total soluble sugars under drought conditions; these compounds may play a role in scavenging reactive oxygen species. Yeast one-hybrid and luciferase activity assays revealed that DREB1A could bind to the DRE/CRT element within the AtTPPF promoter and activate the expression of AtTPPF. A transcriptome analysis of the TPPF-overexpressing plants revealed that the expression levels of drought-repressed genes involved in electron transport activity and cell wall modification were upregulated, while those of stress-related transcription factors related to water deprivation were downregulated. These results indicate that, as well as its involvement in regulating trehalose and soluble sugars, AtTPPF is involved in regulating the transcription of stress-responsive genes. CONCLUSION: AtTPPF functions in regulating levels of trehalose, reactive oxygen species, and sucrose levels during drought stress, and the expression of AtTPPF is activated by DREB1A in Arabidopsis. These findings shed light on the molecular mechanism by which AtTPPF regulates the response to drought stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Secas , Regulação da Expressão Gênica de Plantas/genética , Mutação com Perda de Função/genética , Monoéster Fosfórico Hidrolases/genética , Estresse Fisiológico/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
11.
Mol Plant Microbe Interact ; 32(11): 1547-1556, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31287368

RESUMO

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.


Assuntos
Azorhizobium caulinodans , Sesbania , Simbiose , Azorhizobium caulinodans/enzimologia , Azorhizobium caulinodans/genética , Ativação Enzimática , Mutação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Sesbania/microbiologia , Propriedades de Superfície , Simbiose/genética
12.
J Appl Microbiol ; 127(4): 1113-1124, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31287935

RESUMO

AIMS: Isolation and identification of genes encoding putative phosphatases from Pseudomonas putida strain P13 DSM 23335. METHODS AND RESULTS: By functional screening of a P. putida P13 genomic library, a number of Pho+ clones were identified. Two genes were identified that encoded proteins exhibiting both phytase and sugar phosphatase activities. The proteins were 249 and 462 amino acids, with molecular masses of 26 and 50 kDa respectively. Sequence alignments revealed no significant similarities to representatives of known phosphatase or phytase gene families. However, the genes were found to have a high similarity to members of the major facilitator superfamily (MFS). Both genes were overexpressed in Escherichia coli and the corresponding partially purified recombinant enzymes were found to have significant phytate-dephosphorylating activity. The protein designated P. putida phytase 1 (Ppp1) displayed the highest activity among potential substrates studied on Na phytate, whereas Ppp2 more likely represents a sugar phosphatase than a phytase. The optimal conditions for phytate dephosphorylation were determined as 60°C and pH 4·5 (Ppp1) or pH 5·0 (Ppp2). CONCLUSIONS: Two novel bacterial phosphatase-encoding genes, named ppp1 and ppp2, were isolated from P. putida P13 DSM 23335 by a functional screening procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphatase-encoding genes are of great importance for industrial applications, particularly in agriculture. The identified phosphatase genes represent a new class of acid phosphatases.


Assuntos
Proteínas de Bactérias , Genes Bacterianos/genética , Monoéster Fosfórico Hidrolases , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
13.
Plant Physiol Biochem ; 142: 211-216, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31302410

RESUMO

Choline is a vital metabolite in plant and synthesized from phosphocholine by phosphocholine phosphatase. The Arabidopsis At1g17710 was identified as the first plant gene encoding the phosphatase for both phosphoethanolamine and phosphocholine (PECP) with much higher catalytic efficiency (>10-fold) for former. In betaine accumulating plants, choline is further required for betaine synthesis. In this report, we found three putative PECP genes in sugar beet, betaine accumulating plants. Two genes encode the proteins of 274 amino acid residues and designated as BvPECP1S and BvPECP2S. Another gene encodes the 331 amino acid protein (BvPECP2L) consisted of BvPECP2S with extra C-terminal amino acid. Enzymatic assays of BvPECP1S revealed that BvPECP1S exhibited the phosphatase activity for both phosphoethanolamine and phosphocholine with higher affinity (>1.8-fold) and catalytic efficiency (>2.64-fold) for phosphocholine. BvPECP2L exhibited low activity. RT-PCR experiments for BvPECP1S showed the increased expression in young leaf and root tip under salt-stress whereas the increased expression in all organs under phosphate deficiency. The expression level of BvPECP2L in salt stressed young leaf and root tip was induced by phosphate deficient. Physiological roles of BvPECP1S and BvPECP2L for the betaine synthesis were discussed.


