Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.281
Filtrar
1.
Nat Commun ; 10(1): 3028, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292434

RESUMO

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.


Assuntos
Ataxia Cerebelar/genética , Córtex Cerebelar/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Caseína Quinase Idelta , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Ataxia Cerebelar/patologia , Córtex Cerebelar/citologia , Córtex Cerebelar/patologia , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Proteínas Nucleares/genética , Fosforilação/fisiologia , Cultura Primária de Células , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
2.
Nat Commun ; 10(1): 3005, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285450

RESUMO

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.


Assuntos
Complexos Multienzimáticos/ultraestrutura , Fosfoproteínas/ultraestrutura , Proteínas Serina-Treonina Quinases/ultraestrutura , Estresse Fisiológico , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Listeria monocytogenes/fisiologia , Complexos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Fator sigma/metabolismo , Transdução de Sinais/fisiologia
3.
Arch. bronconeumol. (Ed. impr.) ; 55(6): 306-311, jun. 2019. graf
Artigo em Inglês | IBECS | ID: ibc-181765

RESUMO

Introduction: The endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood. Methods: Rat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1 mg/L, 1 mg/L, and 10 mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway. Results: LPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other. Conclusion: Phosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity


Introducción: La disrupción de la barrera endotelial pulmonar inducida por endotoxina o lipopolisacárido (LPS) es un factor patogénico clave en la lesión pulmonar aguda (LPA) y el síndrome de distrés respiratorio agudo (SDRA). Sin embargo, los mecanismos que subyacen al empeoramiento de la permeabilidad de las células endoteliales de la microvasculatura pulmonar (PMVECs, por sus siglas en inglés) no se conocen. Métodos: Se aislaron y cultivaron en monocapa PMVEC de rata, y se expusieron a diferentes dosis de LPS (0,1, 1 y 10 mg/l). Se utilizó la resistencia eléctrica transendotelial (TER, por sus siglas en inglés) para medir la integridad de la barrera endotelial. Se analizó la actividad del sustrato 1 de la toxina botulínica C3 relacionado con Ras (Rac1) y la fosforilación de las proteínas erzina/raxidina/moesina (ERM) mediante ensayos pulldown y Western blot. Para evaluar la permeabilidad de las PMVEC y las vías relacionadas se inhibieron Rac1 y moesina mediante ARN pequeño de interferencia (siRNA, por sus siglas en inglés). Resultados: El LPS indujo una disminución dependiente de dosis y tiempo de la TER e incrementó la fosforilación en treonina de ERM, al mismo tiempo que inactivó a Rac1 en las PMVEC. El estudio con siRNA demostró que, tanto Rac1 como la moesina estaban implicadas en la mediación de la permeabilidad de las PMVEC en monocapa inducida por LPS, y que Rac1 y la moesina podrían regularse mutuamente. Conclusión: La fosforilación de ERM media la permeabilidad de las PMVECs inducida por LPS mediante la regulación negativa de la actividad de Rac1


Assuntos
Animais , Masculino , Ratos , Polissacarídeos/farmacologia , Fosforilação/fisiologia , Células Endoteliais/metabolismo , Permeabilidade Capilar/efeitos da radiação , Pulmão/irrigação sanguínea , Proteínas rac1 de Ligação ao GTP/metabolismo , Ratos Sprague-Dawley
4.
Nat Commun ; 10(1): 2640, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201334

RESUMO

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , Biossíntese de Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fosforilação/fisiologia , Ligação Proteica/fisiologia , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
5.
Nat Commun ; 10(1): 2574, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189925

RESUMO

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Domínios Proteicos/genética , Adenosina Trifosfatases/genética , Ácido Glutâmico/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Relação Estrutura-Atividade , Tirosina/genética , Tirosina/metabolismo
6.
BMC Bioinformatics ; 20(1): 230, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068129

RESUMO

BACKGROUND: c-Myc plays an important role in cell proliferation, cell growth and in differentiation, making it a key regulator for carcinogenesis and pluripotency. Tight control of c-myc turnover is required by ubiquitin-mediated degradation. This is achieved in the system by two F-box proteins Skp2 and FBXW7. RESULTS: Dynamic modelling technique was used to build two exclusive models for phosphorylation dependent degradation of Myc by FBXW7 (Model 1) and phosphorylation independent degradation by Skp2 (Model 2). Sensitivity analysis performed on these two models revealed that these models were corroborating experimental studies. It was also seen that Model 1 was more robust and perhaps more efficient in degrading c-Myc. These results questioned the existence of the two models in the system and to answer the question a combined model was hypothesised which had a decision making switch. The combined model had both Skp2 and FBXW7 mediated degradation where again the latter played a more important role. This model was able to achieve the lowest levels of ubiquitylated Myc and therefore functioned most efficiently in degradation of Myc. CONCLUSION: In this report, c-Myc degradation by two F-box proteins was mathematically evaluated based on the importance of c-Myc turnover. The study was performed in a homeostatic system and therefore, prompts the exploration of c-Myc degradation in cancer state and in pluripotent state.


