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1.
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
2.
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
3.
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
4.
PLoS Pathog ; 16(1): e1008134, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917826

RESUMO

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.


Assuntos
Proteínas de Caenorhabditis elegans/imunologia , Caenorhabditis elegans/imunologia , Calcineurina/imunologia , Imunidade Inata , Mucosa Intestinal/imunologia , Animais , Bicarbonatos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Calcineurina/genética , Concentração de Íons de Hidrogênio , Mucosa Intestinal/química , Mucosa Intestinal/efeitos dos fármacos , Mutação
5.
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
6.
Arch Microbiol ; 202(4): 921-934, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31807806

RESUMO

We studied a dominant mutant of the Neurospora crassa calcineurin catalytic (cna-1) subunit generated using the repeat-induced point mutation (RIP). The Cna-1RIP mutants showed defects in morphology, aerial hyphae development, carotenoid accumulation, and fertility. The Cna-1RIP mutants also showed sensitivity to osmotic stress and a reduction in acquisition of thermotolerance on exposure to lethal heat shock temperature. The Cna-1RIP allele was dominant over the wild type cna-1 allele, suggesting that the CNA-1RIP mutant protein acts in a dominant-negative manner. In addition, we studied calcineurin regulatory subunit (cnb-1) mutants, which were previously generated using RIP. The cnb-1RIP mutants showed sensitivity to calcium (Ca2+) and heat-shock stress conditions. Thus, calcineurin plays an essential role for vegetative and sexual developments, and tolerance to stress conditions in N. crassa. Moreover, CNA-1 directly interacts with the calmodulin (CaM) and calcineurin-responsive zinc finger-1 (CRZ-1) proteins under high Ca2+ condition in N. crassa.


Assuntos
Calcineurina/genética , Mutação , Neurospora crassa/enzimologia , Neurospora crassa/genética , Proteínas de Protozoários , Estresse Fisiológico/genética , Calmodulina/metabolismo , Domínio Catalítico/genética , Hifas/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
7.
Cancer Lett ; 469: 323-331, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693921

RESUMO

Post-transplant lymphoproliferative disorders (PTLD) represent a severe complication in transplanted patients and Epstein-Barr Virus (EBV) is the main driver. Besides immunodepression, immune activation/chronic inflammation play an important role in both virus reactivation and expansion of EBV-positive B cells. The aim of this study was to assess the impact of immunosuppressive strategies on factors involved in the PTLD's pathogenesis. 124 kidney transplanted patients were enrolled in this study: 71 were treated with mycophenolic acid (MPA) and 53 treated with mTOR inhibitor (mTORi), both in combination with different doses of calcineurin inhibitor. At the time of the transplant (T0), profile of inflammation/immune activation and immune senescence didn't differ between the two groups, but after one year of treatment (T1) markers were significantly higher in MPA-treated patients; their immunosenescence process was supported by the greater erosion of telomeres despite their younger age. Percentages of activated B cells and levels of EBV-DNA significantly increased in MPA-treated patients, and at T1 were significantly higher in MPA- than in mTORi-treated patients. Overall, these findings indicate that mTOR inhibitors constrain the inflammation/immune activation and senescence status, thus reducing the expansion of EBV-infected B cells and the risk of virus-associated PTLD in kidney transplant recipients.


Assuntos
Infecções por Vírus Epstein-Barr/tratamento farmacológico , Transplante de Rim/efeitos adversos , Transtornos Linfoproliferativos/prevenção & controle , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adulto , Linfócitos B/imunologia , Linfócitos B/virologia , Calcineurina/genética , Inibidores de Calcineurina/administração & dosagem , Senescência Celular/efeitos dos fármacos , Senescência Celular/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/patogenicidade , Humanos , Imunossupressores/administração & dosagem , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Transtornos Linfoproliferativos/etiologia , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Serina-Treonina Quinases TOR/genética , Carga Viral
8.
J Ovarian Res ; 12(1): 90, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554511

RESUMO

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 µM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCß1, protein kinase C ß (PKCß), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium-sensitive targets and several downstream genes might take part in the processes.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células da Granulosa/metabolismo , Fosfolipases Tipo C/genética , Animais , Apoptose/genética , Calcineurina/genética , Cálcio/metabolismo , Caspase 3/genética , Proliferação de Células/genética , Estrenos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/patologia , Fosfolipase C beta/genética , Fosfoproteínas Fosfatases/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Suínos , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética
9.
Food Funct ; 10(8): 5166-5173, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31378798

