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1.
PLoS Genet ; 16(6): e1008865, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603360

RESUMO

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.


Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Calcineurina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição Forkhead/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Grupo de Alta Mobilidade/genética , Peptidilprolil Isomerase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética
2.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
3.
Tumour Biol ; 42(7): 1010428320937863, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32686600

RESUMO

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na+/H+ exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na+/H+ exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na+/H+ exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na+/H+ exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pHi, and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Calcineurina/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Nus , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Transplante Heterólogo
4.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Genoma Bacteriano , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
Nature ; 582(7811): 271-276, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499640

RESUMO

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.


Assuntos
Calcineurina/metabolismo , Proliferação de Células , Proteínas de Homeodomínio/metabolismo , Proteína Meis1/metabolismo , Miócitos Cardíacos/citologia , Animais , Animais Recém-Nascidos , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Coração/fisiologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Miocárdio/citologia , Ligação Proteica , Regeneração
6.
Nat Med ; 26(6): 952-963, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32514169

RESUMO

In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.


Assuntos
Apolipoproteína E4/genética , Barreira Hematoencefálica/metabolismo , Calcineurina/metabolismo , Angiopatia Amiloide Cerebral/genética , Fatores de Transcrição NFATC/genética , Pericitos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Barreira Hematoencefálica/citologia , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição NFATC/metabolismo , Permeabilidade , RNA-Seq , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
PLoS One ; 15(6): e0234403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520951

RESUMO

MicroRNAs (miRNAs) are important regulators of gene expression, and their expression is associated with many physiological conditions. Here, we investigated potential associations between expression levels of miRNAs in human placenta and the onset of spontaneous term birth. Using RNA sequencing, we identified 54 miRNAs differentially expressed during spontaneous term labor compared to elective term births. Expression levels of 23 miRNAs were upregulated, whereas 31 were downregulated at least 1.5-fold. The upregulated miRNA miR-371a-5p putatively targets CPPED1, expression of which decreases during spontaneous birth. We used a luciferase reporter-based assay to test whether a miR-371a-5p mimic affected translation when it bound to the 3' untranslated region of CPPED1. In this setting, the miR-371a-5p mimic resulted in lower luciferase activity, which suggests that miR-371a-5p regulates levels of CPPED1. In conclusion, inversely correlated levels of miR-371a-5p and CPPED1 suggest a role for both in spontaneous delivery.


Assuntos
MicroRNAs/genética , Placentação/genética , Nascimento a Termo/genética , Regiões 3' não Traduzidas/genética , Adulto , Calcineurina/genética , Calcineurina/metabolismo , Parto Obstétrico , Feminino , Finlândia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , MicroRNAs/metabolismo , Placenta/metabolismo , Gravidez , Transcriptoma/genética
8.
PLoS Genet ; 16(4): e1008600, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32343701

RESUMO

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.


Assuntos
Calcineurina/metabolismo , Ciclo Celular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação para Baixo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
9.
PLoS One ; 15(3): e0230156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32134989

RESUMO

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Inanição/metabolismo
10.
Cell Prolif ; 53(3): e12774, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32034930

RESUMO

OBJECTIVES: Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. MATERIALS AND METHODS: Sprague Dawley rats were subjected to 28-day hindlimb-unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T-type CaV 3.1 channel was revealed by assessing its blocking effects. MiR-137 was identified as the upstream of CaV 3.1 channel by luciferase assay and investigated by gain/loss-of-function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV 3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. RESULTS: Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T-type CaV 3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR-137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T-type CaV 3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. CONCLUSIONS: The present work demonstrated that miR-137 and its target T-type CaV 3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.


Assuntos
Calcineurina/metabolismo , Canais de Cálcio Tipo T/metabolismo , Artérias Cerebrais/citologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Fatores de Transcrição NFATC/metabolismo , Animais , Encéfalo/irrigação sanguínea , Canais de Cálcio Tipo T/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Simulação de Ausência de Peso
11.
FASEB J ; 34(1): 1247-1269, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914663

RESUMO

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3ß, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.


