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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.
J Physiol Pharmacol ; 70(1)2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31172971

RESUMO

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Mucosa Respiratória/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
3.
mSphere ; 4(1)2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728284

RESUMO

Calcium is a critically important secondary messenger of intracellular signal transduction in eukaryotes but must be maintained at low levels in the cytoplasm of resting cells to avoid toxicity. This is achieved by several pumps that actively transport excess cytoplasmic Ca2+ out of the cell across the plasma membrane and into other intracellular compartments. In fungi, the vacuole serves as the major storage site for excess Ca2+, with two systems actively transporting cytoplasmic calcium ions into the vacuole. The H+/Ca2+ exchanger, Vcx1p, harnesses the proton-motive force across the vacuolar membrane (generated by the V-ATPase) to drive Ca2+ transport, while the P-type ATPase Pmc1p uses ATP hydrolysis to translocate Ca2+ into the vacuole. Ca2+-dependent signaling is required for the prevalent human fungal pathogen Candida albicans to endure exposure to the azole antifungals and to cause disease within the mammalian host. The purpose of this study was to determine if the Pmc1p or Vcx1p Ca2+ pumps are required for C. albicans pathogenicity and if these pumps impact antifungal resistance. Our results indicate that Pmc1p is required by C. albicans to transition from yeast to hyphal growth, to form biofilms in vitro, and to cause disease in a mouse model of disseminated infection. Moreover, loss of Pmc1p function appears to enhance C. albicans azole tolerance in a temperature-dependent manner.IMPORTANCE Maintenance of Ca2+ homeostasis is important for fungal cells to respond to a multitude of stresses, as well as antifungal treatment, and for virulence in animal models. Here, we demonstrate that a P-type ATPase, Pmc1p, is required for Candida albicans to respond to a variety of stresses, affects azole susceptibility, and is required to sustain tissue invasive hyphal growth and to cause disease in a mouse model of disseminated infection. Defining the mechanisms responsible for maintaining proper Ca2+ homeostasis in this important human pathogen can ultimately provide opportunities to devise new chemotherapeutic interventions that dysregulate intracellular signaling and induce Ca2+ toxicity.


Assuntos
Candida albicans/genética , Candida albicans/patogenicidade , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Antifúngicos/farmacologia , Azóis/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candidíase Invasiva/microbiologia , Farmacorresistência Fúngica , Proteínas Fúngicas , Hifas/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Temperatura Ambiente , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Virulência
4.
Cell Commun Signal ; 17(1): 7, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665402

RESUMO

Through a genome-wide screen we have identified calcium-tolerant deletion mutants for five genes in the budding yeast Saccharomyces cerevisiae. In addition to CNB1 and RCN1 that are known to play a role in the calcium signalling pathway, the protein kinase gene CMK2, the sphingolipid homeostasis-related gene ORM2 and the gene SIF2 encoding the WD40 repeat-containing subunit of Set3C histone deacetylase complex are involved in the calcium sensitivity of yeast cells to extracellular calcium. Cmk2 and the transcription factor Crz1 have opposite functions in the response of yeast cells to calcium stress. Deletion of CMK2 elevates the level of calcium/calcineurin signalling and increases the expression level of PMR1 and PMC1, which is dependent on Crz1. Effects of Cmk2 on calcium sensitivity and calcium/calcineurin signalling are dependent on its kinase activity. Therefore, Cmk2 is a negative feedback controller of the calcium/calcineurin signalling pathway. Furthermore, the cmk2 crz1 double deletion mutant is more resistant than the crz1 deletion mutant, suggesting that Cmk2 has an additional Crz1-independent role in promoting calcium tolerance.


Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biocatálise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Deleção de Genes , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Toxicol Lett ; 295: 162-172, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29935216

RESUMO

In pancreatic ß cells, which produce and secrete insulin, Ca2+ signals contribute to insulin production and secretion. Bisphenol A (BPA) and octylphenol (OP) are reported to increase plasma insulin levels and insulin transcription factors, but regulation of plasma glucose levels did not decrease proportionally to the insulin increase. We hypothesized that BPA and OP disrupt calcium homeostasis resulting in insulin resistance through induction of endoplasmic reticulum (ER) stress. BPA and OP treatment leads to survival of pancreatic ß cells against streptozotocin, but despite an increased insulin level, serum glucose regulation is not properly regulated. The expression of genes involved in transporting calcium ions to the cytosol and ER decreased while the expression of those affecting the removal of calcium from the cytosol and ER increased. Depletion of calcium from the ER leads to ER stress and can induce insulin resistance. Insulin resistance is also confirmed by insulin-responsive gene, such as glucose transporter 4 (GLUT4) and IRS2, expression. Taken together, these results imply that disruption of calcium homeostasis by BPA and OP induces ER stress and leads to insulin resistance, especially in a streptozotocin (STZ) -induced type 1 diabetes mellitus model.


Assuntos
Compostos Benzidrílicos/toxicidade , Cálcio/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 1/induzido quimicamente , Disruptores Endócrinos/toxicidade , Resistência à Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Fenóis/toxicidade , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Morte Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/patologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Homeostase , Insulina/sangue , Proteínas Substratos do Receptor de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos ICR , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Estreptozocina , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo
6.
Cell Microbiol ; 20(2)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29113016

RESUMO

Cryptococcus neoformans is a basidiomycetous yeast and the cause of cryptococcosis in immunocompromised individuals. The most severe form of the disease is meningoencephalitis, which is one of the leading causes of death in HIV/AIDS patients. In order to access the central nervous system, C. neoformans relies on the activity of certain virulence factors such as urease, which allows transmigration through the blood-brain barrier. In this study, we demonstrate that the calcium transporter Pmc1 enables C. neoformans to penetrate the central nervous system, because the pmc1 null mutant failed to infect and to survive within the brain parenchyma in a murine systemic infection model. To investigate potential alterations in transmigration pathways in these mutants, global expression profiling of the pmc1 mutant strain was undertaken, and genes associated with urease, the Ca2+ -calcineurin pathway, and capsule assembly were identified as being differentially expressed. Also, a decrease in urease activity was observed in the calcium transporter null mutants. Finally, we demonstrate that the transcription factor Crz1 regulates urease activity and that the Ca2+ -calcineurin signalling pathway positively controls the transcription of calcium transporter genes and factors related to transmigration.


Assuntos
Sistema Nervoso Central/microbiologia , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Calcineurina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Criptococose/metabolismo , Criptococose/microbiologia , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Meningoencefalite/metabolismo , Meningoencefalite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Vacúolos/metabolismo , Vacúolos/microbiologia , Virulência/fisiologia , Fatores de Virulência/metabolismo
7.
Arch Physiol Biochem ; 124(1): 45-53, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28758804

RESUMO

Erythrocyte membrane is a suitable model to study various metabolic and physiological functions as it undergoes variety of biochemical changes during aging. An age-dependent modulatory effect of rapamycin on erythrocyte membrane functions is completely unknown. Therefore, the present study was undertaken to investigate the effect of rapamycin on age-dependent impaired activities of transporters/exchangers, altered levels of redox biomarkers, viz. protein carbonyl (PC), lipid hydroperoxides (LHs), total thiol (-SH), sialic acid (SA) and intracellular calcium ion [Ca2+]i, and osmotic fragility of erythrocyte membrane. A significant reduction in membrane-bound activities of Na+/K+-ATPase (NKA) and Ca2+-ATPase (PMCA), and levels of -SH and SA was observed along with a simultaneous induction in Na+/H+ exchanger (NHE) activity and levels of [Ca2+]i, PC, LH and osmotic fragility in old-aged rats. Rapamycin was found to be a promising age-delaying drug that significantly reversed the aging-induced impaired activities of membrane-bound ATPases and altered levels of redox biomarkers.


Assuntos
Envelhecimento/efeitos dos fármacos , Antioxidantes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Sirolimo/farmacologia , Envelhecimento/sangue , Envelhecimento/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Biomarcadores/sangue , Biomarcadores/metabolismo , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/antagonistas & inibidores , Peróxidos Lipídicos/sangue , Peróxidos Lipídicos/metabolismo , Masculino , Ácido N-Acetilneuramínico/sangue , Ácido N-Acetilneuramínico/metabolismo , Fragilidade Osmótica/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Distribuição Aleatória , Ratos Wistar , Reprodutibilidade dos Testes , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo
8.
Cell Mol Life Sci ; 75(8): 1461-1482, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29085954

RESUMO

Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.