RESUMO

Thymol is a major component of thyme, and it has been reported that thymol administration reduces body weight, plasma insulin and blood glucose in type-2 diabetes. Skeletal muscle is the most important metabolism organs in the body; however, to date, there is no report on the effect of thymol on skeletal muscle. Our goal was to determine whether thymol has an effect on the different types of skeletal muscle fibers and their metabolism characteristics. Hence, we performed in vivo and in vitro experiments. In vivo, SD rats (4 weeks old) were fed with different concentrations of thymol for 4 weeks, and in vitro C2C12 myotubes were directly treated with thymol for 2 days. The rats fed with 0.025% thymol showed a significantly lower body weight, subcutaneous white adipose tissue index and gastrocnemius muscle index (P < 0.05), while their proportion of brown adipose tissue significantly increased (P < 0.05). The protein and mRNA expression of MyHC I and MyHC IIa in the gastrocnemius muscle of the rats significantly increased (P < 0.05), while the protein level of MyHC II and mRNA expression of MyHC IIb decreased (P < 0.05). Furthermore, 0.025% thymol supplement significantly reduced (P < 0.05) the activity of lactate dehydrogenase (LDH) in the gastrocnemius muscle of the rats, but their succinate dehydrogenase (SDH) and hexokinase (HK) activities increased (P < 0.05). Also, the expression of the fatty acid oxidation-related genes in the gastrocnemius muscle of the rats decreased with the thymol supplement (P < 0.05). In vitro, similar results were obtained. Furthermore, the Ca2+-calcineurin-NFAT pathway, which is an important pathway to regulate the transformation of skeletal muscle fiber type, was studied. We found that the effects of thymol on the myosin heavy chain isoforms, genes related to metabolism and the activation of the Ca2+-calcineurin-NFAT pathway were all reversed by a Ca2+ chelator (P < 0.05). Thus, thymol can promote the oxidative metabolism and fiber type switch in skeletal muscle, and the Ca2+-calcineurin-NFAT pathway plays an important role in it.


Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Extratos Vegetais/farmacologia , Timol/farmacologia , Thymus (Planta)/química , Animais , Calcineurina/genética , Linhagem Celular , Masculino , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição NFATC/genética , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
10.
J Ovarian Res ; 12(1): 75, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399054

RESUMO

BACKGROUND: The role of calcineurin/NFAT signaling in ovarian cancer has been unknown. NFAT was significantly overexpressed in ovarian cancer tissues and that overexpression of NFAT was significantly associated with metastasis and poor prognosis on clinical tissue level. To investigate whether NFAT upstream protein, calcineurin (CN), affects the prognosis in various histological subtype of ovarian cancer (OC). METHODS: The association between CN and clinical features was analyzed in 50 OC patients treated from 2007 to 2012. CN expression was examined using immunohistochemistry. We observed the association of CN expression with the prognosis in these patients. RESULTS: CN expression was significantly increased in later-stage tumor tissue of serous carcinoma compared with those with early-stage. The expression of CN positively correlated with the serum cancer antigen 125 (CA125) level in ovarian clear-cell carcinoma and the serum alpha-fetoprotein (AFP) level in papillary serous cystadenocarcinoma. Particularly, higher CN expression in tumor tissues significantly correlated with reduced overall survival among patients with serous carcinoma. In addition, the serum cancer antigen 72-4 (CA72-4) level, serum carcinoembryonic antigen (CEA) levels, pathological stage, lymph node metastasis, and chemotherapeutic resistance were identified as significant prognostic factors in ovarian clear-cell carcinoma, serous carcinoma, or papillary serous cystadenocarcinoma. CONCLUSIONS: CN is upregulated in ovarian cancer tissues with later-stage and that the expression of CN, CA72-4, and CEA was remarkably associated with poor prognosis in unique subtype of ovarian cancer. CN levels may be investigated for use as a prognostic biomarker for risk assessment in unique subtype of OC patients.


Assuntos
Calcineurina/genética , Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Adulto , Idoso , Biomarcadores Tumorais , Calcineurina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Retrospectivos , Carga Tumoral
11.
Am J Physiol Cell Physiol ; 317(5): C1025-C1033, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31433693

RESUMO

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.


Assuntos
Calcineurina/biossíntese , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Neurogranina/biossíntese , Transdução de Sinais/fisiologia , Animais , Calcineurina/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Neurogranina/genética , Adulto Jovem
12.
Genetics ; 213(1): 213-227, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31266771

RESUMO

Limited antifungal diversity and availability are growing problems for the treatment of fungal infections in the face of increasing drug resistance. The echinocandins, one of the newest classes of antifungal drugs, inhibit production of a crucial cell wall component. However, these compounds do not effectively inhibit the growth of the opportunistic fungal pathogen Cryptococcus neoformans, despite potent inhibition of the target enzyme in vitro Therefore, we performed a forward genetic screen to identify cellular processes that mediate the relative tolerance of this organism to the echinocandin drug caspofungin. Through these studies, we identified 14 genetic mutants that enhance caspofungin antifungal activity. Rather than directly affecting caspofungin antifungal activity, these mutations seem to prevent the activation of various stress-induced compensatory cellular processes. For example, the pfa4Δ mutant has defects in the palmitoylation and localization of many of its target proteins, including the Ras1 GTPase and the Chs3 chitin synthase, which are both required for caspofungin tolerance. Similarly, we have confirmed the link between caspofungin treatment and calcineurin signaling in this organism, but we suggest a deeper mechanism in which caspofungin tolerance is mediated by multiple pathways downstream of calcineurin function. In summary, we describe here several pathways in C. neoformans that contribute to the complex caspofungin tolerance phenotype in this organism.


Assuntos
Antifúngicos/farmacologia , Caspofungina/farmacologia , Parede Celular/genética , Cryptococcus neoformans/genética , Farmacorresistência Fúngica/genética , Genes Fúngicos , Calcineurina/genética , Calcineurina/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Estresse Fisiológico , Proteínas ras/genética , Proteínas ras/metabolismo
13.
Biol Pharm Bull ; 42(7): 1230-1235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257299

RESUMO

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase abundantly expressed in the nervous system. To investigate the roles of calcineurin in glial cells, we previously generated glial calcineurin B1-conditional knockout (CKO) mice and found that these mice displayed dysfunction of enteric glial cells, mucosal degeneration and inflammation in the small intestine, and growth retardation and postweaning death, suggesting a novel role of calcineurin in enteric glial cells. Although these findings raised a possibility that abnormalities in calcineurin B1-deficient enteric glial cells may cause dysregulation of gastrointestinal motility and result in maldigestion and/or malabsorption in the CKO mice, these issues remain to be elucidated. In the present study, we showed that gastrointestinal motility was reduced in the CKO mice. Degeneration of mucosal epithelium was observed in the small intestine. Glucose levels were decreased in serum, whereas starch, glucose, and lipid levels were increased in feces. Thus, calcineurin B1 deficiency in glial cells reduces gastrointestinal motility, induces mucosal epithelium degeneration in the small intestine, and results in maldigestion and/or malabsorption in mice, further supporting the important role of calcineurin in enteric glial cells.


Assuntos
Calcineurina/fisiologia , Motilidade Gastrointestinal , Neuroglia/fisiologia , Animais , Calcineurina/genética , Digestão , Fezes/química , Feminino , Glucose/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Intestino Delgado/fisiologia , Metabolismo dos Lipídeos , Camundongos Knockout , Proteínas/metabolismo , Amido/metabolismo
14.
Biochemistry (Mosc) ; 84(6): 686-692, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238868

RESUMO

Resveratrol has been shown to stimulate differentiation of osteoblastic MC3T3-E1 cells in vitro; however, the mechanisms underlying the anabolic effect of resveratrol on osteoblasts remain largely unknown. Our study was aimed to investigate the molecular mechanism of resveratrol-induced differentiation of MC3T3-E1 cells. MC3T3-E1 cells were treated for 8 days with different concentrations of resveratrol (10-8-10-6 M) and 10-6 M cyclosporine A (CsA), a specific inhibitor of the calcineurin/NFAT pathway. According to the results of pilot studies of cell proliferation and alkaline phosphatase activity, 10-7 M concentration of resveratrol was used in subsequent experiments. The levels of mRNA expression of the osteosis-related genes CaN, NFATc1, and Runx2 were analyzed by real-time RT-PCR; the levels of the corresponding proteins were estimated by Western blot analysis. Resveratrol upregulated expression of the CaN, NFATc1, and Runx2 genes at both mRNA and protein levels compared to the control group (p < 0.05), while CsA reduced the effects of resveratrol (p < 0.05). Using immunohistochemical staining, we showed that resveratrol induced NFATc1 accumulation in the cell nuclei, and treatment with CsA inhibited resveratrol-mediated induction of NFATc1, suggesting that the calcineurin/NFATc1 signaling pathway plays an important role in the regulatory effect of resveratrol on osteoblasts.


Assuntos
Antioxidantes/farmacologia , Calcineurina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Resveratrol/farmacologia , Células 3T3 , Animais , Calcineurina/genética , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Fatores de Transcrição NFATC/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
15.
Biomed Res Int ; 2019: 6712536, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183372

RESUMO

Background/Aims: TRPV1 is a nonselective Ca2+ channel which has recently been observed in many cancers, while its effect on cell proliferation, apoptosis, metabolism, and cancer development in colorectal cancer (CRC) is still unclear. In this study, we hypothesized that TRPV1 is a tumor suppressor in CRC development as well as the underlying mechanism. Methods: Immunohistochemistry assay was applied to detect the expression of TRPV1 protein in CRC tissues. HCT116 cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. Cellular Ca2+ concentration was measured by Fluo-4/AM-based flow cytometer. Apoptosis-related proteins were measured by Western blotting. Results: In this study, we found that TRPV1 expression was significantly decreased in CRC tissues, compared with CRC-adjacent tissues and normal tissues, respectively. Then, we found that the TRVP1 agonist capsaicin treatment inhibited CRC growth and induced apoptosis by activating P53. Subsequent mechanistic study revealed that the TRPV1 induced cytosolic Ca2+ influx to regulate cell apoptosis and p53 activation through calcineurin. Conclusions: This study suggests that TRPV1 served as a tumor suppressor in CRC and contributed to the development of novel therapy of CRC.


Assuntos
Apoptose , Calcineurina/metabolismo , Sinalização do Cálcio , Neoplasias Colorretais/metabolismo , Fatores de Transcrição NFATC/metabolismo , Canais de Cátion TRPV/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Calcineurina/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Fatores de Transcrição NFATC/genética , Canais de Cátion TRPV/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
16.
Int J Mol Sci ; 20(11)2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185659

RESUMO

Ganoderic acids (GAs) are a type of highly oxygenated lanostane-type triterpenoids that are responsible for the pharmacological activities of Ganoderma lucidum. They have been investigated for their biological activities, including antibacterial, antiviral, antitumor, anti-HIV-1, antioxidation, and cholesterol reduction functions. Inducer supplementation is viewed as a promising technology for the production of GAs. This study found that supplementation with sodium acetate (4 mM) significantly increased the GAs content of fruiting bodies by 28.63% compared to the control. In order to explore the mechanism of ganoderic acid accumulation, the transcriptional responses of key GAs biosynthetic genes, including the acetyl coenzyme A synthase gene, and the expression levels of genes involved in calcineurin signaling and acetyl-CoA content have been analyzed. The results showed that the expression of three key GAs biosynthetic genes (hmgs, fps, and sqs) were significantly up-regulated. Analysis indicated that the acetate ion increased the expression of genes related to acetic acid assimilation and increased GAs biosynthesis, thereby resulting in the accumulation of GAs. Further investigation of the expression levels of genes involved in calcineurin signaling revealed that Na+ supplementation and the consequent exchange of Na+/Ca2+ induced GAs biosynthesis. Overall, this study indicates a feasible new approach of utilizing sodium acetate elicitation for the enhanced production of valuable GAs content in G. lucidum, and also provided the primary mechanism of GAs accumulation.


Assuntos
Carpóforos/metabolismo , Regulação Fúngica da Expressão Gênica , Reishi/metabolismo , Triterpenos/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reishi/genética , Sódio/metabolismo , Regulação para Cima
17.
Genes (Basel) ; 10(5)2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137880

RESUMO

Salinity is one of the major environment factors that limits the growth of plants and the productivity of crops worldwide. It has been shown that Na+ transporters play a central role in salt tolerance and development of plants. The objective of this study was to identify Na+/H+ antiporter (NHX) genes and investigate their expression patterns in sugar beet (Beta vulgaris L.) subjected to various concentrations of NaCl. A total of five putative NHX genes were identified and distributed on four chromosomes in sugar beet. Phylogenetic analysis revealed that these BvNHX genes are grouped into three major classes, viz Vac- (BvNHX1, -2 and -3), Endo- (BvNHX4), and PM-class NHX (BvNHX5/BvSOS1), and within each class the exon/intron structures are conserved. The amiloride-binding site is found in TM3 at N-terminus of Vac-class NHX proteins. Protein-protein interaction (PPI) prediction suggested that only BvNHX5 putatively interacts with calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK), implying it might be the primary NHX involved in CBL-CIPK pathway under saline condition. It was also found that BvNHX5 contains one abscisic acid (ABA)-responsive element (ABRE), suggesting that BvNHX5 might be involved in ABA signal responsiveness. Additionally, the qRT-PCR analysis showed that all the BvNHX genes in both roots and leaves are significantly up-regulated by salt, and the transcription levels under high salinity are significantly higher than those under either low or moderate salinity. Taken together, this work gives a detailed overview of the BvNHX genes and their expression patterns under salt stress. Our findings also provide useful information for elucidating the molecular mechanisms of Na+ homeostasis and further functional identification of the BvNHX genes in sugar beet.


Assuntos
Beta vulgaris/genética , Filogenia , Estresse Salino/genética , Trocadores de Sódio-Hidrogênio/genética , Beta vulgaris/crescimento & desenvolvimento , Calcineurina/genética , Regulação da Expressão Gênica de Plantas/genética , Folhas de Planta/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Mapas de Interação de Proteínas/genética , Salinidade , Tolerância ao Sal/genética
18.
Cancer Res ; 79(14): 3702-3713, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31142509

RESUMO

Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.


Assuntos
Calcineurina/imunologia , Interleucina-12/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Calcineurina/deficiência , Calcineurina/genética , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/imunologia , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interleucina-12/biossíntese , Interleucina-12/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Evasão Tumoral
19.
Food Funct ; 10(6): 3466-3476, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31140514

RESUMO

Pseudostellaria heterophylla has been becoming a popular research topic because of its functionally active components. The immunomodulatory activity of P. heterophylla peptide (PPH) derived from protein hydrolysate and the molecular mechanism underlying its immunomodulatory effect were investigated in this study. Immunomodulatory PPH achieved the highest stimulation index of 1.53 at a concentration of 100 µg mL-1 for 48 h in spleen lymphocytes and promoted the secretions of tumor necrosis factor-α, interferon-γ, and interleukin-10. Moreover, PPH could elevate the intracellular Ca2+ concentration, calcineurin activity and nuclear factor of activated T cell (NFAT) c1 mRNA expression. Meanwhile these effects could be diminished by the treatment of verapamil and cyclosporin A, suggesting that PPH may activate spleen lymphocytes via the Ca2+/CaN/NFATc1/IFN-γ signaling pathway. These findings demonstrate that the P. heterophylla peptide has the potential to be utilized as a nutraceutical supplement to strengthen the immune system in the human body.


Assuntos
Calcineurina/imunologia , Cálcio/imunologia , Caryophyllaceae/química , Fatores Imunológicos/farmacologia , Interferon gama/imunologia , Linfócitos/efeitos dos fármacos , Fatores de Transcrição NFATC/imunologia , Peptídeos/farmacologia , Animais , Calcineurina/genética , Fatores Imunológicos/química , Interferon gama/genética , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Camundongos , Fatores de Transcrição NFATC/genética , Peptídeos/química , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia
20.
Nat Commun ; 10(1): 2251, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113954

RESUMO

Cerebellar activity supported by PKC-dependent long-term depression in Purkinje cells (PCs) is involved in the stabilization of self-motion based hippocampal representation, but the existence of cerebellar processes underlying integration of allocentric cues remains unclear. Using mutant-mice lacking PP2B in PCs (L7-PP2B mice) we here assess the role of PP2B-dependent PC potentiation in hippocampal representation and spatial navigation. L7-PP2B mice display higher susceptibility to spatial map instability relative to the allocentric cue and impaired allocentric as well as self-motion goal-directed navigation. These results indicate that PP2B-dependent potentiation in PCs contributes to maintain a stable hippocampal representation of a familiar environment in an allocentric reference frame as well as to support optimal trajectory toward a goal during navigation.


Assuntos
Orientação Espacial/fisiologia , Células de Purkinje/fisiologia , Navegação Espacial/fisiologia , Animais , Calcineurina/genética , Calcineurina/metabolismo , Sinais (Psicologia) , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Plasticidade Neuronal/fisiologia , Percepção Espacial/fisiologia
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