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1.
Elife ; 82019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31833473

RESUMO

New methods to directly visualize Rho GTPases reveal how a protein called RhoGDI regulates the activity of these 'molecular switches' at the plasma membrane.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas rho de Ligação ao GTP , Membrana Celular , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
2.
Elife ; 82019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31647414

RESUMO

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane.


Assuntos
Xenopus laevis/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Animais , Extratos Celulares , Membrana Celular/metabolismo , Citocinese , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Exocitose , Proteínas Mutantes/metabolismo , Cicatrização , Proteína cdc42 de Ligação ao GTP/metabolismo
3.
Cells ; 8(9)2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31492019

RESUMO

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.


Assuntos
Neoplasias/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional
4.
Methods Mol Biol ; 2009: 297-306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31152412

RESUMO

The posttranslational lipid modification of Rho GTPases is important for their proper subcellular localization and signal transduction. Rho GTPases terminate in a CaaX motif, in which the cysteine residue is modified with either a farnesyl or geranylgeranyl isoprenoid. RhoGDI renders Rho GTPases soluble by masking their lipid moieties. We recently identified that the brain-specific splice variant of Cdc42 (bCdc42) containing a noncanonical CCaX motif harbors a dual prenyl-palmitoyl modification that prevents its binding to RhoGDI. This chapter describes a method to analyze RhoGDI extraction of Rho GTPases containing different lipid modifications from membranes using a liposome reconstitution assay and click chemistry.


Assuntos
Membrana Celular/química , Química Click , Prenilação de Proteína , Proteína cdc42 de Ligação ao GTP , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Motivos de Aminoácidos , Animais , Células Sf9 , Spodoptera , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/isolamento & purificação , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/isolamento & purificação
5.
Gen Physiol Biophys ; 38(4): 271-280, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31219429

RESUMO

The aim of this study was to investigate the effects of the Rho GDP dissociation inhibitor (RhoGDI) on TGFß1-mediated vascular adventitia myofibroblast transdifferentiation and on the inhibition of ROCK inhibitors. Myofibroblast transdifferentiation and vascular remodeling model were induced by TGFß1 in vitro and by balloon injury in vivo. H&E (Hematoxylin & Eosin) and PSR (Picrosirius Red) staining were used to observe vascular morphology while immunofluorescence, immunohistochemistry, and Western blotting were used to measure protein expression. Fasudil treatment reduced the expression of TGFß1, RhoGDI1, and RhoGDI2 in addition to vascular remodeling in the rat balloon injury model. TGFß1 induced the expression of α-SMA, TGFßRI, phospho-TGFßRI, RhoGDI1, RhoGDI2, and collagen secretion in human aortic adventitial fibroblasts (HAAFs). These effects were diminished after treatment with Y27632. Suppressing both RhoGDI1 and RhoGDI2 expression also blocked TGFß1-induced α-SMA expression and collagen secretion in HAAFs. Moreover, TGFßR inhibition blocked TGFß1-mediated collagen secretion and the expression of α-SMA, RhoGDI1, and RhoGDI2. These data suggested that ROCK inhibitors alleviate myofibroblast transdifferentiation and vascular remodeling by decreasing TGFß1-mediated expression of RhoGDI.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Remodelação Vascular/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/biossíntese , Animais , Humanos , Ratos
6.
Aging (Albany NY) ; 10(12): 4000-4023, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30573703

RESUMO

Naturally-occurring somatic mutations in the estrogen receptor gene (ESR1) have been previously implicated in the clinical development of resistance to hormonal therapies, such as Tamoxifen. For example, the somatic mutation Y537S has been specifically associated with acquired endocrine resistance. Briefly, we recombinantly-transduced MCF7 cells with a lentiviral vector encoding ESR1 (Y537S). As a first step, we confirmed that MCF7-Y537S cells are indeed functionally resistant to Tamoxifen, as compared with vector alone controls. Importantly, further phenotypic characterization of Y537S cells revealed that they show increased resistance to Tamoxifen-induced apoptosis, allowing them to form mammospheres with higher efficiency, in the presence of Tamoxifen. Similarly, Y537S cells had elevated basal levels of ALDH activity, a marker of "stemness", which was also Tamoxifen-resistant. Metabolic flux analysis of Y537S cells revealed a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis. Finally, to understand which molecular signaling pathways that may be hyper-activated in Y537S cells, we performed unbiased label-free proteomics analysis. Our results indicate that TIGAR over-expression and the Rho-GDI/PTEN signaling pathway appear to be selectively activated by the Y537S mutation. Remarkably, this profile is nearly identical in MCF7-TAMR cells; these cells were independently-generated in vitro, suggesting a highly conserved mechanism underlying Tamoxifen-resistance. Importantly, we show that the Y537S mutation is specifically associated with the over-expression of a number of protein markers of poor clinical outcome (COL6A3, ERBB2, STAT3, AFP, TFF1, CDK4 and CD44). In summary, we have uncovered a novel metabolic mechanism leading to endocrine resistance, which may have important clinical implications for improving patient outcomes.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Mitocôndrias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Tamoxifeno/farmacologia , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/genética , Receptor alfa de Estrogênio , Feminino , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Células MCF-7 , Mutação , PTEN Fosfo-Hidrolase/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética
7.
Biochim Biophys Acta Bioenerg ; 1859(9): 984-996, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29626418

RESUMO

Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed "BMF", has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl-CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy.