Assuntos
Oxalato de Cálcio/metabolismo , Cálculos Renais/metabolismo , Rim/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Oxalato de Cálcio/química , Cristalização , Cães , Expressão Gênica , Ontologia Genética , Imunoprecipitação , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/química , Rim/patologia , Cálculos Renais/química , Cálculos Renais/patologia , Células Madin Darby de Rim Canino , Anotação de Sequência Molecular , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
9.
Mycopathologia ; 183(2): 315-327, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29086141

RESUMO

The vacuolar-type H+-ATPase (V-ATPase) is essential for many cell processes. Our previous study has demonstrated that Tfp1 is a putative subunit of V-ATPase, loss of which causes disorders in calcium homeostasis and decreased resistance to oxidative stress. In this study, we found that further deletion of PMC1, a vacuolar calcium pump, in tfp1∆/∆ mutant led to more severe dysregulation of calcium homeostasis. Besides, the tfp1∆/∆pmc1∆/∆ mutant was more sensitive to H2O2 and had a higher ROS level. As is known, V-ATPase mutants are sensitive to NaCl, and PMC1 mutant is resistant against NaCl. However, the tfp1∆/∆pmc1∆/∆ mutant exhibited sensitivity to NaCl. Mechanism study demonstrated that their sensitivity was associated with reduced osmotic resistance caused by relatively low expression of GPD1. In addition, we first found that NaCl addition significantly declined ROS levels in tfp1∆/∆ and tfp1∆/∆pmc1∆/∆ mutants. In tfp1∆/∆ mutant, decreased ROS levels were relevant to enhanced antioxidant activities. However, in tfp1∆/∆pmc1∆/∆ mutant, reduced ROS resulted from decreased total calcium content, revealing that NaCl affected ROS levels in the two mutants through different mechanisms. Taken together, our data indicated that loss of both TFP1 and PMC1 further affected calcium homeostasis and other cellular processes in Candida albicans and provides a potential antifungal target.


Assuntos
Cálcio/metabolismo , Candida albicans/fisiologia , Homeostase , Pressão Osmótica , ATPases Transportadoras de Cálcio da Membrana Plasmática/deficiência , ATPases Translocadoras de Prótons/deficiência , Candida albicans/genética , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/análise , Cloreto de Sódio/metabolismo
10.
Proteomics ; 17(23-24)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29105980

RESUMO

Current treatments for advanced prostate cancer focus on inhibition of the androgen receptor (AR) by androgen deprivation therapy (ADT). However, complex interactions mediated by tumor suppressors, oncogenes, aberrations of AR expression, or de novo androgen production have been shown to induce the adaptive response of prostate cancer, leading to the development of castration resistant prostate cancer. In this study, we report the effects of AR antagonist, enzalutamide on the protein contents of extracellular vesicles (EVs). EVs mediate cell-to-cell communication and increasing evidence shows the role of EVs in promoting cancer survival and metastasis. We found that treatment with enzalutamide alters the secretion of EVs, one of which is a plasma membrane calcium pump, ATP2B1/PMCA ATPase, as an AR-regulated EV protein. We highlight the networks of interactions between AR, Ca2+ , and ATP2B1, where the extracellular proteins thrombospondin-1, gelsolin, and integrinß1 were previously reported as regulators for cancer progression and metastasis, indicating the potential role of EV-derived proteins in mediating calcium homoeostasis under AR inhibition by enzalutamide. Our data further highlight the cross-talk between AR signaling and EV pathways in mediating resistance toward ADT.


Assuntos
Adenocarcinoma/metabolismo , Vesículas Extracelulares/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/química , Transdução de Sinais/efeitos dos fármacos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Gelsolina/metabolismo , Humanos , Integrina beta1/metabolismo , Masculino , Feniltioidantoína/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Trombospondina 1/metabolismo , Células Tumorais Cultivadas
11.
J Biol Chem ; 292(51): 21047-21059, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29042438

RESUMO

Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both in vitro and in vivo murine models of acute pancreatitis to study renalase's effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the in vivo cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, and administering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, plasma membrane calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b-selective inhibitor worsens pancreatitis-induced injury and blocks the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Because exogenous rRNLS reduces the severity of acute pancreatitis, it has potential as a therapeutic agent.


Assuntos
Monoaminoxidase/metabolismo , Pâncreas/metabolismo , Pancreatite/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Biomarcadores/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Linhagem Celular , Ceruletídeo/toxicidade , Ativação Enzimática/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/etiologia , Hipertensão/prevenção & controle , Ligantes , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Monoaminoxidase/sangue , Monoaminoxidase/genética , Monoaminoxidase/uso terapêutico , Pâncreas/efeitos dos fármacos , Pâncreas/imunologia , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologia
12.
Neuron ; 96(4): 827-838.e9, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29056295

RESUMO

Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.


Assuntos
Basigina/metabolismo , Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Subunidades Proteicas/metabolismo
13.
J Physiol ; 595(24): 7383-7398, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29023784

RESUMO

KEY POINTS: The role of plasma membrane Ca2+ -ATPase 1 (PMCA1) in Ca2+ homeostasis and electrical stability in atrial tissue has been investigated at both organ and cellular levels in mice with cardiomyocyte-specific deletion of PMCA1 (PMCA1cko ) The PMCA1cko hearts became more susceptible to atrial arrhythmic stress conditions than PMCA1loxP/loxP hearts. PMCA1 deficiency alters cellular Ca2+ homeostasis under both baseline and stress conditions. PMCA1 is required for maintaining cellular Ca2+ homeostasis and electrical stability in murine atria under stress conditions. ABSTRACT: To determine the role of plasma membrane Ca2+ -ATPase 1 (PMCA1) in maintaining Ca2+ homeostasis and electrical stability in the atrium under physiological and stress conditions, mice with a cardiomyocyte-specific deletion of PMCA1 (PMCA1cko ) and their control littermates (PMCA1loxP/loxP ) were studied at the organ and cellular levels. At the organ level, the PMCA1cko hearts became more susceptible to atrial arrhythmias under rapid programmed electrical stimulation compared with the PMCA1loxP/loxP hearts, and such arrhythmic events became more severe under Ca2+ overload conditions. At the cellular level, the occurrence of irregular-type action potentials of PMCA1cko atrial myocytes increased significantly under Ca2+ overload conditions and/or at higher frequency of stimulation. The decay of Na+ /Ca2+ exchanger current that followed a stimulation protocol was significantly prolonged in PMCA1cko atrial myocytes under basal conditions, with Ca2+ overload leading to even greater prolongation. In conclusion, PMCA1 is required for maintaining Ca2+ homeostasis and electrical stability in the atrium. This is particularly critical during fast removal of Ca2+ from the cytosol, which is required under stress conditions.


Assuntos
Cálcio/metabolismo , Átrios do Coração/metabolismo , Potenciais da Membrana , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Animais , Função Atrial , Células Cultivadas , Átrios do Coração/citologia , Homeostase , Masculino , Camundongos , Miócitos Cardíacos/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
14.
J Clin Invest ; 127(8): 3065-3074, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28714864

RESUMO

The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus. Using CRISPR-Cas9, we fine mapped the genetic signal to an erythroid-specific enhancer of ATP2B4. Erythroid cells with a deletion of the ATP2B4 enhancer had abnormally high intracellular calcium levels. These results illustrate the power of combined transcriptomic, epigenomic, and genome-editing approaches in characterizing noncoding regulatory elements in phenotype-relevant cells. Our study supports ATP2B4 as a potential target for modulating rbc hydration in erythroid disorders and malaria infection.


Assuntos
ATPases Transportadoras de Cálcio/genética , Eritrócitos/citologia , Predisposição Genética para Doença , Malária/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Animais , Sistemas CRISPR-Cas , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Mapeamento Cromossômico , Elementos Facilitadores Genéticos , Epigenômica , Eritroblastos/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Malária/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas
15.
Int J Mol Sci ; 18(7)2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28653986

RESUMO

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is an economically important agricultural pest that is difficult to prevent and control. Scopoletin is a botanical coumarin derivative that targets Ca2+-ATPase to exert a strong acaricidal effect on carmine spider mites. In this study, the full-length cDNA sequence of a plasma membrane Ca2+-ATPase 1 gene (TcPMCA1) was cloned. The sequence contains an open reading frame of 3750 bp and encodes a putative protein of 1249 amino acids. The effects of scopoletin on TcPMCA1 expression were investigated. TcPMCA1 was significantly upregulated after it was exposed to 10%, 30%, and 50% of the lethal concentration of scopoletin. Homology modeling, molecular docking, and three-dimensional quantitative structure-activity relationships were then studied to explore the relationship between scopoletin structure and TcPMCA1-inhibiting activity of scopoletin and other 30 coumarin derivatives. Results showed that scopoletin inserts into the binding cavity and interacts with amino acid residues at the binding site of the TcPMCA1 protein through the driving forces of hydrogen bonds. Furthermore, CoMFA (comparative molecular field analysis)- and CoMSIA (comparative molecular similarity index analysis)-derived models showed that the steric and H-bond fields of these compounds exert important influences on the activities of the coumarin compounds.Notably, the C3, C6, and C7 positions in the skeletal structure of the coumarins are the most suitable active sites. This work provides insights into the mechanism underlying the interaction of scopoletin with TcPMCA1. The present results can improve the understanding on plasma membrane Ca2+-ATPase-mediated (PMCA-mediated) detoxification of scopoletin and coumarin derivatives in T. cinnabarinus, as well as provide valuable information for the design of novel PMCA-inhibiting acaricides.


Assuntos
Acaricidas/toxicidade , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Escopoletina/toxicidade , Tetranychidae/enzimologia , Regulação para Cima/efeitos dos fármacos , Acaricidas/química , Animais , Clonagem Molecular , Regulação da Expressão Gênica/efeitos dos fármacos , Simulação de Acoplamento Molecular , Filogenia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Relação Quantitativa Estrutura-Atividade , Escopoletina/química , Tetranychidae/efeitos dos fármacos , Tetranychidae/genética
16.
Physiol Rev ; 97(3): 1089-1125, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28566538

RESUMO

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/enzimologia , Doença/etiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Doença/genética , Predisposição Genética para Doença , Variação Genética , Homeostase , Humanos , Especificidade de Órgãos , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Conformação Proteica , Relação Estrutura-Atividade
17.
EMBO J ; 36(12): 1770-1787, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28487411

RESUMO

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Fatores de Processamento de RNA/metabolismo , Sinapses/fisiologia , Animais , Biologia Computacional , Perfilação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Picrotoxina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteoma/análise
18.
Int Rev Cell Mol Biol ; 332: 259-296, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28526135

RESUMO

The plasma membrane Ca2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It has 10 transmembrane helices and 2 cytosolic loops, one of which contains the catalytic center. Its most distinctive feature is a C-terminal tail that contains most of the regulatory sites including that for calmodulin. The pump is also regulated by acidic phospholipids, kinases, a dimerization process, and numerous protein interactors. In mammals, four genes code for the four basic isoforms. Isoform complexity is increased by alternative splicing of primary transcripts. Pumps 2 and 3 are expressed preferentially in the nervous system. The pumps coexist with more powerful systems that clear Ca2+ from the bulk cytosol: their role is thus the regulation of Ca2+ in selected subplasma membrane microdomains, where a number of important Ca2+-dependent enzymes interact with them. Malfunctions of the pump lead to disease phenotypes that affect the nervous system preferentially.