Assuntos
Calcineurina/metabolismo , Fertilização , Quinase 3 da Glicogênio Sintase/metabolismo , Motilidade Espermática , Espermatozoides/enzimologia , Animais , Calcineurina/genética , Inibidores de Calcineurina/farmacologia , Epididimo/metabolismo , Epididimo/patologia , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Masculino , Camundongos , Camundongos Knockout , Tacrolimo/farmacologia
12.
J Neurosci ; 40(6): 1344-1354, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31862855

RESUMO

Interfering with memory reconsolidation or inducing memory extinction are two approaches for weakening maladaptive memories in disorders such as addiction and post-traumatic stress disorder. Both extinction and reconsolidation are regulated by intracellular protein kinases and phosphatases, and interfering with these signaling molecules can alter memory strength. The calcium-dependent protein phosphatase, calcineurin (CaN), has been implicated in both the consolidation and extinction of fear memories. However, the role of CaN in regulating drug-cue associative memories has not been investigated. Prior studies have demonstrated that plasticity at thalamo-lateral amygdala (T-LA) synapses is critically involved in the regulation of cocaine-cue memories. Therefore, in the present study, we tested the effects of LA administration of an activator of CaN, chlorogenic acid (CGA), on behavioral and electrophysiological indices of cocaine cue memory reconsolidation and extinction. Male, Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. The cue memory was then either briefly reactivated, extinguished, or not manipulated, followed immediately by LA infusion of CGA. Rats were tested 24 h later for cue-induced reinstatement, or LA slices were prepared for electrophysiological recordings. We found that intra-LA infusions of CGA following cue extinction or reconsolidation reduced cue-induced reinstatement, which was blocked by co-infusion of the CaN inhibitor, FK-506. Similarly, CGA infusions following cue re-exposure significantly attenuated EPSC amplitude at T-LA synapses, suggesting that CaN affects cocaine-cue memory reconsolidation and extinction by altering T-LA synaptic strength. Therefore, CaN signaling in the LA may represent a novel target for disrupting cocaine-associated memories to reduce relapse.SIGNIFICANCE STATEMENT Repetitive drug use induces synaptic plasticity that underlies the formation of long-lasting associative memories for environmental cues paired with the drug. We previously identified thalamo-amygdala synapses (T-LA) that project via the interal capsule, as an important locus for the regulation of cocaine-cue memories. These synapses are strengthened by repeated cocaine-cue pairings, but this is reversed by extinction training or by optogenetic induction of in vivo long-term depression (LTD). Here, we demonstrate that activating calcineurin, a calcium-dependent phosphatase, following the reactivation or extinction of a cocaine-cue memory, induces LTD-like changes at T-LA synapses, and a corresponding decrease in cue-induced reinstatement, suggesting that calcineurin may be a potential therapeutic target for relapse prevention.


Assuntos
Tonsila do Cerebelo/fisiologia , Calcineurina/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Sinais (Psicologia) , Masculino , Ratos , Ratos Sprague-Dawley
13.
Adv Exp Med Biol ; 1131: 471-487, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646521

RESUMO

All seven canonical transient potential receptor (TRPC1-7) channel members are expressed in mammalian airway smooth muscle cells (ASMCs). Among this family, TRPC3 channel plays an important role in the control of the resting [Ca2+]i and agonist-induced increase in [Ca2+]i. This channel is significantly upregulated in molecular expression and functional activity in airway diseases. The upregulated channel significantly augments the resting [Ca2+]i and agonist-induced increase in [Ca2+]i, thereby exerting a direct and essential effect in airway hyperresponsiveness. The increased TRPC3 channel-mediated Ca2+ signaling also results in the transcription factor nuclear factor-κB (NF-κB) activation via protein kinase C-α (PKCα)-dependent inhibitor of NFκB-α (IκBα) and calcineurin-dependent IκBß signaling pathways, which upregulates cyclin-D1 expression and causes cell proliferation, leading to airway remodeling. TRPC3 channel may further interact with intracellular release Ca2+ channels, Orai channels and Ca2+-sensing stromal interaction molecules, mediating important cellular responses in ASMCs and the development of airway diseases.


