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1.
Biochem Biophys Res Commun ; 524(4): 929-935, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32059846

RESUMO

Bone is consisted of osteoblast-linage cells, bone-forming cells in various differentiation stages. However, it is not fully understood how communicate and interact these cells immigrated from bone marrow. In this study, we showed that prostaglandin E2 (PGE2) had a role in autonomous modification of matrix mineralization in osteoblastic cell line, MC3T3-E1, and interactions across the cells in different differentiation stages. Analysis using LC-MS/MS and inhibitors showed the autonomous secretion of PGE2 among the prostanoids in differentiation stages and that depend on COX-2, a key enzyme for production of PGE2. Treatment with inhibitors of PGE2 receptors and COX-2 indicated that secreted PGE2 regulates matrix mineralization in an autocrine/paracrine manner. In addition, we showed that the expression profile of PGE2 receptors (EP1-EP4) and PGE2 effects on matrix mineralization derived from it changed during cell differentiation. Treatment with inhibitors of PGE2 signaling in the early differentiation stage of MC3T3-E1 cells induced significant changes in matrix mineralization several days after. Stimulation with the extracts from culture medium of the matured cells including PGE2 and co-culture with the matured cells secreting PGE2 significantly promoted matrix mineralization of the early stage cells, in contrast, treatment with inhibitor of COX-2 and PGE2 receptors failed to do so. These results support that PGE2 plays important roles in the interaction system of osteoblast-linage cells in bone tissue to regulate matrix mineralization reflecting condition of bone-forming cells, that is, population and maturation.


Assuntos
Matriz Óssea/metabolismo , Calcificação Fisiológica , Dinoprostona/metabolismo , Osteoblastos/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Camundongos , Osteoblastos/citologia , Osteogênese , Receptores de Prostaglandina E/metabolismo
2.
Am J Physiol Cell Physiol ; 316(2): C162-C174, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30462540

RESUMO

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTß), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.


Assuntos
Adipogenia/fisiologia , Glicerilfosforilcolina/metabolismo , Líquido Intracelular/metabolismo , Mitose/fisiologia , Fosfolipases/antagonistas & inibidores , Fosfolipases/metabolismo , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Líquido Intracelular/efeitos dos fármacos , Camundongos , Mitose/efeitos dos fármacos , Células NIH 3T3 , RNA Interferente Pequeno/farmacologia
3.
Biochem Biophys Res Commun ; 514(2): 393-400, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31047639

RESUMO

Actin cytoskeleton is reported to be related in various functions of osteoblast, bone-forming cell. However the function of actin cytoskeleton in osteoblasts is not fully understood, since bone formation is derived from extracellular interactions of functional proteins produced from osteoblasts, including osteocalcin (Ocn), and it is a result of closely and complex organized sequence of biochemical events. In this study, we showed that actin cytoskeleton of MC3T3-E1 cells functioned in recognition of cell condition and regulation of extracellular matrix mineralization, bone formation. Maturation of MC3T3-E1 cells by 14 days of culture reduced F-actin filaments, while induced expression of Ocn mRNA known as late stage differentiation marker and matrix mineralization, terminal stage of cell differentiation. The disruption of actin cytoskeleton with Cyto D in immature MC3T3-E1 cells significantly increased expression of Ocn mRNA in 24 h. Both PTX-induced inhibition of signal transduction through GPCRs and celecoxib-induced suppression of lipid mediators in immature MC3T3-E1 cells reduced actin filaments and suppressed matrix mineralization. Furthermore, addition of lipid mediators extracted from culture mediums of differentiated MC3T3-E1 cells by Bligh-Dyer method induced actin cytoskeleton reorganization and matrix mineralization change in MC3T3-E1 cells. Taken together, our data suggest that actin cytoskeleton of MC3T3-E1 cells regulates activation of developmental pathway reflecting cell differentiation stages through lipid mediators. The function we identified is important for bone formation tightly regulated by mechanical stress, since actin cytoskeleton is also known as a mechanosensor of osteoblasts.


