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1.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
2.
Chemistry ; 25(44): 10505-10510, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173420

RESUMO

Precision cell-selective surface glycan remodeling is of vital importance for modulation of cell surface dynamics, tissue-specific imaging, and immunotherapy, but remains an unsolved challenge. Herein, we report a switchable enzymatic accessibility (SEA) strategy for highly specific editing of carbohydrate moieties of interest on the target cell surface. We demonstrate the blocking of enzyme in the inaccessible state with a metal-organic framework (MOF) cage and instantaneous switching to the accessible state through disassembly of MOF. We further show that this level of SEA regulation enables initial guided enzyme delivery to the target cell surface for subsequent cell-specific glycan remodeling, thus providing a temporally and spatially controlled tool for tuning the glycosylation architectures. Terminal galactose/N-acetylgalactosamine (Gal/GalNAc) remodeling and terminal sialic acid (Sia) desialylation have been precisely achieved on target cells even with other cell lines in close spatial proximity. The SEA protocol features a modular and generically adaptable design, a very short protocol duration (ca. 30 min or shorter), and a very high spatial resolving power (ability to differentiate immediately neighboring cell lines).


Assuntos
Membrana Celular/enzimologia , Polissacarídeos/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Aptâmeros de Nucleotídeos/química , Biocatálise , Membrana Celular/química , Ativação Enzimática , Galactose/química , Galactose/metabolismo , Galactose Oxidase/antagonistas & inibidores , Galactose Oxidase/metabolismo , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Estruturas Metalorgânicas/química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Imagem Óptica/métodos , Polissacarídeos/química , Propriedades de Superfície
3.
Subcell Biochem ; 92: 337-366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214992

RESUMO

The inner membrane of Gram-negative bacteria is a ~6 nm thick phospholipid bilayer. It forms a semi-permeable barrier between the cytoplasm and periplasm allowing only regulated export and import of ions, sugar polymers, DNA and proteins. Inner membrane proteins, embedded via hydrophobic transmembrane α-helices, play an essential role in this regulated trafficking: they mediate insertion into the membrane (insertases) or complete crossing of the membrane (translocases) or both. The Gram-negative inner membrane is equipped with a variety of different insertases and translocases. Many of them are specialized, taking care of the export of only a few protein substrates, while others have more general roles. Here, we focus on the three general export/insertion pathways, the secretory (Sec) pathway, YidC and the twin-arginine translocation (TAT) pathway, focusing closely on the Escherichia coli (E. coli) paradigm. We only briefly mention dedicated export pathways found in different Gram-negative bacteria. The Sec system deals with the majority of exported proteins and functions both as a translocase for secretory proteins and an insertase for membrane proteins. The insertase YidC assists the Sec system or operates independently on membrane protein clients. Sec and YidC, in common with most export pathways, require their protein clients to be in soluble non-folded states to fit through the translocation channels and grooves. The TAT pathway is an exception, as it translocates folded proteins, some loaded with prosthetic groups.


Assuntos
Membrana Celular/enzimologia , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Canais de Translocação SEC/metabolismo , Sistema de Translocação de Argininas Geminadas/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Transporte Proteico
4.
Science ; 364(6438): 389-394, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31023924

RESUMO

Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. Signals received by the G protein-coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo-electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.


Assuntos
Adenilil Ciclases/química , Membrana Celular/enzimologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Proteínas de Membrana/química , Adenilil Ciclases/ultraestrutura , Animais , Domínio Catalítico , Bovinos , Microscopia Crioeletrônica , AMP Cíclico/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Proteínas de Membrana/ultraestrutura , Transdução de Sinais
5.
Mol Cell ; 74(2): 393-408.e20, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30956043

RESUMO

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.


Assuntos
Domínio Catalítico/genética , Mutagênese/genética , Conformação Proteica , Quinases da Família src/química , Regulação Alostérica/genética , Membrana Celular/química , Membrana Celular/enzimologia , Células HEK293 , Humanos , Fosforilação , Quinases da Família src/genética
6.
Molecules ; 24(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818768

RESUMO

Phenols are the most abundant naturally accessible antioxidants present in a human normal diet. Since numerous beneficial applications of phenols as preventive agents in various diseases were revealed, the evaluation of phenols bioavailability is of high interest of researchers, consumers and drug manufacturers. The hydrophilic nature of phenols makes a cell membrane penetration difficult, which imply an alternative way of uptake via membrane transporters. However, the structural and functional data of membrane transporters are limited, thus the in silico modelling is really challenging and urgent tool in elucidation of transporter ligands. Focus of this research was a particular transporter bilitranslocase (BTL). BTL has a broad tissue expression (vascular endothelium, absorptive and excretory epithelia) and can transport wide variety of poly-aromatic compounds. With available BTL data (pKi [mmol/L] for 120 organic compounds) a robust and reliable QSAR models for BTL transport activity were developed and extrapolated on 300 phenolic compounds. For all compounds the transporter profiles were assessed and results show that dietary phenols and some drug candidates are likely to interact with BTL. Moreover, synopsis of predictions from BTL models and hits/predictions of 20 transporters from Metrabase and Chembench platforms were revealed. With such joint transporter analyses a new insights for elucidation of BTL functional role were acquired. Regarding limitation of models for virtual profiling of transporter interactions the computational approach reported in this study could be applied for further development of reliable in silico models for any transporter, if in vitro experimental data are available.


Assuntos
Membrana Celular/enzimologia , Ceruloplasmina/metabolismo , Simulação por Computador , Fenóis/metabolismo , Transporte Biológico , Transporte Biológico Ativo , Bases de Dados de Produtos Farmacêuticos , Humanos
7.
Arch Pharm Res ; 42(5): 436-445, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30919268

RESUMO

Mitochondrial dysfunction caused by oxidative stress appears at early stages of aging and age-related diseases. Plasma membrane redox enzymes act in a compensatory manner to decrease oxidative stress and supply reductive capacity to ensure cell survival. Plasma membrane redox enzymes transfer electrons from NAD(P)H to oxidized ubiquinone and α-tocopherol, resulting in inhibition of further oxidative damage. Plasma membrane redox enzymes and their partners are affected by aging, leading to progression of neurodegenerative disease pathogenesis. Up-regulating plasma membrane redox enzymes via calorie restriction and phytochemicals make cells more resistant to oxidative damage under stress conditions by maintaining redox homeostasis and improving mitochondrial function. Investigation into plasma membrane redox enzymes can provide mechanistic details underlying the relationships between plasma membrane redox enzymes and mitochondrial complexes and provide a good therapeutic target for prevention and delay of neurodegenerative disorders.


Assuntos
Membrana Celular/enzimologia , Mitocôndrias/enzimologia , NADH NADPH Oxirredutases/metabolismo , Doenças Neurodegenerativas/terapia , Fármacos Neuroprotetores/farmacologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Animais , Restrição Calórica , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Progressão da Doença , Regulação para Baixo/fisiologia , Radicais Livres/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Modelos Animais , NADH NADPH Oxirredutases/antagonistas & inibidores , Doenças Neurodegenerativas/patologia , Fármacos Neuroprotetores/uso terapêutico , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/uso terapêutico , Regulação para Cima/efeitos dos fármacos , alfa-Tocoferol/metabolismo
8.
Gene ; 702: 133-142, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30904717

RESUMO

Phosphofructokinase plays an essential role in sugar metabolism in plants. Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP), and the ATP-dependent phosphofructokinase (PFK). Until now, the gene evolution, expression patterns, and functions of phosphofructokinase proteins were unknown in pear. In this report, 14 phosphofructokinase genes were identified in pear. The phylogenetic tree indicated that the phosphofructokinase gene family could be grouped into two subfamilies, with 10 genes belonging to the PbPFK subfamily, and 4 genes belonging to the PbPFP subfamily. Conserved motifs and exon numbers of the phosphofructokinase were found in pear and other six species. The evolution analysis indicated that WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes expansion in pear and other six species. Analysis of cis-regulatory element sequences of all phosphofructokinase genes identified light regulation and the MYB binding site in the promoter of all pear phosphofructokinase genes, suggesting that phosphofructokinase might could be regulated by light and MYB transcription factors (TFs). Gene expression patterns revealed that PbPFP1 showed similar pattern with sorbitol contents, suggesting important contributions to sugar accumulation during fruit development. Further functional analysis indicated that the phosphofructokinase gene PbPFP1 was localized on plasma membrane compartment, indicating that PbPFP1 had function in plasma membrane. Transient transformation of PbPFP1 in pear fruits led to significant increases of fructose and sorbitol compared to controls. Overall, our study provides important insights into the gene expression patterns and important potential functions of phosphofructokinase for sugar accumulation in pear fruits, which will help to enrich understanding of sugar-related bio-pathways and lay the molecular basis for fruit quality improvement.


Assuntos
Família Multigênica , Fosfofrutoquinases/classificação , Fosfofrutoquinases/genética , Pyrus/enzimologia , Motivos de Aminoácidos , Membrana Celular/enzimologia , Mapeamento Cromossômico , Éxons , Frutas/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Íntrons , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Pyrus/classificação , Pyrus/genética , Pyrus/crescimento & desenvolvimento , Rosaceae/classificação , Açúcares/metabolismo , Transcrição Genética
9.
Plant Sci ; 280: 408-415, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824019

RESUMO

Using various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds.


Assuntos
Helianthus/enzimologia , Dormência de Plantas , Reguladores de Crescimento de Planta/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Abscísico/metabolismo , Membrana Celular/enzimologia , Etilenos/metabolismo , Germinação , Helianthus/genética , Helianthus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia
10.
Molecules ; 24(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857224

RESUMO

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.


Assuntos
Membrana Celular/enzimologia , Kluyveromyces/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Western Blotting , Cromatografia em Gel , Substâncias Macromoleculares/metabolismo
11.
Biosci Biotechnol Biochem ; 83(6): 1171-1179, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30777491

RESUMO

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2-5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase. Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection.


Assuntos
Acetobacteraceae/enzimologia , Dissacarídeos/metabolismo , Glucose Desidrogenase/metabolismo , Membrana Celular/enzimologia , Glucose/metabolismo , Lactose/metabolismo , Oxirredução , Especificidade por Substrato
12.
J Biol Chem ; 294(15): 5956-5969, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30770471

RESUMO

Cholesterol's effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-ß-cyclodextrin (mßCD). The mßCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3 + → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2 + → E1Na3 +). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K2 +). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.


Assuntos
Membrana Celular/enzimologia , Colesterol , Simulação de Dinâmica Molecular , ATPase Trocadora de Sódio-Potássio , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Colesterol/química , Colesterol/metabolismo , Estabilidade Enzimática , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , beta-Ciclodextrinas/química
13.
J Biol Chem ; 294(14): 5720-5734, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723156

RESUMO

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.


Assuntos
Membrana Celular/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Animais , Membrana Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Técnicas de Silenciamento de Genes , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Sódio/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidade
14.
Cell Physiol Biochem ; 52(1): 76-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790506

RESUMO

BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.


Assuntos
Membrana Celular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteína Quinase C-alfa/metabolismo , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Dibutirato de 12,13-Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Quinases Associadas a rho/metabolismo
15.
Methods Mol Biol ; 1927: 47-72, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788785

RESUMO

Eukaryotic membrane bound cytochrome P450s are expressed in bacterial systems to produce large yields of catalytically active protein for structure function studies. Recently, there have been several instances of expressing eukaryotic membrane bound CYPs in bacteria after making various modifications to both the N-terminus membrane binding domains of the protein and to noncontiguous F-G membrane binding loop that is also implicated in substrate binding. These modifications have been shown not to disturb the function of the protein of interest. The major factors that have been key to express the membrane bound cytochrome P450s in bacteria have been the following: (a) exon optimization (b) selection of the appropriate vector and host strain, and (c) growth and expression conditions with respect to temperature and speed of shaking the media flask. Herein, we describe methods to express and purify eukaryotic membrane bound cytochrome P450s. We also describe the measurement of the activity of the cytochrome P450 expressed by taking the example of cytochrome P450 2J2, the primary P450 expressed in the human heart and CYP725A4, the primary cytochrome P450 expressed in the first step of taxol synthesis. Additionally, we discuss the pros and cons of the different modifications done in order to express the membrane bound cytochrome P450s.


Assuntos
Membrana Celular/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Bacteriana da Expressão Gênica , Nanotecnologia , Animais , Membrana Celular/enzimologia , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Ativação Enzimática , Família Multigênica , Mutação , Nanotecnologia/métodos , Ratos , Proteínas Recombinantes de Fusão , Espectrofotometria/métodos
16.
Cancer Res ; 79(2): 301-310, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30610085

RESUMO

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Receptor PAR-2/metabolismo , Serina Proteases/metabolismo , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Progressão da Doença , Glicosilfosfatidilinositóis/metabolismo , Humanos , Neoplasias/enzimologia , Transdução de Sinais
17.
Environ Sci Pollut Res Int ; 26(5): 4975-4986, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604360

RESUMO

Acid rain is a global environmental problem that threatens agricultural production. Calcium (Ca), as a signal substance for physiological activities, has been known to regulate plant growth under abiotic stresses. To clarify whether calcium could be one of possible ways to alleviate the reduction caused by acid rain in agricultural production and investigate its regulating mechanism on adaptation of plants under acid rain stress, we studied the effect of exogenous Ca2+ (5 mM CaCl2) on growth of soybean at different growth stages (seedling, flowering-podding, and filling stages) as well as yield and grain quality of soybean under simulated acid rain (pH 4.5 or pH 3.0) stress. We found that the application of Ca2+ could regulate the activity of plasma membrane H+-ATPase, for mitigating the increase of ammonium and the decrease of nitrate and phosphorus in soybean roots, which mitigated the inhibition on growth and improved the yield and grain quality of soybean under simulated acid rain stress. In addition, the alleviating effect of exogenous Ca2+ on soybean was the most significant at seedling stage. The results indicate that the exogenous Ca2+ could enhance the adaptation of soybean and facilitate the recovery of soybean productivity and grain quality under simulated acid rain stress by maintaining the uptake of nitrate, ammonium, and phosphorus.


Assuntos
Chuva Ácida/análise , Cálcio/farmacologia , Soja/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Modelos Teóricos , Nitratos/metabolismo , Fósforo/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Soja/efeitos dos fármacos , Soja/metabolismo
18.
Plant Cell Physiol ; 60(5): 935-944, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649552

RESUMO

Brassinosteroids (BRs) are steroid phytohormones that regulate plant growth and development, and promote cell elongation at least in part via the acid-growth process. BRs have been suggested to induce cell elongation by the activating plasma membrane (PM) H+-ATPase. However, the mechanism by which BRs activate PM H+-ATPase has not been clarified. In this study, we investigated the effects of BR on hypocotyl elongation and the phosphorylation status of a penultimate residue, threonine, of PM H+-ATPase, which affects the activation, in the etiolated seedlings of Arabidopsis thaliana. Brassinolide (BL), an active endogenous BR, induced hypocotyl elongation, phosphorylation of the penultimate, threonine residue of PM H+-ATPase, and binding of the 14-3-3 protein to PM H+-ATPase in the endogenous BR-depleted seedlings. Changes in both BL-induced elongation and phosphorylation of PM H+-ATPase showed similar concentration dependency. BL did not induce phosphorylation of PM H+-ATPase in the BR receptor mutant bri1-6. In contrast, bikinin, a specific inhibitor of BIN2 that acts as a negative regulator of BR signaling, induced its phosphorylation. Furthermore, BL accumulated the transcripts of SMALL AUXIN UP RNA 9 (SAUR9) and SAUR19, which suppress dephosphorylation of the PM H+-ATPase penultimate residue by inhibiting D-clade type 2C protein phosphatase in the hypocotyls of etiolated seedlings. From these results, we conclude that BL-induced phosphorylation of PM H+-ATPase penultimate residue is mediated via the BRI1-BIN2 signaling pathway, together with the accumulation of SAURs during hypocotyl elongation.


Assuntos
Brassinosteroides/farmacologia , Membrana Celular/enzimologia , Hipocótilo/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos
19.
J Biol Chem ; 294(10): 3432-3443, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622140

RESUMO

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.


Assuntos
Membrana Celular/enzimologia , Coanoflagelados/enzimologia , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Mutação , Domínios Proteicos , Rodopsina/genética , Análise Espectral
20.
J Plant Physiol ; 232: 248-256, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30537611

RESUMO

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2- scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2-, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.


Assuntos
NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , ATPases Translocadoras de Prótons/metabolismo , Superóxido Dismutase-1/metabolismo , Vigna/enzimologia , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/fisiologia , Peroxidases/fisiologia , Proteínas de Plantas/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Superóxido Dismutase-1/fisiologia , Superóxidos/metabolismo , Vigna/crescimento & desenvolvimento
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