Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.028
Filtrar
1.
Nat Commun ; 11(1): 1077, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103024

RESUMO

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.


Assuntos
Caseína Quinase II/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Divisão Celular Assimétrica/fisiologia , Sítios de Ligação/fisiologia , Camelídeos Americanos , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenvolvimento Embrionário/fisiologia , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/ultraestrutura , Fosforilação , Ligação Proteica/fisiologia , Conformação Proteica
2.
Mol Pharmacol ; 97(3): 226-236, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900312

RESUMO

Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein ßγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.


Assuntos
Descoberta de Drogas/métodos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células Cultivadas , Cristalografia por Raios X/métodos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Neutrófilos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Peixe-Zebra
3.
Nat Commun ; 11(1): 180, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924779

RESUMO

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pinocitose/fisiologia , Linfócitos T/fisiologia , Aminoácidos , Animais , Linfócitos T CD4-Positivos/fisiologia , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Linfócitos T/citologia
4.
Nat Cell Biol ; 22(1): 120-134, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31871319

RESUMO

Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Drosophila , Humanos , Ligação Proteica/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Biochem Soc Trans ; 47(5): 1415-1427, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31671184

RESUMO

The cellular signalling enzymes, EPAC1 and EPAC2, have emerged as key intracellular sensors of the secondary messenger cyclic 3',5'-adenosine monophosphate (cyclic adenosine monophosphate) alongside protein kinase A. Interest has been galvanised in recent years thanks to the emergence of these species as potential targets for new cardiovascular disease therapies, including vascular inflammation and insulin resistance in vascular endothelial cells. We herein summarise the current state-of-the-art in small-molecule EPAC activity modulators, including cyclic nucleotides, sulphonylureas, and N-acylsulphonamides.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Humanos , Ligantes , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/metabolismo
6.
Hum Genet ; 138(11-12): 1391-1407, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691004

RESUMO

FGFR signaling is critical to development and disease pathogenesis, initiating phosphorylation-driven signaling cascades, notably the RAS-RAF-MEK-ERK and PI3 K-AKT cascades. PTEN antagonizes FGFR signaling by reducing AKT and ERK activation. Mouse lenses lacking FGFR2 exhibit microphakia and reduced ERK and AKT phosphorylation, widespread apoptosis, and defective lens fiber cell differentiation. In contrast, simultaneous deletion of both Fgfr2 and Pten restores ERK and AKT activation levels as well as lens size, cell survival and aspects of fiber cell differentiation; however, the molecular basis of this "rescue" remains undefined. We performed transcriptomic analysis by RNA sequencing of mouse lenses with conditional deletion of Fgfr2, Pten or both Fgfr2 and Pten, which reveal new molecular mechanisms that uncover how FGFR2 and PTEN signaling interact during development. The FGFR2-deficient lens transcriptome demonstrates overall loss of fiber cell identity with deregulated expression of 1448 genes. We find that ~ 60% of deregulated genes return to normal expression levels in lenses lacking both Fgfr2 and Pten. Further, application of customized filtering parameters to these RNA-seq data sets identified 68 high-priority candidate genes. Bioinformatics analyses showed that the cis-binding motif of a high-priority homeodomain transcription factor, NKX6-1, was present in the putative promoters of ~ 78% of these candidates. Finally, biochemical reporter assays demonstrate that NKX6-1 activated the expression of the high-priority candidate Rasgrp1, a RAS-activating protein. Together, these data define a novel regulatory module in which NKX6-1 directly activates Rasgrp1 expression to restore the balance of ERK and AKT activation, thus providing new insights into alternate regulation of FGFR downstream events.


Assuntos
Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Homeodomínio/metabolismo , Microftalmia/prevenção & controle , PTEN Fosfo-Hidrolase/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Transcriptoma , Animais , Diferenciação Celular , Proliferação de Células , Fatores de Troca do Nucleotídeo Guanina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Microftalmia/etiologia , Microftalmia/patologia , Fosforilação , Transdução de Sinais
7.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31583084

RESUMO

Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF-effector feedback modules and survey recent developments in this exciting area of cell biology.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Retroalimentação , Humanos
8.
PLoS Biol ; 17(10): e3000484, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622333

RESUMO

Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.


Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Feromônios/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Fator de Acasalamento/genética , Receptores de Fator de Acasalamento/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo
9.
Med Sci Monit ; 25: 7826-7835, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31626606

RESUMO

BACKGROUND Previous studies have reported that ARHGEF39 might be frequently upregulated in different cancer types and relevant to cancer progression. However, the expression pattern and clinicopathological features of ARHGEF39 in patients with hepatocellular carcinoma (HCC) needs further exploration. MATERIAL AND METHODS ARHGEF39 expression level of HCC in The Cancer Genome Atlas (TCGA) dataset was analyzed. Quantitative real-time polymerase chain reaction and immunohistochemistry were employed to determine ARHGEF39 mRNA and protein levels in our own study collected HCC tissues and matched non-cancerous tissues. Moreover, the association of ARHGEF39 expression with the clinicopathological factors and prognosis of HCC were investigated. RESULTS The level of ARHGEF39 in HCC tissues was significantly higher than that in adjacent normal tissues (P<0.05) from TCGA database. High level of ARHGEF39 was a significant prognostic factor of poor overall survival (OS) (TCGA, P=0.006). Consistently, the expression levels of ARHGEF39 mRNA and protein in HCC specimens were significantly higher than those in adjacent liver specimens (P<0.05) from our cohort. Further analysis revealed that high ARHGEF39 level was significantly associated with poor OS (P<0.001) and short disease-free survival (DFS) (P<0.001). Cox multivariate analysis indicated that ARHGEF39 was an independent, unfavorable prognostic factor (P=0.000) of OS and DFS. CONCLUSIONS ARHGEF39 might act as an oncogene in the progression of HCC and might serve as a promising potential prognostic indicator and therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Bases de Dados Genéticas , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Sobrevida , Transcriptoma/genética
10.
Med Sci Monit ; 25: 6554-6562, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31473760

RESUMO

BACKGROUND In previous studies, higher expression of PREX1 (PtdIns (3,4,5)P3-dependent Rac exchanger 1) has been detected in some subsets of breast cancer, and activation of PREX1 has been associated with tumor progression in vivo. However, an association between PREX1 and breast cancer prognosis has not been examined. MATERIAL AND METHODS In this study, we investigated the expression and correlation of PREX1 with important clinical factors and prognosis of patients with breast cancer. Immunohistochemical staining was performed for 121 tumor tissue specimens obtained from primary breast cancer lesions. RESULTS We found that 55 tissues exhibited positive staining for PREX1. Moreover, tumors positive for PREX1 were found to have significant association with recurrence rate (P=0.000) and metastasis rate (P=0.001). Univariate and multivariate regression analyses also identified PREX1 expression as an independent variable of disease-free survival. Our analyses indicate that high levels of PREX1 expression were related to longer disease-free survival in patients with breast cancer (P=0.013). CONCLUSIONS PREX1 is a favorable variable of prognosis for breast cancer patients, these study results need to be confirmed in larger research studies.


Assuntos
Neoplasias da Mama/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Prognóstico , Estudos Retrospectivos , Adulto Jovem
11.
Molecules ; 24(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514408

RESUMO

Small GTPases are key regulators of cellular events, and their dysfunction causes many types of cancer. They serve as molecular switches by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. GTPases are deactivated by GTPase-activating proteins (GAPs) and are activated by guanine-nucleotide exchange factors (GEFs). The intrinsic GTP hydrolysis activity of small GTPases is generally low and is accelerated by GAPs. GEFs promote GDP dissociation from small GTPases to allow for GTP binding, which results in a conformational change of two highly flexible segments, called switch I and switch II, that enables binding of the gamma phosphate and allows small GTPases to interact with downstream effectors. For several decades, crystal structures of many GEFs and GAPs have been reported and have shown tremendous structural diversity. In this review, we focus on the latest structural studies of GEFs. Detailed pictures of the variety of GEF mechanisms at atomic resolution can provide insights into new approaches for drug discovery.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática , Retroalimentação Fisiológica , Humanos , Filogenia , Dobramento de Proteína
12.
Genes (Basel) ; 10(9)2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514484