Assuntos
Simulação por Computador/normas , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proliferação de Células , Humanos
7.
Nat Commun ; 10(1): 2136, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086188

RESUMO

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Simulação por Computador , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA de Transferência de Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , eIF-2 Quinase/metabolismo
8.
Nat Commun ; 10(1): 2193, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097705

RESUMO

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Morfogênese/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/diagnóstico por imagem , Epitélio/diagnóstico por imagem , Epitélio/crescimento & desenvolvimento , Fibroblastos , Microscopia Intravital , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/genética
9.
Biomed Res Int ; 2019: 8952414, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31080833

RESUMO

Introduction: Hyperglycemia is a major factor in influencing the patency rate of arteriovenous shunts, potentially associated with the RhoA/Rho-associated protein kinase (ROCK) pathway. Besides, galectin-3 mediates thrombotic mechanisms in venous thrombosis and peripheral artery disease. We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenous shunt dysfunction. Methods: We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction. 29 patients received a complete follow-up and each provided two blood samples, which were collected at the first visit for occluded status of arteriovenous shunts and 1 month later for patent status. A Western blot assay for a myosin phosphatase target subunit (MYPT) was performed to examine Rho-kinase activity. A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbent assay (ELISA) for circulating galectin-3 were completed. Results: Higher platelet MYPT ratios and galectin-3 levels were identified at occluded arteriovenous shunts (MYPT ratio: 0.5 [0.3-1.4] vs. 0.4 [0.3-0.6], p = 0.01; galectin-3: 1.2 [0.4-1.6] vs. 0.7 [0.1-1.2], p = 0.0004). The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (8.4 [6.0-9.7] µg/mL vs. 7.1 [4.5-9.1] µg/mL, p = 0.009). Biomarker ratios (occluded/patent status) trended high in patients with poorly controlled diabetes (MYPT ratio: 1.7 [1.0-3.0] vs. 1.1 [0.7-1.3], p = 0.06; galectin-3: 1.6 [1.3-3.4] vs. 1.1 [0.8-1.9], p = 0.05). Conclusion: High platelet ROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction, especially in patients with poorly controlled diabetes.


Assuntos
Fístula Arteriovenosa/metabolismo , Plaquetas/metabolismo , Diabetes Mellitus/metabolismo , Galectina 3/metabolismo , Quinases Associadas a rho/metabolismo , Idoso , Derivação Arteriovenosa Cirúrgica/métodos , Feminino , Humanos , Masculino , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação/fisiologia , Estudos Prospectivos , Diálise Renal/métodos , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo
10.
Plant Cell Physiol ; 60(8): 1804-1810, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31119298

RESUMO

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Quitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fosforilação/genética , Fosforilação/fisiologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
11.
Nat Commun ; 10(1): 1676, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976006

RESUMO

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.


Assuntos
Divisão Celular/fisiologia , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Cristalografia por Raios X , Ciclina A/isolamento & purificação , Quinase 2 Dependente de Ciclina/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/isolamento & purificação , Proteínas de Fusão bcr-abl/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Treonina/metabolismo , Tirosina/metabolismo , Quinases da Família src/isolamento & purificação , Quinases da Família src/metabolismo
12.
Nat Commun ; 10(1): 1804, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000703

RESUMO

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Domínios PDZ/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Técnicas Biossensoriais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Fosforilação/fisiologia , Análise de Célula Única/métodos , Xenopus laevis
13.
Folia Histochem Cytobiol ; 57(1): 28-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30957871

RESUMO

INTRODUCTION: CD44H is a transmembrane molecule important for cell-cell and cell-extracellular matrix interactions. In monocytes, CD44H is implicated in phagocytosis of particles coated by hyaluronan (HA). HA fragments were shown to induce chemokine secretion by monocytes. Tumour derived microvesicles (TMVs), which are small membrane fragments derived from tumour cells can carry fragments of HA. The aim of the study was to examine whether monocyte's CD44H is involved in the engulfment of pancreatic adenocarcinoma-derived microvesicles and in the production of chemokines induced by TMVs. MATERIALS AND METHODS: TMVs engulfment and chemokines' secretion stimulated with TMVs were determined in control human monocytes and cells incubated with anti-CD44H monoclonal antibody (mAb) by flow cytometry and ELISA, respectively. Phosphorylation of STAT3, transcription factor essential for chemokines' production and CD44 signal transduction, was determined by Western blotting. RESULTS: Blocking of CD44H by anti-CD44H mAb on monocytes decreased the engulfment of TMVs and the secretion of CCL4 and CCL5, but had no effect on CCL2, CCL3 and CXCL8. STAT-3 phosphorylation in monocytes incubated with TMVs after CD44 blocking was also reduced. CONCLUSION: The results suggest that tumour-derived microvesicles (TMVs) may carry bioactive cargo(s) which induces STAT3 dependent signalling pathway in human monocytes via CD44 molecules.