Assuntos
Beta vulgaris/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Beta vulgaris/enzimologia , Beta vulgaris/genética , Beta vulgaris/fisiologia , Colina/metabolismo , Etanolaminas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Monoéster Fosfórico Hidrolases/genética , Filogenia , Proteínas de Plantas/genética , Proteínas Recombinantes , Estresse Salino , Alinhamento de Sequência
14.
Int J Mol Sci ; 20(11)2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31212584

RESUMO

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT-mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT-mTOR signaling pathway to modulate cell proliferation.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Imunoprecipitação , Insulina/metabolismo , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Monoéster Fosfórico Hidrolases/genética , Fosforilação/genética , Fosforilação/fisiologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
15.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213552

RESUMO

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/enzimologia , Lipídeo A/metabolismo , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Bactérias Gram-Negativas/genética , Lipídeo A/genética , Proteínas de Membrana/genética , Antígenos O/genética , Antígenos O/metabolismo , Peptidoglicano/genética , Peptidoglicano/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosfatos de Poli-Isoprenil/metabolismo , Homologia de Sequência de Aminoácidos
16.
Gene ; 710: 210-217, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31176733

RESUMO

Low temperature is a key stress factor for the growth and development of wheat (Triticum aestivum L.), and glycometabolism plays an important role in plant cold tolerance. Our previous study identified trehalose 6-phosphate synthase 11 gene (TaTPS11), which had a significantly different expression pattern between a high freezing-tolerant wheat cultivar and a low freezing-tolerant wheat cultivar. In this study, TaTPS11 was isolated from a winter-hardy wheat cultivar (D1) and overexpressed in Arabidopsis thaliana to study its effect on cold tolerance in plants. Transgenic plants expressing TaTPS11 had lower sucrose content, higher starch content, and higher activity of key enzyme (sucrose phosphate synthase, sucrose synthase, and invertase) involved in sucrose metabolism. In addition, the expression level of sucrose non-fermenting 1-related kinase 1 (SnRK1), which catalyzes the sucrose in plants, increased in the TaTPS11-overexpressed plants. These results indicated that heterologous expression of TaTPS11 influenced carbohydrate metabolism in Arabidopsis plants. The resultant plants had a significantly higher survival rate after -5 °C treatment for 2 h and exhibited enhanced cold tolerance without unfavorable phenotypes compared to wild-type. Our findings indicated that manipulation of TaTPS11 improved cold tolerance in plants and TaTPS11 had potential values in wheat cold-tolerance breeding.


Assuntos
Arabidopsis/genética , Resposta ao Choque Frio , Monoéster Fosfórico Hidrolases/genética , Triticum/enzimologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Regulação da Expressão Gênica de Plantas , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase em Tempo Real , Sacarose/metabolismo , Triticum/genética
17.
Appl Microbiol Biotechnol ; 103(15): 6129-6139, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31172205

RESUMO

Phosphatases, which catalyze the dephosphorylation of compounds containing phosphate groups, are important members of the haloacid dehalogenase (HAD)-like superfamily. Herein, a thermostable phosphatase encoded by an open reading frame of Trd_1070 from Thermomicrobium roseum was enzymologically characterized. This phosphatase showed promiscuous activity against more than ten sugar phosphates, with high specific activity toward ribose 5-phosphate, followed by ribulose 5-phosphate and fructose 6-phosphate. The half-life of Trd_1070 at 70 °C and pH 7.0 was about 14.2 h. Given that the catalytic efficiency of Trd_1070 on fructose 6-phosphate was 49-fold higher than that on glucose 6-phosphate, an in vitro synthetic biosystem containing alpha-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, and Trd_1070 was constructed for the production of fructose from maltodextrin by whole-cell catalysis, resulting in 21.6 g/L fructose with a ratio of fructose to glucose of approximately 2:1 from 50 g/L maltodextrin. This in vitro biosystem provides an alternative method to produce fructose with higher fructose content compared with the traditional production method using glucose isomerization. Further discovery and enzymologic characterization of phosphatases may promote further production of alternative monosaccharides through in vitro synthetic biosystems.


Assuntos
Chloroflexi/enzimologia , Frutose/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Polissacarídeos/metabolismo , Biotransformação , Estabilidade Enzimática/efeitos da radiação , Temperatura Alta , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Especificidade por Substrato
18.
Crit Rev Oncol Hematol ; 140: 8-16, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31158800

RESUMO

Biliary tract carcinoma (BTC) comprises gallbladder and intra-/extrahepatic cholangiocarcinoma (GBC, ICC, EHC), which are currently classified by anatomical origin. Better understanding of the mutational profile of BTCs might refine classification and improve treatment. We performed a systematic review of studies reporting on mutational profiling of BTC. We included articles reporting on whole-exome/whole-genome-sequencing (WES/WGS) and targeted sequencing (TS) of BTC, published between 2000-2017. Pooled mutation proportions were calculated, stratified by anatomical region and sequencing technique. A total of 25 studies with 1806 patients were included. Overall, TP53 was the most commonly mutated gene in BTC. GBC was associated with mutations in PFKFB3, PLXN2 and PGAP1. Mutations in IDH1, IDH2 and FGFR fusions almost exclusively occurred in ICC patients. Mutations in APC, GNAS and TGFBR2 occurred exclusively in EHC patients. In conclusion, subtypes of BTCs exhibit minor differences in mutational profile, which is likely influenced by the cell of origin.


Assuntos
Neoplasias do Sistema Biliar/genética , Mutação , Proteínas de Neoplasias/genética , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Sistema Biliar/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Isocitrato Desidrogenase/genética , Proteínas de Membrana/genética , Fosfofrutoquinase-2/genética , Monoéster Fosfórico Hidrolases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética
19.
Acta Biochim Biophys Sin (Shanghai) ; 51(7): 688-696, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31168624

RESUMO

In this study, the mechanism of DNA cleavage by cationic peroxidase from proso millet (PmPOD) was investigated. PmPOD cleaved supercoiled circular DNA into both nicked circular and linear forms via a cleavage mechanism that resembles those of native endonucleases. Inhibition and ligation studies demonstrated that reactive oxygen species and the ferriprotoporphyrin IX moiety in PmPOD are not involved in PmPOD-mediated DNA cleavage. Similar to other endonucleases, Mg ions considerably enhance the DNA cleavage activity of PmPOD. Further studies suggested that PmPOD can disrupt phosphodiester bonds in DNA and mononucleotides, indicating that it is a phosphatase. The phosphatase activity of PmPOD is higher than that of horseradish peroxidase (HRP), but the peroxidase activity of PmPOD was lower than that of HRP. PmPOD-mediated hydrolytic cleavage of DNA observed in this study is different from those reported for heme proteins. This study provides valuable insights into the distinct mechanisms underlying DNA cleavage by heme proteins.


Assuntos
DNA Super-Helicoidal/metabolismo , Endonucleases/metabolismo , Panicum/enzimologia , Peroxidase/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Clivagem do DNA , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Magnésio/metabolismo , Panicum/genética , Peroxidase/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/genética
20.
EBioMedicine ; 45: 220-230, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31202813

RESUMO

BACKGROUND: The molecular mechanism underlying clear cell renal cell carcinoma (ccRCC) metastasis remains unclear. We therefore aimed to elucidate the role of IMPA2 in ccRCC metastatic progression. METHODS: Using the Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC) staining, we investigated differences in IMPA2 mRNA and protein expression, as well as their clinical relevance, in ccRCC. To investigate the function of IMPA2 in ccRCC metastasis, we performed in vitro migration and in vivo lung colony-forming assays. We further explored the effect of microRNA (miR)-25 on IMPA2 expression by performing a luciferase reporter assay. FINDINGS: We show that ccRCC expresses relatively lower transcript levels of IMPA2 than normal kidney tissue. IMPA2 downregulation was greater in high-grade ccRCC than in low-grade ccRCC and was correlated with a poor prognosis in ccRCC patients. Importantly, we demonstrate that IMPA2 expression is inversely associated with the metastatic potential of ccRCC cells. We found that IMPA2 knockdown promotes, but overexpression suppresses, the cellular migration and lung colony-forming abilities of ccRCC cells. By using in silico and luciferase reporter assays, we found that IMPA2 expression is primarily influenced by miR-25 in ccRCC cells. Significantly, the inhibition of miR-25 function restored IMPA2 expression, thereby diminishing the metastatic potential of ccRCC cells. INTERPRETATION: We conclude that miR-25-mediated IMPA2 downregulation constitutes a novel signature for cancer metastasis and poor outcomes in ccRCC. We further postulate that the therapeutic targeting of miR-25 can be useful for preventing the metastatic progression of ccRCC associated with IMPA2 downregulation. FUND: This study was supported by the Ministry of Science and Technology, Taiwan (MOST 107-2314-B-038-094, MOST 106-2314-B-038-069-MY3, MOST 105-2320-B-038-021-MY3 and MOST 107-2320-B-038-056).


Assuntos
Carcinoma de Células Renais/genética , MicroRNAs/genética , Monoéster Fosfórico Hidrolases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Transdução de Sinais/genética , Taiwan
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