RESUMO

Existing methods often fail to recognize the conversions for the biological roles of the pairs of genes and microRNAs (miRNAs) between the tumor and normal samples. We have developed a novel cluster scoring method to identify messenger RNA (mRNA) and miRNA interaction pairs and clusters while considering tumor and normal samples jointly. Our method has identified 54 significant clusters for 15 cancer types selected from The Cancer Genome Atlas project. We also determined the shared clusters across tumor types and/or subtypes. In addition, we compared gene and miRNA overlap between lists identified in our liver hepatocellular carcinoma (LIHC) study and regulatory relationships reported from human and rat nonalcoholic fatty liver disease studies (NAFLD). Finally, we analyzed biological functions for the single significant cluster in LIHC and uncovered a significantly enriched pathway (phospholipase D signaling pathway) with six genes represented in the cluster, symbols: DGKQ, LPAR2, PDGFRB, PIK3R3, PTGFR and RAPGEF3.


Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , RNA Mensageiro/genética , Algoritmos , Carcinoma Hepatocelular/genética , Genoma Humano , Genômica/métodos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo
13.
Cell Host Microbe ; 26(4): 551-563.e6, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31540829

RESUMO

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.


Assuntos
Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Fagocitose/imunologia , Proteínas/genética , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Células U937 , Fatores de Virulência/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
Immunol Rev ; 291(1): 134-153, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31402496

RESUMO

T cells play important roles in autoimmune diseases and cancer. Following the cloning of the T cell receptor (TCR), the race was on to map signaling proteins that contributed to T cell activation downstream of the TCR as well as co-stimulatory molecules such as CD28. We term this "canonical TCR signaling" here. More recently, it has been appreciated that T cells need to accommodate increased metabolic needs that stem from T cell activation in order to function properly. A central role herein has emerged for mechanistic/mammalian target of rapamycin (mTOR). In this review we briefly cover canonical TCR signaling to set the stage for discussion on mTOR signaling, mRNA translation, and metabolic adaptation in T cells. We also discuss the role of mTOR in follicular helper T cells, regulatory T cells, and other T cell subsets. Our lab recently uncovered that "tonic signals", which pass through proximal TCR signaling components, are robustly and selectively transduced to mTOR to promote baseline translation of various mRNA targets. We discuss insights on (tonic) mTOR signaling in the context of T cell function in autoimmune diseases such as lupus as well as in cancer immunotherapy through CAR-T cell or checkpoint blockade approaches.


Assuntos
Ativação Linfocitária/imunologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Sinalização do Cálcio , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ativação Linfocitária/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Sistemas do Segundo Mensageiro
15.
Elife ; 82019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31433293

RESUMO

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of ß1 adrenergic receptors (ßARs) on cardiac myocytes. Blocking these receptors is part of the basis for ß-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including ßARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi ßARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface ßAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident ß1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious ß-blocker therapies.


Assuntos
Cardiomegalia/fisiopatologia , Complexo de Golgi/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Animais , Complexo de Golgi/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hidrólise , Miócitos Cardíacos/enzimologia , Ratos
16.
Oncogene ; 38(37): 6429-6444, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332286

RESUMO

Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase and a critical regulator of a variety of cellular processes as well as tumor progression. Therefore, more substrates must be identified in the physiology or disease context. Here, we found that TRIM65 is upregulated and associated with poor survival in colorectal cancer (CRC). More specifically, high expression of TRIM65 is associated with CRC metastasis and recurrence. Ectopic overexpression of TRIM65 in CRC cell lines enhanced proliferation, invasion, and migration, while knockdown of TRIM65 expression had the opposite effects. Furthermore, we identified a new substrate of TRIM65, namely ARHGAP35, a Rho GTPase-activating protein (GAP) that is involved in polarized cell migration. Phenotypically, forced expression of TRIM65 induces increased production of migration-related structures, focal adhesions, and/or filopodia and enhances CRC metastasis to the liver or the lung in a mouse model. Mechanistic studies revealed that TRIM65 mediates ubiquitination of ARHGAP35, whose degradation leads to elevated Rho GTPase activity. In addition, we identified several phosphorylation sites on TRIM65. In sum, we reveal a novel TRIM65-GAP-Rho regulatory axis that modulates the actin cytoskeleton and the migration behavior of CRC cells, and the TRIM65-ARHGAP35 interaction might be a valuable therapeutic target in CRC.