Assuntos
Adenocarcinoma/metabolismo , Receptores de Hialuronatos/metabolismo , Monócitos/metabolismo , Neoplasias Pancreáticas/metabolismo , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Humanos , Receptores de Hialuronatos/imunologia , Fosforilação/fisiologia , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia
14.
Nat Commun ; 10(1): 1791, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996251

RESUMO

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Zinco/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Encéfalo/patologia , Encéfalo/cirurgia , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutagênese , Mutação , Fosforilação/fisiologia , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Int J Mol Sci ; 20(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974739

RESUMO

STK16 (Ser/Thr kinase 16, also known as Krct/PKL12/MPSK1/TSF-1) is a myristoylated and palmitoylated Ser/Thr protein kinase that is ubiquitously expressed and conserved among all eukaryotes. STK16 is distantly related to the other kinases and belongs to the NAK kinase family that has an atypical activation loop architecture. As a membrane-associated protein that is primarily localized to the Golgi, STK16 has been shown to participate in the TGF-ß signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. This review aims to provide a comprehensive summary of the progress made in recent research about STK16, ranging from its distribution, molecular characterization, post-translational modification (fatty acylation and phosphorylation), interactors (GlcNAcK/DRG1/MAL2/Actin/WDR1), and related functions. As a relatively underexplored kinase, more studies are encouraged to unravel its regulation mechanisms and cellular functions.


Assuntos
Ciclo Celular/fisiologia , Complexo de Golgi/enzimologia , Lipoilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Humanos , Fosforilação/fisiologia , Transporte Proteico/fisiologia
16.
C R Biol ; 342(3-4): 90-96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028003

RESUMO

The objective of our study was to elucidate the role of the transcription factor CREB-1 in controlling ovarian cell proliferation, apoptosis, and hormone release and the significance of CREB-1 phosphorylation in these processes. Human ovarian granulosa cells were transfected with a gene construct encoding wild-type CREB-1 (CREB-1 WT) or CREB-1 nonphosphorylatable mutant (CREB-1 M1). The expression of total and phosphorylated CREB-1, markers of proliferation (PCNA) and apoptosis (bax), as well as the release of progesterone, oxytocin, prostaglandin F2 alpha (PGF2), prostaglandin E2 (PGE2), and insulin-like growth factor I (IGF-I) were compared by immunocytochemistry, enzyme immunoassay (EIA), and immunoradiometric assay (IRMA). Transfection with CREB-1 WT or CREB-1 M1 increased total CREB-1 expression and proliferation and decreased the release of oxytocin, PGE2, and IGF-I by ovarian cells. CREB-1 M1, not CREB-1 WT, promoted apoptosis and inhibited progesterone output. PGF2 release was inhibited by CREB-1 WT but stimulated by CREB-1 M1 construct. Phosphorylated CREB-1 was undetected in any cell group. These observations confirm the involvement of CREB-1 in the control of ovarian cell proliferation, apoptosis, and steroid hormone release. This is the first demonstration of the involvement of CREB-1 in the regulation of the ovarian non-steroidal hormones such as oxytocin, PGF2, PGE2, and IGF-I. The absence of CREB-1 phosphorylation, similar effects exerted by CREB-1 WT and CREB-1 M1 on cell proliferation and release of oxytocin, PGE2, and IGF-I, and the influence of CREB-1 M1 on apoptosis and progesterone suggest that phosphorylation plays no role in the action of CREB-1 on the majority of analyzed functions of human ovarian cells.


Assuntos
Proliferação de Células/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ovário/fisiologia , Fosforilação/fisiologia , Adulto , Animais , Apoptose/fisiologia , Células Cultivadas , Feminino , Células da Granulosa/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Ocitocina/metabolismo , Progesterona/metabolismo
17.
Nat Commun ; 10(1): 1689, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976076

RESUMO

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.


Assuntos
Inflamassomos/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/fisiologia , Piroptose/fisiologia , Receptores Estrogênicos/metabolismo , Neoplasias Cutâneas/patologia , Animais , Caspase 3/metabolismo , Citocromos c/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Mutação , Fosforilação/fisiologia , Cultura Primária de Células , Domínios Proteicos/genética , Receptores Estrogênicos/genética , Treonina/metabolismo
18.
Nat Commun ; 10(1): 1231, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874556

RESUMO

The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.


Assuntos
Mycobacterium tuberculosis/fisiologia , Fosforilação/fisiologia , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Homeostase/fisiologia , Ligantes , Mutação , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
19.
Nat Commun ; 10(1): 1261, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890705

RESUMO

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.


Assuntos
Arrestina/metabolismo , Fosforilação/fisiologia , Rodopsina/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Arrestina/química , Arrestina/genética , Arrestina/isolamento & purificação , Bioensaio , Bovinos , Membrana Celular/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodopsina/química , Segmento Externo da Célula Bastonete/metabolismo , beta-Arrestina 1/química , beta-Arrestina 1/isolamento & purificação , beta-Arrestina 2/química , beta-Arrestina 2/isolamento & purificação
20.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871006

RESUMO

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.


Assuntos
Anáfase/fisiologia , Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Metáfase/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Linhagem Celular , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA