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1.
Food Chem ; 350: 129212, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33609939

RESUMO

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.


Assuntos
Aspergillus oryzae/enzimologia , Temperatura Baixa , Engenharia Genética/métodos , Chaperonas Moleculares/genética , Petróleo/análise , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/metabolismo , Clonagem Molecular , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/genética
2.
Methods Mol Biol ; 2251: 91-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481233

RESUMO

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.


Assuntos
Membrana Celular/química , Fosfatidilinositóis/análise , Imagem Individual de Molécula/métodos , Animais , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Humanos , Membranas/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Transporte Proteico/fisiologia , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo
3.
Methods Mol Biol ; 2251: 225-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481244

RESUMO

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.


Assuntos
Ensaios Enzimáticos/métodos , Fosfatidilinositol 4,5-Difosfato/análogos & derivados , Fosfolipases Tipo C/análise , Fluoresceína-5-Isotiocianato/química , Hidrólise , Isoenzimas/análise , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase C gama/análise , Fosfolipase C gama/metabolismo , Ligação Proteica/fisiologia , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo
4.
Med Hypotheses ; 143: 110148, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32759016

RESUMO

Estrogen hormone acts as a potential key player in providing immunity against certain viral infection. It is found to be associated in providing immunity against acute lungs inflammation and influenza virus by modulating cytokines storm and mediating adaptive immune alterations respectively. Women are less affected by SARS-CoV-2 infection because of the possible influence of estrogen hormone as compared to men. We hypothesized that SARS-CoV-2 causes stress in endoplasmic reticulum (ER) which in turn aggravates the infection, estrogen hormone might play key role in decreasing ER stress by activating estrogen mediated signaling pathways, results in unfolded protein response (UPR). Estrogen governs degradation of phosphotidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3) with the help of phospholipase C. IP3 start in-fluxing Ca+2 ions that helps in UPR activation. To support our hypothesis, we analyzed the data of 162,392 COVID-19 patients to determine the relation of this disease with gender. We observed that 26% of women and 74% of men were affected by SARS-CoV-2. It indicated that women are less affected because of the possible influence of estrogen hormone in women.


Assuntos
Betacoronavirus , Infecções por Coronavirus/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Estrogênios/fisiologia , Modelos Biológicos , Pandemias , Pneumonia Viral/fisiopatologia , Adulto , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/metabolismo , Conjuntos de Dados como Assunto/estatística & dados numéricos , Diglicerídeos/metabolismo , Resistência à Doença , Feminino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Pneumonia Viral/epidemiologia , Pneumonia Viral/metabolismo , Caracteres Sexuais , Distribuição por Sexo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Resposta a Proteínas não Dobradas , Proteínas Virais/biossíntese , Proteínas Virais/genética
5.
Arterioscler Thromb Vasc Biol ; 40(9): 2212-2226, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640908

RESUMO

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade do Canal Arterial/metabolismo , Canal Arterial/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Canal Arterial/anormalidades , Permeabilidade do Canal Arterial/genética , Permeabilidade do Canal Arterial/patologia , Células Endoteliais/patologia , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Proteína Quinase C/metabolismo , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(20): 11097-11108, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358199

RESUMO

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.


Assuntos
Potenciais de Ação/fisiologia , Axônios/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Células de Purkinje/metabolismo , Cálcio/metabolismo , Cerebelo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Purinérgicos P2Y1 , Fosfolipases Tipo C/metabolismo
7.
Gene ; 753: 144797, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32454180

RESUMO

Phospholipase C proteins are phospholipid hydrolysing enzymes and crucial components of abiotic stress triggered lipid signaling in plants. PLCs are implicated in plant reaction to drought, salinity, and cold stress responses, however, characterization of the PLC family in the legume crop chickpea is missing. Here, we identify and describe nine PLC encoding genes in the chickpea genome. Phylogenetic analysis showed that the chickpea PLC family has evolved through a common path in dicots. Subcellular localization of fluorescence tagged proteins confirmed cytoplasmic and plasma membrane bound forms of PLCs in chickpea. The promoters of all the PLC genes are comprised of several hormone response related, development and abiotic stress related cis-regulatory elements. Expression analysis in five developmental stages (germination, seedling, vegetative, reproductive and senescence) showed significant expression of multiple PLCs in germination, vegetative and reproductive stages, suggesting their diverse role in various developmental processes. qRT-PCR expression analysis of the entire PLC gene family under drought, salt and cold stresses revealed that most PLC genes are differentially expressed in multiple abiotic stresses. These observations indicate the involvement of PLC gene family in abiotic stress signaling and responses in important legume crop. The present study opens new avenues for utilizing PLC- related information in biotechnological programs for abiotic stress tolerance and legume crop improvement.


Assuntos
Cicer/genética , Estresse Fisiológico/genética , Fosfolipases Tipo C/genética , Resposta ao Choque Frio , Secas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas , Genômica , Germinação , Filogenia , Proteínas de Plantas/genética , Salinidade , Cloreto de Sódio/metabolismo , Transcriptoma/genética , Fosfolipases Tipo C/metabolismo
8.
Biochem Pharmacol ; 177: 113985, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311347

RESUMO

The 5-HT2A receptor is a homodimeric G protein-coupled receptor implied in multiple diseases, including schizophrenia. Recently, its co-crystallisation with the antipsychotic drugs zotepine and risperidone has revealed the importance of its extracellular domains in its pharmacology. Previous studies have shown that the non-specific disruption of extracellular disulphide bridges in the 5-HT2A receptor decreases ligand binding and receptor activation. There is enough evidence to hypothesize that this decrease may be due to a reduction of the disulphide bridge that links transmembrane domain 3 (TM-3) and extracellular loop 2 (ECL-2) of the 5-HT2A receptor via cysteine 148 (C148) and C227. Thus, to study the influence of the C148-C227 disulphide bridge on 5-HT2A receptor pharmacology, we substituted C148 and C227 in the human 5-HT2A receptor (WT) with alanines, to obtain two single mutants (C148A and C227A) and a double mutant (C148A/C227A), and the resultant DNA constructs were used to generate four stable cell lines. These substitutions reduced the binding of the 5-HT2A receptor to [3H]lysergic acid diethylamide ([3H]LSD) and impeded the 5-HT2A receptor-mediated activation of phospholipase C (PLC). Furthermore, bioluminescence resonance energy transfer (BRET) and western blotting analysis revealed that these mutations did not alter the homodimeric nature of the 5-HT2A receptor. However, fluorescence microscopy showed that these mutations hindered receptor trafficking to the cell membrane. These results illustrate the importance of the disulphide bridge between TM-3 and ECL-2 in maintaining the correct 5-HT2A receptor conformation to allow ligand binding and migration of the homodimeric receptor to the cell membrane.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Dissulfetos/química , Receptor 5-HT2A de Serotonina/química , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Efeito Fundador , Expressão Gênica , Células HEK293 , Humanos , Ligantes , Dietilamida do Ácido Lisérgico/farmacologia , Mutação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Proteínas Recombinantes , Serotonina/farmacologia , Fosfolipases Tipo C/genética
9.
PLoS Comput Biol ; 16(4): e1007708, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32255775

RESUMO

Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.


Assuntos
Ácidos Fosfatídicos/metabolismo , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Quimiotaxia/fisiologia , Diglicerídeos/metabolismo , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Modelos Teóricos , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Ácidos Fosfatídicos/fisiologia , Fosfolipase D/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Quinase C/fisiologia , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/fisiologia
10.
Mol Cell Biol ; 40(12)2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32205408

RESUMO

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1 The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1 Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.


Assuntos
Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Proteína Fosfatase 2/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fosfolipases Tipo C/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Deleção de Genes , Redes Reguladoras de Genes , Genes Fúngicos , Proteína Fosfatase 2/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fosfolipases Tipo C/metabolismo
11.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211893

RESUMO

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.


Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
12.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211894

RESUMO

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.


Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
13.
Life Sci ; 246: 117419, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32045592

RESUMO

AIMS: Although resistin-like molecule ß (RELM-ß) is involved in the pathological processes of various lung diseases, such as pulmonary inflammation, asthma and fibrosis, its potential roles in hypoxic pulmonary arterial hypertension (PAH) remain largely unknown. The study aims to investigate whether RELM-ß contributes to hypoxia-induced excessive proliferation of human pulmonary artery smooth muscle cells (PASMCs) and to explore the potential mechanisms of this process. MAIN METHODS: Human PASMCs were exposed to normoxia or hypoxia (1% O2) for 24 h. siRNA targeting RELM-ß was transfected into cells. Protein levels of KCNK3, RELM-ß, pSTAT3 and STAT3 were determined by immunoblotting. The translocation of NFATc2 and expression of KCNK3 were visualized by immunofluorescence. 5-ethynyl-2'-deoxyuridine assays and cell counting kit-8 assays were performed to assess the proliferation of PASMCs. KEY FINDINGS: (1) Chronic hypoxia significantly decreased KCNK3 protein levels while upregulating RELM-ß protein levels in human PASMCs, which was accompanied by excessive proliferation of cells. (2) RELM-ß could promote human PASMCs proliferation and activate the STAT3/NFAT axis by downregulating KCNK3 protein under normoxia. (3) Inhibition of RELM-ß expression effectively prevented KCNK3-mediated cell proliferation under hypoxia. (4) Phospholipase C (PLC) inhibitor U-73122 could not only prevent the hypoxia/RELM-ß-induced decrease in KCNK3 protein, but also inhibit the enhanced cell viability caused by hypoxia/RELM-ß. (5) Both hypoxia and RELM-ß could downregulate membrane KCNK3 protein levels by enhancing endocytosis. SIGNIFICANCE: RELM-ß activation is responsible for hypoxia-induced excessive proliferation of human PASMCs. Interfering with RELM-ß may alleviate the progression of hypoxic PAH by upregulating PLC-dependent KCNK3 expression.


Assuntos
Hipóxia/complicações , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Imunofluorescência , Humanos , Hipóxia/tratamento farmacológico , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , Artéria Pulmonar/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
14.
Biochem Biophys Res Commun ; 524(3): 589-594, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32029276

RESUMO

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.


Assuntos
Acetilcolina/metabolismo , Neurônios Motores/metabolismo , Óxido Nítrico/metabolismo , Fosfolipídeos/metabolismo , Ranidae/metabolismo , Receptor Muscarínico M3/metabolismo , Sinapses/metabolismo , Animais , Canabinoides/metabolismo , Neurônios Motores/efeitos dos fármacos , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Sinapses/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo
15.
Eur J Med Chem ; 191: 112162, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101781

RESUMO

Phospholipases are enzymes that are involved in the hydrolysis of acyl and phosphate esters of phospholipids, generating secondary messengers that have implications in various cellular processes including proliferation, differentiation and motility. As such inhibitors of phospholipases have been widely studied for their use as anti-cancer therapeutics. Phosphatidylcholine-specific phospholipase C (PC-PLC) is implicated in the progression of a number of cancer cell lines including aggressing triple-negative breast cancers. Most current studies on PC-PLC have utilised D609 as the standard inhibitor however it is known to have multiple failings, including poor stability in aqueous media. 2-Morpholinobenzoic acids were recently identified using vHTS as a potential class of lead compounds, with improvements over D609. In this work 129 analogues in this class were prepared and their PC-PLC inhibitory activity was assessed. It was found that the majority of these novel compounds had improved activity when compared to D609 with the most potent inhibitors completely inhibiting enzyme activity. It was determined that the best compound/s contained a morpholino and 2-substituted N-benzyl moieties with these findings explained using molecular modelling. The compounds reported here will allow for improved study of PC-PLC activity.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Norbornanos , Relação Estrutura-Atividade , Tiocarbamatos , Tionas/síntese química , Tionas/química , Fosfolipases Tipo C/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-31954174

RESUMO

Perinatal and long-term offspring morbidities are strongly dependent on the preservation of placental vascular homeostasis during pregnancy. In adults, the HDL-apoM-S1P complex protects the endothelium and maintains vascular integrity. However, the metabolism and biology of cord blood-derived HDLs (referred to as neonatal HDL, nHDL) strikingly differ from those in adults. Here, we investigate the role of neonatal HDLs in the regulation of placental vascular function. We show that nHDL is a major carrier of sphingosine-1-phosphate (S1P), which is anchored to the particle through apoM (rs = 0.90, p < 0.0001) in the fetal circulation. Furthermore, this complex interacts with S1P receptors on the feto-placental endothelium and activates specifically extracellular signal-regulated protein kinases 1 and 2 (ERK) and phospholipase C (PLC) downstream signaling, promotes endothelial cell proliferation and calcium flux. Notably, the nHDL-S1P complex triggers actin filaments reorganization, leading to an enhancement of placental endothelial barrier function. Additionally, nHDL induces vasorelaxation of isolated placental chorionic arteries. Taken together, these results suggest that circulating nHDL exerts vasoprotective effects on the feto-placental endothelial barrier mainly via S1P signaling.


Assuntos
Sangue Fetal/metabolismo , Lipoproteínas HDL/metabolismo , Lisofosfolipídeos/metabolismo , Placenta/irrigação sanguínea , Esfingosina/análogos & derivados , Apolipoproteínas M/sangue , Apolipoproteínas M/metabolismo , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Humanos , Lipoproteínas HDL/sangue , Lisofosfolipídeos/sangue , Sistema de Sinalização das MAP Quinases , Gravidez , Esfingosina/sangue , Esfingosina/metabolismo , Fosfolipases Tipo C/metabolismo
17.
Anticancer Res ; 40(1): 213-220, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892569