Assuntos
Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Proteínas com Motivo Tripartido/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CACO-2 , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
17.
EMBO J ; 38(14): e99299, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304629

RESUMO

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.


Assuntos
Adenocarcinoma/metabolismo , Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Quinases Associadas a rho/metabolismo , Adenocarcinoma/genética , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Organoides/citologia , Organoides/metabolismo , RNA Interferente Pequeno/farmacologia , Quinases Associadas a rho/genética
18.
Int J Clin Oncol ; 24(11): 1350-1358, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31309383

RESUMO

BACKGROUND: PLEKHG5, a Rho-specific guanine-nucleotide exchange factor, is involved in tumor cell migration, invasion and angiogenic potential. In this study, the expression pattern, prognostic value and function of PLEKHG5 in gliomas were investigated. METHODS: Immunohistochemistry was used to determine the expression pattern of PLEKHG5 in 61 glioma patients after curative resection. Statistical analysis was performed to evaluate the diagnostic and prognostic significance of PLEKHG5. Gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis and Gene set enrichment analysis (GSEA) were used to predict potential functions of PLEKHG5. Migration assay and western blot analysis determined PLEKHG5 function in glioma migration and invasion. RESULTS: Increased PLEKHG5 expression levels were associated with higher glioma grades (P < 0.05). In addition, glioblastomas multiforme have higher ratio and stronger intensity of PLEKHG5 expression compared with low-grade gliomas. High expression level of PLEKHG5 indicated poorer prognosis and shorter survival time in all glioma patients (P < 0.001). GO analysis, KEGG pathway analysis and GSEA analysis suggested that PLEKHG5 was involved in glioma migration, invasion and epithelial-mesenchymal transition. Migration assay and western blot analysis revealed PLEKHG5 promoted glioma migration and invasion. CONCLUSION: Our results demonstrated PLEKHG5 could be used as a novel prognostic biomarker and anti-tumor target for glioma patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Adulto , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioma/genética , Glioma/mortalidade , Glioma/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico
19.
Nat Commun ; 10(1): 3084, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300652

RESUMO

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.


Assuntos
Proteínas do Domínio Armadillo/ultraestrutura , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Bovinos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X
20.
Eur J Pharmacol ; 858: 172514, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31265841

RESUMO

Recently, we reported the role of coixol (6-methoxy-2(3H)-benzoxazolone), an alkaloid from Scoparia dulcis, in glucose-dependent insulin secretion; however, its insulin secretory mechanism(s) remained unknown. Here, we explored the insulinotropic mechanism(s) of coixol in vitro and in vivo. Mice islets were batch incubated, perifused with coixol in the presence of agonists/antagonists, and insulin secretion was measured by ELISA. Intracellular cAMP levels were measured using enzyme immunoassay. K+- and Ca2+-currents were recorded in MIN6 cells using whole-cell patch-clamp technique. The in vivo glucose tolerance and the insulinogenic index were evaluated in diabetic rats treated with coixol at 25 and 50 mg/kg, respectively. Coixol, unlike sulfonylurea, enhanced insulin secretion in batch incubated and perifused islets at high glucose, with no effect at basal glucose concentrations. Coixol showed no pronounced effect on the inward rectifying K+- and Ca2+-currents in whole-cell patch recordings. Moreover, coixol-induced insulin secretion was further amplified in the depolarized islets. Coixol showed an additive effect with forskolin (10 µM)-induced cAMP level, and in insulin secretion; however, no additive effect was observed with isobutylmethylxanthine (IBMX, 100 µM)-induced cAMP level, nor in insulin secretion. The PKA inhibitor H-89 (50 µM), and Epac2 inhibitor MAY0132 (50 µM) significantly inhibited the coixol-induced insulin secretion (P < 0.01). Furthermore, insulin secretory kinetics revealed that coixol potentiates insulin secretion in both early and late phases of insulin secretion. In diabetic animals, coixol showed significant improvement in glucose tolerance and on fasting blood glucose levels. These data suggest that coixol amplifies glucose-stimulated insulin secretion by cAMP-mediated signaling pathways.


Assuntos
Benzoxazóis/farmacologia , AMP Cíclico/metabolismo , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Glicemia/metabolismo , Canais de Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Potássio/metabolismo , Ratos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA