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1.
Sci Rep ; 13(1): 11694, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37474559

RESUMO

Fungal pathogens are a major cause of death, especially among immunocompromised patients. Therapies against invasive fungal infections are restricted to a few antifungals; therefore, novel therapies are necessary. Nutritional signaling and regulation are important for pathogen establishment in the host. In Cryptococcus neoformans, the causal agent of fungal meningitis, amino acid uptake and biosynthesis are major aspects of nutritional adaptation. Disruptions in these pathways lead to virulence attenuation in an animal model of infection, especially for sulfur uptake and sulfur amino acid biosynthesis. Deletion of Cys3, the main transcription factor that controls these pathways, is the most deleterious gene knockout in vitro and in vivo, making it an important target for further application. Previously, we demonstrated that Cys3 is part of a protein complex, including calcineurin, which is necessary to maintain high Cys3 protein levels during sulfur uptake and sulfur amino acid biosynthesis. In the current study, other aspects of Cys3 regulation are explored. Two lines of evidence suggest that C. neoformans Cys3 does not interact with the F-box WD40 protein annotated as Met30, indicating another protein mediates Cys3 ubiquitin degradation. However, we found another level of Cys3 regulation, which involves protein interactions between Cys3 and ATP sulfurylase (MET3 gene). We show that an atypical leucine zipper at the N-terminus of ATP sulfurylase is essential for physical interaction with Cys3 and calcineurin. Our data suggests that Cys3 and ATP sulfurylase interact to regulate Cys3 transcriptional activity. This work evidences the complexity involved in the regulation of a transcription factor essential for the sulfur metabolism, which is a biological process important to nutritional adaptation, oxidative stress response, nucleic acid stability, and methylation. This information may be useful in designing novel therapies against fungal infections.


Assuntos
Aminoácidos Sulfúricos , Criptococose , Cryptococcus neoformans , Animais , Calcineurina/metabolismo , Zíper de Leucina , Sulfato Adenililtransferase/metabolismo , Fatores de Transcrição/metabolismo , Criptococose/microbiologia , Aminoácidos Sulfúricos/metabolismo , Enxofre/metabolismo , Proteínas Fúngicas/metabolismo
2.
Plant Cell Environ ; 45(11): 3305-3321, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36041917

RESUMO

Freezing stress is a major limiting factor in crop production. To increase frost-hardiness of crops via breeding, deciphering the genes conferring freezing-tolerance is vital. Potato cultivars (Solanum tuberosum) are generally freezing-sensitive, but some potato wild species are freezing-tolerant, including Solanum commersonii, Solanum malmeanum and Solanum acaule. However, the underlying molecular mechanisms conferring the freezing-tolerance to the wild species remain to be deciphered. In this study, five representative genotypes of the above-mentioned species with distinct freezing-tolerance were investigated. Comparative transcriptomics analysis showed that SaCBL1-like (calcineurin B-like protein) was upregulated substantially in all of the freezing-tolerant genotypes. Transgenic overexpression and known-down lines of SaCBL1-like were examined. SaCBL1-like was shown to confer freezing-tolerance without significantly impacting main agricultural traits. A functional mechanism analysis showed that SaCBL1-like increases the expression of the C-repeat binding factor-regulon as well as causes a prolonged higher expression of CBF1 after exposure to cold conditions. Furthermore, SaCBL1-like was found to only interact with SaCIPK3-1 (CBL-interacting protein kinase) among all apparent cold-responsive SaCIPKs. Our study identifies SaCBL1-like to play a vital role in conferring freezing tolerance in potato, which may provide a basis for a targeted potato breeding for frost-hardiness.


Assuntos
Solanum tuberosum , Solanum , Calcineurina/genética , Calcineurina/metabolismo , Congelamento , Proteínas Quinases/metabolismo , Solanum/metabolismo , Solanum tuberosum/metabolismo , Transcriptoma/genética
3.
PLoS One ; 17(3): e0265777, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35303047

RESUMO

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.


Assuntos
Candida glabrata , Equinocandinas , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Sistemas CRISPR-Cas/genética , Calcineurina/metabolismo , Candida glabrata/genética , Candida glabrata/metabolismo , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Equinocandinas/uso terapêutico , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zinco/metabolismo , Dedos de Zinco
4.
J Alzheimers Dis ; 84(4): 1415-1430, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34719501

RESUMO

One of the changes found in the brain in Alzheimer's disease (AD) is increased calpain, derived from calcium dysregulation, oxidative stress, and/or neuroinflammation, which are all assumed to be basic pillars in neurodegenerative diseases. The role of calpain in synaptic plasticity, neuronal death, and AD has been discussed in some reviews. However, astrocytic calpain changes sometimes appear to be secondary and consequent to neuronal damage in AD. Herein, we explore the possibility of calpain-mediated astroglial reactivity in AD, both preceding and during the amyloid phase. We discuss the types of brain calpains but focus the review on calpains 1 and 2 and some important targets in astrocytes. We address the signaling involved in controlling calpain expression, mainly involving p38/mitogen-activated protein kinase and calcineurin, as well as how calpain regulates the expression of proteins involved in astroglial reactivity through calcineurin and cyclin-dependent kinase 5. Throughout the text, we have tried to provide evidence of the connection between the alterations caused by calpain and the metabolic changes associated with AD. In addition, we discuss the possibility that calpain mediates amyloid-ß clearance in astrocytes, as opposed to amyloid-ß accumulation in neurons.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Calpaína/metabolismo , Plasticidade Neuronal , Doença de Alzheimer/patologia , Animais , Astrócitos/patologia , Calcineurina/metabolismo , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Humanos , Doenças Neuroinflamatórias/metabolismo
5.
Biomolecules ; 11(9)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34572535

RESUMO

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.


Assuntos
Calcineurina/metabolismo , Simulação por Computador , Espaço Intracelular/parasitologia , Leishmania/patogenicidade , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Sequência de Aminoácidos , Calcineurina/química , Calpaína/metabolismo , Sequência Conservada , Humanos , Imunofilinas/metabolismo , Imunossupressores/farmacologia , Ácido Mirístico/metabolismo , Fosforilação , Domínios Proteicos , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Frações Subcelulares/metabolismo
6.
Int J Sports Med ; 42(8): 694-702, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33321522

RESUMO

Digoxin is a cardiotonic that increases the cardiac output without causing deleterious effects on heart, as well as improves the left ventricular performance during physical exercise. We tested whether the association between chronic digoxin administration and aerobic interval training (AIT) promotes beneficial cardiovascular adaptations by improving the myocardial contractility and calcium (Ca2+) handling. Male Wistar rats were randomly assigned to sedentary control (C), interval training (T), sedentary digoxin (DIGO) and T associated to digoxin (TDIGO). AIT was performed on a treadmill (1h/day, 5 days/week) for 60 days, consisting of successive 8-min periods at 80% and 20% of VO2máx for 2 min. Digoxin was administered by orogastric gavage for 60 days. Left ventricle samples were collected to analysis of Ca2+ handling proteins; contractility and Ca2+ handling were performed on isolated cardiomyocytes. TDIGO group had a greater elevation in fractional shortening (44%) than DIGO, suggesting a cardiomyocyte contractile improvement. In addition, T or TDIGO groups showed no change in cardiomyocytes properties after Fura2-acetoxymethyl ester, as well as in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban and calcineurin expressions. The main findings indicate that association of digoxin and aerobic interval training improved the cardiomyocyte contractile function, but these effects seem to be unrelated to Ca2+ handling.


Assuntos
Cálcio/metabolismo , Cardiotônicos/farmacologia , Digoxina/farmacologia , Treinamento Intervalado de Alta Intensidade/métodos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/administração & dosagem , Digoxina/administração & dosagem , Teste de Esforço/métodos , Ventrículos do Coração/metabolismo , Ácido Láctico/sangue , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Condicionamento Físico Animal/métodos , Distribuição Aleatória , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Comportamento Sedentário
7.
Dev Biol ; 469: 86-95, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33058830

RESUMO

During digit development, the correct balance of chondrogenic signals ensures the recruitment of undifferentiated cells into the cartilage lineage or the maintenance of cells at the undifferentiated stage. WNT/ß catenin maintains the pool of progenitor cells, whereas TGFß signalling promotes cartilage differentiation by inducing Sox9 expression. Moreover, WNT5A promotes the degradation of ß catenin during mouse limb development. Although these mechanisms are well established, it is still unknown whether the signalling pathway downstream WNT5A is also involved in early chondrogenesis during digit formation. Thus, the aim of this study was to determine the role of WNT5A during the recruitment of progenitor cells during digit development. Our results showed that WNT5A activated calcium (Ca2+) release in the undifferentiated region during digit development. Further, the blockade of Ca2+ release or calcineurin (CaN) or nuclear factor of activated T-cells (NFAT) functions resulted in an inhibition of cartilage differentiation. Together, our results demonstrate that non canonical WNT5A-Ca2+-CaN-NFAT signalling plays a key role during embryonic digit development in vivo promoting the competence for chondrogenic signals and also acts as a permissive factor for chondrogenesis independently of cell death mechanisms.


Assuntos
Sinalização do Cálcio , Condrogênese , Fatores de Transcrição NFATC/metabolismo , Dedos do Pé/embriologia , Proteína Wnt-5a/fisiologia , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Embrião de Galinha , Extremidades/embriologia , Fatores de Transcrição SOX9/metabolismo
8.
Sci Rep ; 10(1): 9019, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488204

RESUMO

Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the ß isoform of CaMKII controls the bidirectional inversion of plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in cerebellar cortex. Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how ß CaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that ß CaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the ß isoform of the kinase to regulate bidirectional plasticity at these synapses.


Assuntos
Actinas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebelar/citologia , Plasticidade Neuronal/fisiologia , Células de Purkinje/fisiologia , Animais , Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Córtex Cerebelar/fisiologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos Knockout , Modelos Biológicos , Fosforilação , Células de Purkinje/citologia , Receptores de AMPA/metabolismo
9.
Cell Calcium ; 89: 102211, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32422433

RESUMO

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.


Assuntos
Anoctamina-1/metabolismo , Calcineurina/metabolismo , Cálcio/farmacologia , Proteína 1A de Ligação a Tacrolimo/metabolismo , Ciclosporina/farmacologia , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Sirolimo/farmacologia , Tacrolimo/farmacologia
10.
Mol Neurobiol ; 57(1): 502-512, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31388960

RESUMO

Although the pathogenesis of Alzheimer's disease (AD) remains unclear, some molecular aspects that precede or accompany the deposit of ß-amyloid in senile plaques attract attention, such as calcium dysregulation and neuroinflammation. It has been suggested that the Ca2+/calmodulin-dependent protein phosphatase, calcineurin (CaN), plays an important role in AD pathogenesis. We hypothesized that CaN activation is involved in the inflammatory changes observed in the streptozotocin (STZ)-induced model of AD. We investigated hippocampal inflammatory and CaN changes in Wistar rats in two moments after intracerebroventricular STZ administration: in the first week (early) and fourth week (later on). We found an early (at 1 week) and persistent (at fourth week) increment in the subunit A of CaN, as well as an increase in the major 48 kDa fragment of this subunit. Glial and inflammatory activation were confirmed by changes of IBA-1, TLR-4, glial fibrillary acidic protein (GFAP), and S100B in the hippocampus. Augmented CaN activity was accompanied by reduced phosphorylation of the pro-apoptotic protein BAD, at Ser 136. Importantly, we found an increase in the nuclear translocation of NFAT4 (more associated to astroglial reactivity) in the hippocampus at 1 and 4 weeks in this model. NFAT3 (more associated with neuronal activation) exhibited an early increase, but decreased later on. Taken together, these data contribute to the understanding of neurochemical changes in the STZ model of sporadic AD, and may explain the persistent inflammatory response in AD, which might occur via the proteolytic activation of CaN, and signaling of NFAT mediated by isoform 4, in activated astrocytes.


Assuntos
Calcineurina/metabolismo , Demência/induzido quimicamente , Demência/patologia , Hipocampo/patologia , Inflamação/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Demência/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/metabolismo , Injeções Intraventriculares , Masculino , Microglia/patologia , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Estreptozocina/administração & dosagem , Proteína de Morte Celular Associada a bcl/metabolismo
11.
Sci Rep ; 9(1): 11923, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417135

RESUMO

Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.


Assuntos
Aminoácidos Sulfúricos/biossíntese , Vias Biossintéticas , Calcineurina/metabolismo , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Vias Biossintéticas/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/patogenicidade , Regulação Fúngica da Expressão Gênica , Ontologia Genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Estado Nutricional , Transporte Proteico , Proteômica , Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Virulência/genética
12.
Biochem Pharmacol ; 161: 26-36, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615862

RESUMO

The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110 min of perfusion; 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10 min of R; 4) SB + a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca2+ response (CaR), Ca2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations.


Assuntos
Calcineurina/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anticorpos/farmacologia , Preparação de Coração Isolado/métodos , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
13.
Mol Neurobiol ; 55(10): 7949-7960, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29488138

RESUMO

Failure in fear extinction is one of the more troublesome characteristics of posttraumatic stress disorder (PTSD). Cotinine facilitates fear memory extinction and reduces depressive-like behavior when administered 24 h after fear conditioning in mice. In this study, it was investigated the behavioral and molecular effects of cotinine, and other antidepressant preparations infused intranasally. Intranasal (IN) cotinine, IN krill oil, IN cotinine plus krill oil, and oral sertraline were evaluated on depressive-like behavior and fear retention and extinction after fear conditioning in C57BL/6 mice. Since calcineurin A has been involved in facilitating fear extinction in rodents, we also investigated changes of calcineurin in the hippocampus, a region key on contextual fear extinction. Short-term treatment with cotinine formulations was superior to krill oil and oral sertraline in reducing depressive-like behavior and fear consolidation and enhancing contextual fear memory extinction in mice. IN krill oil slowed the extinction of fear. IN cotinine preparations increased the levels of calcineurin A in the hippocampus of conditioned mice. In the light of the results, the future investigation of the use of IN cotinine preparations for the extinction of contextual fear memory and treatment of treatment-resistant depression (TRD) in PTSD is discussed.


Assuntos
Calcineurina/metabolismo , Cotinina/farmacologia , Depressão/tratamento farmacológico , Depressão/psicologia , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Hipocampo/metabolismo , Óleos/farmacologia , Administração Intranasal , Animais , Comportamento Animal , Condicionamento Psicológico , Cotinina/administração & dosagem , Cotinina/uso terapêutico , Euphausiacea/química , Camundongos Endogâmicos C57BL , Modelos Biológicos , Óleos/administração & dosagem , Sertralina/farmacologia
14.
Biochim Biophys Acta Mol Basis Dis ; 1864(5 Pt A): 1653-1662, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29486284

RESUMO

Skeletal muscle plays a central role in insulin-controlled glucose homeostasis. The molecular mechanisms related to insulin resistance in this tissue are incompletely understood. Herpud1 is an endoplasmic reticulum membrane protein that maintains intracellular Ca2+ homeostasis under stress conditions. It has recently been reported that Herpud1-knockout mice display intolerance to a glucose load without showing altered insulin secretion. The functions of Herpud1 in skeletal muscle also remain unknown. Based on these findings, we propose that Herpud1 is necessary for insulin-dependent glucose disposal in skeletal muscle. Here we show that Herpud1 silencing decreased insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane, and Akt Ser473 phosphorylation in cultured L6 myotubes. A decrease in insulin-induced Akt Ser473 phosphorylation was observed in soleus but not in extensor digitorum longus muscle samples from Herpud1-knockout mice. Herpud1 knockdown increased the IP3R-dependent cytosolic Ca2+ response and the activity of Ca2+-dependent serine/threonine phosphatase calcineurin in L6 cells. Calcineurin decreased insulin-dependent Akt phosphorylation and glucose uptake. Moreover, calcineurin inhibition restored the insulin response in Herpud1-depleted L6 cells. Based on these findings, we conclude that Herpud1 is necessary for adequate insulin-induced glucose uptake due to its role in Ca2+/calcineurin regulation in L6 myotubes.


Assuntos
Calcineurina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Calcineurina/genética , Glucose/genética , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética
15.
Neuroscience ; 372: 74-86, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29292080

RESUMO

Striatal dopamine D2 receptors activate the PLC → IP3 → Calcineurin-signaling pathway to modulate the neural excitability of En+ Medium-sized Spiny GABAergic neurons (MSN) through the regulation of L-type Ca2+ channels. Presynaptic dopaminergic D2 receptors modulate GABA release at striatopallidal terminals through L-type Ca2+ channels as well, but their signaling pathway is still undetermined. Since D2 receptors are Gi/o-coupled and negatively modulate adenylyl cyclase (AC), we investigated whether presynaptic D2 receptors modulate GABA release through the same signaling cascade that controls excitability in the striatum or by the inhibition of AC and decreased PKA activity. Activation of D2 receptors stimulated formation of [3H]IP1 and decreased Forskolin-stimulated [3H]cAMP accumulation in synaptosomes from rat Globus Pallidus. D2 receptor activation with Quinpirole in the presence of L 745,870 decreased, in a dose-dependent manner, K+-induced [3H]GABA release in pallidal slices. The effect was prevented by the pharmacological blockade of Gi/o ßγ subunit effects with Gallein, PLC with U 73122, IP3 receptor activation with 4-APB, Calcineurin with FK506. In addition, when release was stimulated with Forskolin to activate AC, D2 receptors also decreased K+-induced [3H]GABA release, an effect occluded with the effect of the blockade of PKA with H89 or stimulation of release with the cAMP analog 8-Br-cAMP. These data indicate that D2 receptors modulate [3H]GABA release at striatopallidal terminals by activating the PLC → IP3 → Calcineurin-signaling cascade, the same one that modulates excitability in soma. Additionally, D2 receptors inhibit release when AC is active. Both mechanisms appear to converge to regulate the activity of presynaptic L-type Ca2+ channels.


Assuntos
Corpo Estriado/metabolismo , Globo Pálido/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D2/metabolismo , Ácido gama-Aminobutírico/metabolismo , Adenilil Ciclases/metabolismo , Animais , Calcineurina/metabolismo , Corpo Estriado/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Globo Pálido/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Fosfoinositídeo Fosfolipase C/metabolismo , Potássio/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Trítio
16.
J Photochem Photobiol B ; 178: 61-68, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29112891

RESUMO

The use of a broad spectrum sunscreen is considered one of the main and most popular measures for preventing the damaging effects of ultraviolet radiation (UVR) on the skin. In this study we have developed a novel in vitro method to assess sunscreens efficacy to protect calcineurin enzyme activity, a skin cell marker. The photoprotective efficacy of sunscreen products was assessed by measuring the UV-A1 radiation-induced depletion of calcineurin (Cn) enzyme activity in primary neonatal human dermal fibroblast (HDFn) cell lysates. After exposure to 24J/cm2 UV-A1 radiation, the sunscreens containing larger amounts of UV-A1 filters (brand B), the astaxanthin (UV-A1 absorber) and the Tinosorb® M (UV-A1 absorber) were capable of preventing loss of Cn activity when compared to the sunscreens formulations of brand A (low concentration of UV-A1 filters), with the Garcinia brasiliensis extract (UV-B absorber) and with the unprotected cell lysate and exposed to irradiation (Irradiated Control - IC). The Cn activity assay is a reproducible, accurate and selective technique for evaluating the effectiveness of sunscreens against the effects of UV-A1 radiation. The developed method showed that calcineurin activity have the potential to act as a biological indicator of UV-A1 radiation-induced damages in skin and the assay might be used to assess the efficacy of sunscreens agents and plant extracts prior to in vivo tests.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Protetores Solares/farmacologia , Raios Ultravioleta/efeitos adversos , Biomarcadores/metabolismo , Calcineurina/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Recém-Nascido , Pele/citologia , Pele/efeitos da radiação
17.
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
18.
J Mol Neurosci ; 64(1): 111-116, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29214423

RESUMO

In this study, we evaluated the expression profile changes of genes that intervene in the calcium signaling pathway, in young and adult Wistar rats, using the animal model of neonatal lesion in ventral hippocampus (NLVH) (a recognized animal model for schizophrenia) and compared to the group of control animals (Sham). Through microarray technology, gene expression profiles were obtained from the three brain areas (nucleus accumbens, prefrontal cortex, and hippocampus) of young male Wistar rats (45 days) and adults (90 days) whether or not subjected to NLVH. The calcium signaling pathway reported a greater number of differentially expressed genes with z-score two values, > 2 (over-expression) and < - 2 (under-expression), in the three evaluated areas. The comparative analyses of this approach were performed in juvenile and adult rats with ventral hippocampal lesion in neonate rats (NLVH). NLVH influenced change expressions in various genes involved in Ca2+ homeostasis, including Cacna1d, Atp2a2, Adcy2, Ppp3cb, and Ptk2b. The expression of Adcy2, Ppp3cb, and Ptk2b genes changed in both age groups; therefore, the study of gene expression profiles between juvenile and adult rats may help to understand the molecular mechanisms of schizophrenia.


Assuntos
Sinalização do Cálcio/genética , Esquizofrenia/genética , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Esquizofrenia/metabolismo
19.
PLoS One ; 12(9): e0184724, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28886186

RESUMO

Calsequestrin-2 (CASQ2) is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. Previously, we demonstrated that MEF-2 and SRF binding sites within the human CASQ2 gene (hCASQ2) promoter region are functional in neonatal cardiomyocytes. In this work, we investigated if the calcineurin/NFAT pathway regulates hCASQ2 expression in neonatal cardiomyocytes. The inhibition of NFAT dephosphorylation with CsA or INCA-6, reduced both the luciferase activity of hCASQ2 promoter constructs (-3102/+176 bp and -288/+176 bp) and the CASQ2 mRNA levels in neonatal rat cardiomyocytes. Additionally, NFATc1 and NFATc3 over-expressing neonatal cardiomyocytes showed a 2-3-fold increase in luciferase activity of both hCASQ2 promoter constructs, which was prevented by CsA treatment. Site-directed mutagenesis of the -133 bp MEF-2 binding site prevented trans-activation of hCASQ2 promoter constructs induced by NFAT overexpression. Chromatin Immunoprecipitation (ChIP) assays revealed NFAT and MEF-2 enrichment within the -288 bp to +76 bp of the hCASQ2 gene promoter. Besides, a direct interaction between NFAT and MEF-2 proteins was demonstrated by protein co-immunoprecipitation experiments. Taken together, these data demonstrate that NFAT interacts with MEF-2 bound to the -133 bp binding site at the hCASQ2 gene promoter. In conclusion, in this work, we demonstrate that the Ca2+-calcineurin/NFAT pathway modulates the transcription of the hCASQ2 gene in neonatal cardiomyocytes.


Assuntos
Calsequestrina/genética , Calsequestrina/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Western Blotting , Calcineurina/genética , Calcineurina/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
J Mol Cell Cardiol ; 103: 121-136, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28007541

RESUMO

The calcium-activated protein phosphatase, calcineurin, lies at the intersection of protein phosphorylation and calcium signaling cascades, where it provides an essential nodal point for coordination between these two fundamental modes of intracellular communication. In excitatory cells, such as neurons and cardiomyocytes, that experience rapid and frequent changes in cytoplasmic calcium, calcineurin protein levels are exceptionally high, suggesting that these cells require high levels of calcineurin activity. Yet, it is widely recognized that excessive activation of calcineurin in the heart contributes to pathological hypertrophic remodeling and the progression to failure. How does a calcium activated enzyme function in the calcium-rich environment of the continuously contracting heart without pathological consequences? This review will discuss the wide range of calcineurin substrates relevant to cardiovascular health and the mechanisms calcineurin uses to find and act on appropriate substrates in the appropriate location while potentially avoiding others. Fundamental differences in calcineurin signaling in neonatal verses adult cardiomyocytes will be addressed as well as the importance of maintaining heterogeneity in calcineurin activity across the myocardium. Finally, we will discuss how circadian oscillations in calcineurin activity may facilitate integration with other essential but conflicting processes, allowing a healthy heart to reap the benefits of calcineurin signaling while avoiding the detrimental consequences of sustained calcineurin activity that can culminate in heart failure.


Assuntos
Calcineurina/genética , Calcineurina/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Transdução de Sinais , Animais , Calcineurina/química , Inibidores de Calcineurina , Proteínas de Transporte/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Coração/efeitos dos fármacos , Humanos , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Fatores de Transcrição/metabolismo
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