Assuntos
Cálcio/metabolismo , Células/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Sinalização do Cálcio , Humanos , Modelos Biológicos , Isoformas de Proteínas/metabolismo
19.
BMC Mol Biol ; 18(1): 14, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28532435

RESUMO

BACKGROUND: Along with sodium/calcium (Ca2+) exchangers, plasma membrane Ca2+ ATPases (ATP2Bs) are main regulators of intracellular Ca2+ levels. There are four ATP2B paralogs encoded by four different genes. Atp2b2 encodes the protein pump with the fastest activation, ATP2B2. In mice, the Atp2b2 transcript has several alternate transcriptional start site variants: α, ß, µ and δ. These variants are expressed in developmental and tissue specific manners. The α and ß Atp2b2 transcripts are equally expressed in the brain. αAtp2b2 is the only transcript found in the outer hair cells of young mice (Silverstein RS, Tempel BL. in Neuroscience 141:245-257, 2006). Mutations in the coding region of the mouse Atp2b2 gene indicate a narrow window for tolerated dysfunction of the ATP2B2 protein, specifically in the auditory system. This highlights the necessity of tight regulation of this gene for normal cell physiology. RESULTS: Although ATP2Bs are important regulators of Ca2+ in many cell types, little is known about their transcriptional regulation. This study identifies the proximal promoter of the αAtp2b2 transcript. Further investigations indicate that ATOH1 and EGR1 modulate promoter activity. Additionally, we report that EGR1 increases endogenous expression of Atp2b2 transcript in two cell lines. Electrophoretic mobility shift assays (EMSA) indicate that EGR1 binds to a specific site in the CpG island of the αAtp2b2 promoter. CONCLUSION: This study furthers our understanding of Atp2b2 regulation by: (I) elucidating transcriptional regulatory mechanisms for Atp2b2, and (II) identifying transcription factors that modulate expression of Atp2b2 in the brain and peripheral auditory system and (III) allows for future studies modulating gene expression of Atp2b2.


Assuntos
Córtex Auditivo/metabolismo , Encéfalo/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Regiões Promotoras Genéticas , Animais , Cálcio , Linhagem Celular , Cerebelo/metabolismo , Ilhas de CpG , Haploinsuficiência , Camundongos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcrição Genética
20.
J Bioinform Comput Biol ; 15(3): 1750011, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28506097

RESUMO

Many cellular events including electrical activity and muscle contraction are regulated and coordinated by intracellular [Formula: see text] concentration ([[Formula: see text]][Formula: see text]. In detrusor smooth muscle (DSM) cells, [[Formula: see text]]i is normally maintained at very low levels and rises transiently during signalling processes as a result of (i) influx from the extracellular space (mainly via L-type and T-type [Formula: see text] channels) and (ii) [Formula: see text] release from sarcoplasmic reticulum (SR) into the cytoplasm. Intracellular [Formula: see text] buffers, both fixed and diffusible, play a vital role in shaping the radial distribution of free [Formula: see text]. Our aim, in the work presented here, is to develop a mathematical model of [Formula: see text] buffering and diffusion and to generate [Formula: see text] transient in guinea pig DSM cells. The [Formula: see text] transient is generated using inward [Formula: see text] current that arises following voltage clamp and mediated by L-type and T-type [Formula: see text] channels. [Formula: see text] transient is obtained for different radial locations (or shells) of the DSM cytosol. This modeling study explores the levels of [[Formula: see text]]i achieved near the plasma membrane and in deeper locations. The [Formula: see text] transient generated in our model shows a high degree of similarity with experimental findings in terms of amplitude, duration and half-decay time. A number of different buffer properties such as concentration and mobility are tested for their effect on amplitude and shape of [Formula: see text] transient. The presence of fast buffer concentration in the cytosol markedly delays the rise of [[Formula: see text]]i in the core of the cell. Increase in the mobility of fast buffer slightly speeds up the redistribution of [Formula: see text]. To explore the model further, the role of plasma membrane [Formula: see text]-ATPase (PMCA) pump, sarcoplasmic/endoplasmic reticulum [Formula: see text]-ATPase (SERCA) pump and sodium calcium exchanger (NCX) on [Formula: see text] transient is studied and it is suggested that NCX may be of primary importance for the immediate lowering of [[Formula: see text]]i during the falling phase of a [Formula: see text] transient in DSM cells.


Assuntos
Sinalização do Cálcio , Modelos Biológicos , Miócitos de Músculo Liso/metabolismo , Bexiga Urinária/citologia , Animais , Tampões (Química) , Canais de Cálcio/metabolismo , Difusão , Cobaias , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Bexiga Urinária/fisiologia
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