Assuntos
Cálcio , Miócitos de Músculo Liso , Canais de Cátion TRPC , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Proliferação de Células/genética , Humanos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Sistema Respiratório/fisiopatologia , Canais de Cátion TRPC/metabolismo
14.
Int J Biochem Cell Biol ; 119: 105680, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866508

RESUMO

It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPSc-neurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106-126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Calcineurina/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Calcineurina/farmacologia , Linhagem Celular Tumoral , Humanos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Tacrolimo/farmacologia
15.
Exp Cell Res ; 386(1): 111716, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734152

RESUMO

BACKGROUND: Emerging evidence demonstrates that epoxyeicosatrienoic acids (EETs) as important active eicosanoids that regulate cardiovascular homeostasis, but the mechanisms underlying its favorable anti-hypertrophic benefits in overpressure model remain obscure. METHODS AND RESULTS: Four weeks after transverse aortic constriction (TAC), TAC mice developed maladaptive cardiac hypertrophy and consequent cardiac failure. Conversely, a cardiotropic adeno-associated viral vector (AAV9) encoding CYP2J2 prevented transverse aortic constriction-induced cardiac hypertrophy with preserved ejection fraction. EET also conferred protection against phenylephrine-induced hypertrophy in H9c2 cardiomyoblasts. Further investigations indicate CYP2J2/EET exerts protection against cardiac hypertrophy through opposing the increase of intracellular Ca2+ level and Ca2+-mediated calcineurin/NFATc3 signaling. Meanwhile, extended myocardial fibrosis in TAC mice was also effectively abolished with the administration of AAV9-2J2. Intriguingly, TAC mice display activated TGF-ß/Samd-3 signaling with decreased Smad-7 expression, whereas AAV9-2J2 attenuated the phosphorylation of Smad-3 without altering TGF-ß expression, whilst preservation of Smad-7. Subsequently, the differentiation of cardiac fibroblasts into myofibroblasts in the presence of TGF-ß1 stimulation was significantly disrupted with EET treatment, accompanied by declined Smad-3 activation and collagen production, whereas inhibition of Smad-7 with SiRNA Smad-7 substantially abrogated these effects of EET on cardiac fibroblasts. CONCLUSIONS: EET has synergistic actions on cardiomyocytes and cardiac fibroblasts, preventing cardiac hypertrophy through inhibition of Ca2+-mediated calcineurin/NFATc3 signaling cascades, and ameliorating myocardial fibrosis dependent on Smad-7. This work further extends the potential mechanisms of EET, providing a novel therapeutic approach for the treatment of pathological remodeling and heart failure.


Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Calcineurina/metabolismo , Cardiomegalia/prevenção & controle , Cardiotônicos/farmacologia , Fatores de Transcrição NFATC/metabolismo , Proteína Smad7/metabolismo , Ácido 8,11,14-Eicosatrienoico/uso terapêutico , Animais , Cálcio/metabolismo , Cardiomegalia/tratamento farmacológico , Cardiotônicos/uso terapêutico , Linhagem Celular , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
16.
J Immunol ; 204(3): 569-585, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31871019

RESUMO

Calcium ion (Ca2+) is a widespread and primitive second messenger that regulates physiological cell functions in almost all life beings. Ca2+ influx-induced NFAT activation is essential for T cell function and adaptive immunity. However, whether and how Ca2+ signaling modulates T cell immunity in early vertebrates, especially in nontetrapods, remains largely unknown. To address these questions, a Nile tilapia (Oreochromis niloticus) model was employed to investigate the regulation of ancestral T cell immunity by Ca2+-NFAT signaling in jawed fish. In Nile tilapia, an evolutionarily conserved Ca2+-NFAT signaling pathway is involved in the primary adaptive immune response during Streptococcus agalactiae infection. Meanwhile, T cell signals trigger several events along the Ca2+-NFAT axis in this early vertebrate, including Ca2+ influx, calcineurin activation, and NFAT nuclear import. More critically, suppression of Ca2+-NFAT signaling by the calcineurin inhibitor cyclosporine A impairs primordial T cell activation, clonal expansion, and infection clearance. Mechanistically, Nile tilapia NFAT interacts with several other transcription factors for potent gene expression, and T cells in this nontetrapod employ Cabin1 and DYRK1A to regulate NFAT nuclear import and export, respectively. To the best of our knowledge, this study is the first to demonstrate the regulatory mechanism of Ca2+-NFAT signaling on T cell immunity in a nontetrapod species. We suggest that modulation of T cell immunity by Ca2+-NFAT signaling is a primitive strategy that already existed prior to the divergence of bony fish from the tetrapod lineage. The findings of this study provide valuable perspectives for understanding the evolution of adaptive immune system.


Assuntos
Núcleo Celular/metabolismo , Ciclídeos/imunologia , Proteínas de Peixes/metabolismo , Fatores de Transcrição NFATC/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus agalactiae/fisiologia , Linfócitos T/imunologia , Transporte Ativo do Núcleo Celular , Animais , Evolução Biológica , Calcineurina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Proteínas de Peixes/genética , Imunidade Celular , Fatores de Transcrição NFATC/genética , Filogenia , Vertebrados
17.
mBio ; 10(6)2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822582

RESUMO

Echinocandins show fungicidal activity against common invasive mycoses but are ineffective against cryptococcosis. The underlying mechanism for echinocandin resistance in Cryptococcus neoformans remains poorly understood but has been shown to involve Cdc50, the regulatory subunit of lipid flippase. In a forward genetic screen for cdc50Δ suppressor mutations that are caspofungin resistant, we identified Crm1 (caspofungin resistant mutation 1), a homolog of mechanosensitive channel proteins, and showed that crm1Δ restored caspofungin resistance in cdc50Δ cells. Caspofungin-treated cdc50Δ cells exhibited abnormally high intracellular calcium levels ([Ca2+]c) and heightened activation of the calcineurin pathway. Deletion of CRM1 in the cdc50Δ background normalized the abnormally high [Ca2+]c. Cdc50 interacts with Crm1 to maintain cellular calcium homeostasis. Analysis of chitin/chitosan content showed that deleting CRM1 reversed the decreased chitosan production of cdc50Δ cells. Together, these results demonstrate that Cdc50 and Crm1 regulation of the calcineurin pathway and cytoplasmic calcium homeostasis may underlie caspofungin resistance in C. neoformans IMPORTANCE Cryptococcus neoformans is the leading cause of fungal meningitis, accounting for ∼15% of HIV/AIDS-related deaths, but treatment options for cryptococcosis are limited. Echinocandins are the newest fungicidal drug class introduced but are ineffective in treating cryptococcosis. Our previous study identified the lipid flippase subunit Cdc50 as a contributor to echinocandin resistance in C. neoformans Here, we further elucidated the mechanism of Cdc50-mediated caspofungin drug resistance. We discovered that Cdc50 interacts with the mechanosensitive calcium channel protein Crm1 to regulate calcium homeostasis and caspofungin resistance via calcium/calcineurin signaling. These results provide novel insights into echinocandin resistance in this pathogen, which may lead to new treatment options, as well as inform echinocandin resistance mechanisms in other fungal organisms and, hence, advance our understanding of modes of antifungal drug susceptibility and resistance.


Assuntos
Antifúngicos/farmacologia , Proteínas de Transporte/metabolismo , Criptococose/tratamento farmacológico , Cryptococcus neoformans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Equinocandinas/farmacologia , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Quitina/química , Quitosana/química , Criptococose/metabolismo , Cryptococcus neoformans/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Homeostase/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos
18.
ACS Chem Biol ; 14(12): 2672-2682, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31633908

RESUMO

The Phosphoprotein Phosphatase Calcineurin (CN, PP2B, PP3) recognizes and binds to two short linear motifs (SLiMs), PxIxIT and LxVP, in its regulators and substrates. These interactions enable CN function in many key biological processes. The identification of SLiMs is difficult because of their short, degenerate sequence and often low binding affinity. Here we combine Structure Based Shape Complementarity (SBSC) analysis and proteome-wide affinity purification-mass spectrometry to identify PxIxIT and LxVP containing CN interactors to expand and thereby redefine the LxVP motif. We find that the new πφ-LxVx primary sequence defines an ensemble of binding competent confirmations and thus the binding on-rate, making it difficult to predict the LxVP binding strength from its sequence. Our analysis confirms existing and, more importantly, identifies novel CN interactors, substrates, and thus biological functions of CN.


Assuntos
Calcineurina/efeitos dos fármacos , Calcineurina/metabolismo , Humanos , Espectrometria de Massas/métodos , Transporte Proteico , Proteoma , Especificidade por Substrato
19.
Cell Host Microbe ; 26(4): 453-462, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600499

RESUMO

Calcium is an abundant intracellular ion, and calcium homeostasis plays crucial roles in several cellular processes. The calcineurin signaling cascade is one of the major pathways governed by intracellular calcium. Calcineurin, a conserved protein from yeast to humans, is a calcium-calmodulin-dependent serine-threonine-specific phosphatase that orchestrates cellular stress responses. In eukaryotic microbial pathogens, calcineurin controls essential virulence pathways, such as the ability to grow at host temperature, morphogenesis to enable invasive hyphal growth, drug tolerance and resistance, cell wall integrity, and sexual development. Therefore, the calcineurin cascade is an attractive target in drug development against eukaryotic pathogens. In the present review, we summarize and discuss the current knowledge on the roles of calcineurin in eukaryotic microbial pathogens, focusing on fungi and parasitic protists.


Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Fungos/patogenicidade , Parasitos/patogenicidade , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Humanos , Parasitos/crescimento & desenvolvimento , Parasitos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência
20.
Mol Biol Rep ; 46(6): 6197-6204, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31486013

RESUMO

Post-traumatic stress disorder (PTSD) is a mental disease associated with the exposure of traumatic stress, and results in the structural and functional changes of hippocampus. Calcineurin (CaN), a calcium/calmodulin-regulated protein phosphatase ubiquitously expressed in brain, has a very important role in the fear extinction, neuronal structure and neuronal excitability. With CaN activation, its down target nuclear factor of activated T cells (NFATs) dephosphorylated and then translocated from the cytoplasm to the nucleus to affect neuronal function, resulting in the function changes of brain structure such as hippocampus. Increasing evidence has suggested that CaN/NFATs signaling are involved in the regulation of mental disorders like Alzheimer's disease, depression, while little is known about its effects on the molecular mechanisms on PTSD. This study seek to know the relationship between PTSD and CaN/NFATc4 pathway, and to detect whether CaN/NFATc4 pathway are involved in the hippocampus dysfunctions in a single-prolonged stress (SPS)-based rat model of PTSD. Our results have showed that after 4 days exposed to SPS, the protein expression of CaN up-regulated and the NFATc4 dephosphorylated and imported into the nucleus; while at the 7 and 14 day exposed to SPS, with the down-regulation of CaN, the expression of phosphorylate-NFATc4 increased. Our results show that CaN/NFATc4 pathway were involved in the development of PTSD model, which suggested that the changes of CaN/NFATc4 pathway may be one of the pathological molecular mechanism in the dysfunction of hippocampus in PTSD.


Assuntos
Calcineurina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Transtornos de Estresse Pós-Traumáticos/etiologia , Transtornos de Estresse Pós-Traumáticos/metabolismo , Estresse Psicológico , Animais , Comportamento Animal , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Imuno-Histoquímica , Aprendizagem em Labirinto , Fosforilação , Ratos , Transtornos de Estresse Pós-Traumáticos/psicologia
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