Assuntos
Citoesqueleto de Actina/metabolismo , Diferenciação Celular , Lipídeos , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Celecoxib/farmacologia , Linhagem Celular , Forma Celular , Matriz Extracelular , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteocalcina/genética , Osteogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
4.
J Biol Chem ; 290(7): 4260-71, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25528375

RESUMO

The known mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) hydrolyze glycerophosphodiesters. In this study, two novel members of the mammalian GP-PDE family, GDE4 and GDE7, were isolated, and the molecular basis of mammalian GP-PDEs was further explored. The GDE4 and GDE7 sequences are highly homologous and evolutionarily close. GDE4 is expressed in intestinal epithelial cells, spermatids, and macrophages, whereas GDE7 is particularly expressed in gastro-esophageal epithelial cells. Unlike other mammalian GP-PDEs, GDE4 and GDE7 cannot hydrolyze either glycerophosphoinositol or glycerophosphocholine. Unexpectedly, both GDE4 and GDE7 show a lysophospholipase D activity toward lysophosphatidylcholine (lyso-PC). We purified the recombinant GDE4 and GDE7 proteins and show that these enzymes can hydrolyze lyso-PC to produce lysophosphatidic acid (LPA). Further characterization of purified recombinant GDE4 showed that it can also convert lyso-platelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PAF) to alkyl-LPA. These data contribute to our current understanding of mammalian GP-PDEs and of their physiological roles via the control of lyso-PC and lyso-PAF metabolism in gastrointestinal epithelial cells and macrophages.


Assuntos
Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fator de Ativação de Plaquetas/análogos & derivados , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Obesos , Microscopia de Fluorescência , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/genética , Filogenia , Fator de Ativação de Plaquetas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
5.
J Chromatogr A ; 1720: 464769, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38442499

RESUMO

In this study, we introduce a novel approach for the analysis of salivary ions using capillary electrophoresis (CE) with a triple-layer coated capillary. The capillary is sequentially coated with cationic silylating reagents, poly(vinylsulfonate), and polybrene to form a custom designed surface that effectively inhibits adsorption of protein matrix on the capillary inner wall and allows for reproducible ion analysis. For the CE with capacitively coupled contactless conductivity detection, we used suitable background electrolytes (BGEs) for salivary ion analysis. Anions were separated using a mixture of 2-(N-morpholino)ethanesulfonic acid and l-arginine, and cations were separated using that with 18-crown-6. This setup enabled rapid separation, within 4 min, together with sensitive detection. We quantified nine common anions and five cations typically found in saliva samples using this CE method, both before and after a cold pressure test (CPT, a standard stress test). The CE system demonstrated consistent ion separation across 30 consecutive measurements without requiring capillary replacement. Notably, the salivary ion balance remained predominantly anion-rich, regardless of the CPT. Cold water exposure induced greater variation in the total anion concentration than in the total cation concentration. Further analysis using multiple regression analysis revealed strong relationships between nitrate and nitrite, formate and phosphate, and potassium and nitrate, before and after the CPT. Notably, potassium and nitrate ions exhibited variations in response to stress. These results provided a method for assessing salivary ion composition and insights into the potential of salivary ions as biomarkers for stress.


Assuntos
Eletroforese Capilar , Nitratos , Cátions/análise , Ânions/análise , Eletroforese Capilar/métodos , Água , Potássio
6.
Commun Biol ; 7(1): 604, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769369

RESUMO

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.


Assuntos
Camundongos Knockout , Contração Muscular , Músculo Esquelético , Fosfatidilcolinas , Animais , Músculo Esquelético/metabolismo , Camundongos , Fosfatidilcolinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Diester Fosfórico Hidrolases
7.
Microbiol Spectr ; 11(3): e0506922, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37199605

RESUMO

Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum ß-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.


Assuntos
Infecções por Escherichia coli , Fosfomicina , Infecções Urinárias , Humanos , Fosfomicina/farmacologia , Escherichia coli , Antibacterianos/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/metabolismo
8.
ACS Appl Mater Interfaces ; 15(16): 19892-19903, 2023 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-37046176

RESUMO

Silicon nanowire (SiNW) biosensors have attracted a lot of attention due to their superior sensitivity. Recently, the dependence of biomolecule detection sensitivity on the nanowire (NW) width, number, and doping density has been partially investigated. However, the primary reason for achieving ultrahigh sensitivity has not been elucidated thus far. In this study, we designed and fabricated SiNW biosensors with different widths (10.8-155 nm) by integrating a complementary metal-oxide-semiconductor process and electron beam lithography. We aimed to investigate the detection limit of SiNW biosensors and reveal the critical effect of the 10-nm-scaled SiNW width on the detection sensitivity. The sensing performance was evaluated by detecting antiovalbumin immunoglobulin G (IgG) with various concentrations (from 6 aM to 600 nM). The initial thickness of the depletion region of the SiNW and the changes in the depletion region due to biomolecule binding were calculated. The basis of this calculation are the resistance change ratios as functions of IgG concentrations using SiNWs with different widths. The calculation results reveal that the proportion of the depletion region over the entire SiNW channel is the essential reason for high-sensitivity detection. Therefore, this study is crucial for an indepth understanding on how to maximize the sensitivity of SiNW biosensors.


Assuntos
Técnicas Biossensoriais , Nanofios , Silício , Imunoglobulina G , Óxidos , Impressão
9.
J Biochem ; 173(5): 393-411, 2023 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-36779417

RESUMO

HNF4α regulates various genes to maintain liver function. There have been reports linking HNF4α expression to the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. In this study, liver-specific Hnf4a-deficient mice (Hnf4aΔHep mice) developed hepatosteatosis and liver fibrosis, and they were found to have difficulty utilizing glucose. In Hnf4aΔHep mice, the expression of fatty acid oxidation-related genes, which are PPARα target genes, was increased in contrast to the decreased expression of PPARα, suggesting that Hnf4aΔHep mice take up more lipids in the liver instead of glucose. Furthermore, Hnf4aΔHep/Ppara-/- mice, which are simultaneously deficient in HNF4α and PPARα, showed improved hepatosteatosis and fibrosis. Increased C18:1 and C18:1/C18:0 ratio was observed in the livers of Hnf4aΔHep mice, and the transactivation of PPARα target gene was induced by C18:1. When the C18:1/C18:0 ratio was close to that of Hnf4aΔHep mouse liver, a significant increase in transactivation was observed. In addition, the expression of Pgc1a, a coactivator of PPARs, was increased, suggesting that elevated C18:1 and Pgc1a expression could contribute to PPARα activation in Hnf4aΔHep mice. These insights may contribute to the development of new diagnostic and therapeutic approaches for NAFLD by focusing on the HNF4α and PPARα signaling cascade.


Assuntos
Fator 4 Nuclear de Hepatócito , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/genética , PPAR alfa/metabolismo
10.
J Biochem ; 170(6): 713-727, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34523685

RESUMO

GDE4 and GDE7 are membrane-bound enzymes that exhibit lysophospholipase D activities. We found that GDE7 produced not only lysophosphatidic acid (LPA) but also cyclic phosphatidic acid (cPA) from lysophospholipids by a transphosphatidylation reaction. In contrast, GDE4 produced only LPA. The analysis of substrate specificity showed that 1-alkyl-lysophosphospholipids were preferred substrates for both enzymes rather than 1-alkyl-lysophospholipids and 1-alkenyl-lysophospholipids. Among the various lysophospholipids with different polar head groups that were tested, lysophosphatidylglycerol and lysophosphatidylserine were preferred substrates for GDE4 and GDE7, respectively. The detailed analysis of the dependency of the enzyme activities of GDE4 and GDE7 on divalent cations suggested multiple divalent cations were bound in the active sites of both enzymes. Taken together, these results suggest the possibility that GDE7 functions as a cPA-producing enzyme in the body.


Assuntos
Lisofosfolipídeos/química , Diester Fosfórico Hidrolases/química , Animais , Camundongos , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
11.
J Biol Chem ; 285(36): 27652-63, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20576599

RESUMO

Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5DeltaC471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5DeltaC471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism.


Assuntos
Desenvolvimento Muscular , Músculo Esquelético/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Atrofia Muscular/enzimologia , Atrofia Muscular/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos
12.
J Chromatogr A ; 1635: 461647, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33291035

RESUMO

This paper describes the simultaneous capillary electrophoresis (CE) of anions and cations using an anion exchange-modified capillary, which was prepared by chemical coating with a cationic silylating reagent, and its application to saliva analysis. The CE method provides high-throughput (5 min for a single sample injection) analysis by generating a high-velocity electroosmotic flow at pH 3.0-3.5. The detection limits at a signal-to-noise ratio of 3 ranged from 1.2 to 18 µM for anions and 1.0 to 2.7 µM for cations. The relative standard deviations for the migration times and peak areas of analytes (n = 4) ranged from 0.05% to 0.40% and 0.94% to 4.7%, respectively. The CE system was used to analyze 11 common ions in saliva samples collected from long-distance runners and sedentary university students before and after running for a set distance or a set time. Interestingly, the SCN- concentrations decreased in the saliva samples of all 14 athletes and 16 sedentary students after running. Furthermore, when the concentrations of the analyzed ions were compared with that of cortisol as a typical stress marker by multiple regression analysis, SCN- and NO3- in saliva samples from the two subject groups strongly correlated with cortisol levels, as determined by an electrochemiluminescence immunoassay. This study improves our knowledge of both the analytical methodology for CE and statistical methods for identifying common ions that could be used as physical stress markers.


Assuntos
Ânions/isolamento & purificação , Cátions/isolamento & purificação , Eletroforese Capilar/métodos , Saliva/química , Biomarcadores/análise , Eletro-Osmose , Humanos , Indicadores e Reagentes , Limite de Detecção
13.
Biochim Biophys Acta ; 1791(8): 797-805, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19439193

RESUMO

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.


Assuntos
Tioléster Hidrolases/metabolismo , Linhagem Celular , Cromatografia Líquida , Humanos , Cinética , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/metabolismo , Espectrometria de Massas , Proteínas Mutantes/metabolismo , Peptídeos/química , Transporte Proteico , Frações Subcelulares/enzimologia , Tioléster Hidrolases/isolamento & purificação
14.
ACS Appl Mater Interfaces ; 12(46): 51808-51819, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33142064

RESUMO

As critical factors affecting the sensing performance of silicon nanowire (SiNW) biosensors, the structure, functional interface, and detection target were analyzed and designed to improve sensing performance. For an improved understanding of the dependence of sensor structure on sensitivity, a simple theoretical analysis was proposed to predict the sensitivity of biosensors with different SiNW types, widths, and doping concentrations. Based on the theoretical analysis, a biosensor integrating optimized critical factors was designed and fabricated. Optimizations focusing on the following aspects are considered: (1) employing n-type SiNW and controlling the impurity doping concentration of SiNW at approximately 2 × 1016-6 × 1016 atoms/cm3 to obtain a suitable charge density, (2) minimizing the SiNW width to 16.0 nm to increase the surface area-to-volume ratio, (3) using a native oxide layer on SiNW as a gate insulator to transport the captured charge molecules closer to the SiNW surface, (4) modifying the SiNW surface by 2-aminoethylphosphonic acid coupling to form a high-density self-assembled monolayer for enhancing the stability bound molecules, and (5) functionalizing the SiNW with ovalbumin molecules for specifically capturing the target immunoglobulin G (IgG) molecules. The sensing performance was evaluated by detecting IgG with concentrations ranging from 6 aM to 600 nM and control experiments. The SiNW biosensor revealed ultrahigh sensitivity and specific detection of target IgG with a measured limit of detection of 6 aM. The integration of the critical SiNW biosensor factors provides a significant possibility of a rapid and ultrasensitive diagnosis of diseases at their early stages.


Assuntos
Técnicas Biossensoriais/métodos , Imunoglobulina G/análise , Nanofios/química , Silício/química , Ácido Aminoetilfosfônico/química , Animais , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Imunoglobulina G/imunologia , Limite de Detecção , Ovalbumina/imunologia , Coelhos , Propriedades de Superfície , Transistores Eletrônicos
15.
Nat Commun ; 11(1): 1922, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321922

RESUMO

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic ß-cells are destroyed. Intestinal helminths can cause asymptomatic chronic and immunosuppressive infections and suppress disease in rodent models of T1D. However, the underlying regulatory mechanisms for this protection are unclear. Here, we report that CD8+ regulatory T (Treg) cells prevent the onset of streptozotocin -induced diabetes by a rodent intestinal nematode. Trehalose derived from nematodes affects the intestinal microbiota and increases the abundance of Ruminococcus spp., resulting in the induction of CD8+ Treg cells. Furthermore, trehalose has therapeutic effects on both streptozotocin-induced diabetes and in the NOD mouse model of T1D. In addition, compared with healthy volunteers, patients with T1D have fewer CD8+ Treg cells, and the abundance of intestinal Ruminococcus positively correlates with the number of CD8+ Treg cells in humans.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Linfócitos T Reguladores/imunologia , Animais , Clostridiales , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Modelos Animais de Doenças , Faecalibacterium prausnitzii , Feminino , Microbioma Gastrointestinal , Humanos , Imunossupressores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , RNA Ribossômico 16S/metabolismo , Ruminococcus , Trealose/farmacologia
16.
J Bacteriol ; 190(4): 1219-23, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18083802

RESUMO

Escherichia coli cytosolic glycerophosphodiester phosphodiesterase, UgpQ, functions in the absence of other proteins encoded by the ugp operon and requires Mg2+, Mn2+, or Co2+, in contrast to Ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophosphodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metais/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Magnésio/metabolismo , Manganês/metabolismo , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/genética , Prótons , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
17.
J Mol Biol ; 362(3): 490-501, 2006 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-16934832

RESUMO

Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia.


Assuntos
1-Pirrolina-5-Carboxilato Desidrogenase/química , Thermus thermophilus/enzimologia , 1-Pirrolina-5-Carboxilato Desidrogenase/deficiência , 1-Pirrolina-5-Carboxilato Desidrogenase/genética , 1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Prolina/sangue , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Thermus thermophilus/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-17554163

RESUMO

Delta(1)-Pyrroline-5-carboxylate dehydrogenase (P5CDh) is known to preferentially use NAD(+) as a coenzyme. The k(cat) value of Thermus thermophilus P5CDh (TtP5CDh) is four times lower for NADP(+) than for NAD(+). The crystal structure of NADP(+)-bound TtP5CDh was solved in order to study the structure-activity relationships for the coenzymes. The binding mode of NADP(+) is essentially identical to that in the previously solved NAD(+)-bound form, except for the regions around the additional 2'-phosphate group of NADP(+). The coenzyme-binding site can only accommodate this group by the rotation of a glutamate residue and subtle shifts in the main chain. The 2'-phosphate of NADP(+) increases the number of hydrogen bonds between TtP5CDh and NADP(+) compared with that between TtP5CDh and NAD(+). Furthermore, the phosphate of the bound NADP(+) would restrict the ;bending' of the coenzyme because of steric hindrance. Such bending is important for dissociation of the coenzymes. These results provide a plausible explanation of the lower turnover rate of NADP(+) compared with NAD(+).


Assuntos
1-Pirrolina-5-Carboxilato Desidrogenase/metabolismo , Coenzimas/metabolismo , Desidrogenase de Glutamato (NADP+)/metabolismo , Thermus thermophilus/enzimologia , 1-Pirrolina-5-Carboxilato Desidrogenase/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/fisiologia , Coenzimas/química , Cristalografia por Raios X , Desidrogenase de Glutamato (NADP+)/química , NADP/química , NADP/metabolismo
19.
J Mol Biol ; 352(4): 905-17, 2005 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-16126223

RESUMO

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.


Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , NADP/metabolismo , Estrutura Quaternária de Proteína , Thermus thermophilus/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Sulfatos/metabolismo , Valina/metabolismo
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