RESUMO

BACKGROUND/AIM: Kisspeptin produced from the KISS1 gene is secreted from the living cells, binds to endogenous receptor KISS1R (also called G protein-coupled receptor 54, GPR54), and has various functions in normal physiological conditions. Although an anti-metastatic role of kisspeptin in cancer is well known in several cancer types, its role in brain tumors is not yet understood. Herein, we investigated a the role of kisspeptin in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells were treated with kisspeptin and subjected to proliferation, migration, and invasion assays. KISS1R dependency was tested by KISS1R silencing with KISS1R siRNAs. RESULTS: Kisspeptin inhibited migratory and invasive abilities of U87-MG, U-251-MG and U373-MG glioblastoma cells with no effect on cell viability. KISS1R gene silencing with KISS1R siRNAs blocked kisspeptin-induced glioblastoma cell invasiveness. Moreover, chemical inhibitors against Gq, PLC or PKC blocked kisspeptin-induced glioblastoma cell invasiveness. CONCLUSION: Kisspeptin induces glioblastoma cell invasiveness via the KISS1R-Gq-PLC-PKC signaling pathway.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Kisspeptinas/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Invasividade Neoplásica , Metástase Neoplásica
18.
BMC Biotechnol ; 20(1): 7, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992276

RESUMO

BACKGROUND: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.


Assuntos
Anticorpos Antibacterianos/metabolismo , Toxinas Bacterianas/genética , Baculoviridae/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio/genética , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/metabolismo , Proteínas Recombinantes/administração & dosagem , Fosfolipases Tipo C/genética , Animais , Anticorpos Monoclonais/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Células CACO-2 , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Infecções por Clostridium/metabolismo , Clostridium perfringens/genética , Clostridium perfringens/imunologia , Sequência Consenso , Humanos , Imunização , Camundongos , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/química , Fosfolipases Tipo C/imunologia , Fosfolipases Tipo C/metabolismo
19.
Eur J Med Chem ; 187: 111919, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31810783

RESUMO

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a promising target for new anticancer treatment. Herein, we report our work in the discovery of novel drug-like PC-PLC inhibitors. Virtual screening led to the identification of promising hits from four different structural series that contain the molecular scaffold of benzenesulphonamides (10), pyrido[3,4-b]indoles (22), morpholinobenzoic acid (84) and benzamidobenzoic acid (80). 164 structural analogues were tested to investigate the chemical space around the hit series and to generate preliminary structurally activity relationships (SAR). Two of the pyrido[3,4-b]indoles (22_10 and 22_15) had comparable or better potency as D609, an established but non-drug-like PC-PLC inhibitor. Furthermore, three morpholinobenzoic acids (84, 84_4 and 84_5) had superior potency than D609. Therefore, this study paves the way towards the development of drug-like PL-PLC inhibitors as potential anticancer agents.


Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Ácido Benzoico/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Ácido Benzoico/síntese química , Ácido Benzoico/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismo
20.
Am J Physiol Regul Integr Comp Physiol ; 318(1): R38-R48, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31596114

RESUMO

Astrocytes generate robust cytoplasmic calcium signals in response to reductions in extracellular glucose. This calcium signal, in turn, drives purinergic gliotransmission, which controls the activity of catecholaminergic (CA) neurons in the hindbrain. These CA neurons are critical to triggering glucose counter-regulatory responses (CRRs) that, ultimately, restore glucose homeostasis via endocrine and behavioral means. Although the astrocyte low-glucose sensor involvement in CRR has been accepted, it is not clear how astrocytes produce an increase in intracellular calcium in response to a decrease in glucose. Our ex vivo calcium imaging studies of hindbrain astrocytes show that the glucose type 2 transporter (GLUT2) is an essential feature of the astrocyte glucosensor mechanism. Coimmunoprecipitation assays reveal that the recombinant GLUT2 binds directly with the recombinant Gq protein subunit that activates phospholipase C (PLC). Additional calcium imaging studies suggest that GLUT2 may be connected to a PLC-endoplasmic reticular-calcium release mechanism, which is amplified by calcium-induced calcium release (CICR). Collectively, these data help outline a potential mechanism used by astrocytes to convert information regarding low-glucose levels into intracellular changes that ultimately regulate the CRR.


Assuntos
Astrócitos/fisiologia , Cálcio/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Rombencéfalo/citologia , Fosfolipases Tipo C/metabolismo , Anilidas/farmacologia , Animais , Antioxidantes/farmacologia , Compostos de Boro/farmacologia , Cálcio/farmacologia , Dantroleno/farmacologia , Estrenos/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Florizina/farmacologia , Pró-Fármacos , Pirrolidinonas/farmacologia , Quercetina/farmacologia , Ratos , Ratos Long-Evans , Fosfolipases Tipo C/antagonistas & inibidores
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