Assuntos
Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Óleos Vegetais/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida
8.
Radiother Oncol ; 129(1): 30-37, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29519627

RESUMO

BACKGROUND AND PURPOSE: SKLB-163 is a novel benzothiazole-2-thiol derivative with antitumor activities. This study investigated the effects of SKLB-163 on nasopharyngeal carcinoma (NPC) and its mechanisms. MATERIALS AND METHODS: Rho GDP-dissociation inhibitor (RhoGDI) expression was examined in NPC cell lines by western blot. Effects of SKLB-163 on proliferation, migration and radiosensitivity were assessed by MTT, wound healing and colony formation assays in vitro. Anti-tumor and anti-metastatic effects, and radiosensitizing effects of SKLB-163 were evaluated in a NPC lung metastatic nude mouse model and a subcutaneous xenograft mouse model. Effects of SKLB-163 on proliferation and apoptosis were assessed by PCNA immunohistochemistry and TUNEL assay in vivo. Key molecules in RhoGDI/c-Jun N-terminal kinases-1 (JNK-1) pathway were examined by western blot. RESULTS: RhoGDI was up-regulated in NPC cell lines. SKLB-163 inhibited proliferation and migration, and increased radiosensitivity of NPC cells. SKLB-163 inhibited tumor growth and metastases, and sensitized tumor to irradiation. The radiosensitizing effects were correlated with induction of apoptosis and suppression of proliferation. The molecular mechanism was the down-regulation of RhoGDI and activation of JNK-1 signaling, and the subsequent activation of caspase-3 and the decrease in phosphorylated AKT. CONCLUSIONS: SKLB-163 shows strong anti-tumor activities against NPC and sensitizes NPC to irradiation by affecting the RhoGDI/JNK-1 pathway.


Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/patologia , Transplante de Neoplasias/métodos , Fosforilação , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
9.
Nat Commun ; 9(1): 90, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311697

RESUMO

Isoprenylated proteins are associated with membranes and their inter-compartmental distribution is regulated by solubilization factors, which incorporate lipid moieties in hydrophobic cavities and thereby facilitate free diffusion during trafficking. Here we report the crystal structure of a solubilization factor, the prenyl-binding protein (PrBP/δ), at 1.81 Å resolution in its ligand-free apo-form. Apo-PrBP/δ harbors a preshaped, deep hydrophobic cavity, capacitating apo-PrBP/δ to readily bind its prenylated cargo. To investigate the molecular mechanism of cargo solubilization we analyzed the PrBP/δ-induced membrane dissociation of rod photoreceptor phosphodiesterase (PDE6). The results suggest that PrBP/δ exclusively interacts with the soluble fraction of PDE6. Depletion of soluble species in turn leads to dissociation of membrane-bound PDE6, as both are in equilibrium. This "solubilization by depletion" mechanism of PrBP/δ differs from the extraction of prenylated proteins by the similar folded solubilization factor RhoGDI, which interacts with membrane bound cargo via an N-terminal structural element lacking in PrBP/δ.


Assuntos
Proteínas de Transporte/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Neopreno/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Proteínas de Transporte/química , Bovinos , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Neopreno/química , Ligação Proteica , Domínios Proteicos , Prenilação de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo
10.
Sci Rep ; 8(1): 761, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335599

RESUMO

Peripheral ischemia is associated with higher degree of endothelial dysfunction and a worse prognosis after percutaneous coronary interventions (PCI). However, the role of peripheral ischemia on vascular remodeling in remote districts remains poorly understood. Here we show that the presence of hindlimb ischemia significantly enhances neointima formation and impairs endothelial recovery in balloon-injured carotid arteries. Endothelial-derived microRNAs are involved in the modulation of these processes. Indeed, endothelial miR-16 is remarkably upregulated after vascular injury in the presences of hindlimb ischemia and exerts a negative effect on endothelial repair through the inhibition of RhoGDIα and nitric oxide (NO) production. We showed that the repression of RhoGDIα by means of miR-16 induces RhoA, with consequent reduction of NO bioavailability. Thus, hindlimb ischemia affects negative carotid remodeling increasing neointima formation after injury, while systemic antagonizzation of miR-16 is able to prevent these negative effects.


Assuntos
Artérias Carótidas/patologia , Células Endoteliais/patologia , Membro Posterior/patologia , Isquemia/patologia , Neointima , Animais , Modelos Animais de Doenças , MicroRNAs/metabolismo , Óxido Nítrico/metabolismo , Ratos , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo
11.
Sci Rep ; 7(1): 17262, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29222525

RESUMO

Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.


Assuntos
Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Sintenia , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Citoesqueleto/metabolismo , Evolução Molecular , Genômica , Humanos , Filogenia , Regiões Promotoras Genéticas/genética , Ligação Proteica
12.
Int J Radiat Biol ; 93(9): 920-928, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28697312

RESUMO

PURPOSE: Epidemiological studies indicate that radiation doses as low as 0.5 Gy increase the risk of cardiovascular disease decades after the exposure. The aim of the present study was to investigate whether this radiation dose causes late molecular alterations in endothelial cells that could support the population-based data. MATERIALS AND METHODS: Human coronary artery endothelial cells were irradiated at 0.5 Gy (X-ray) and radiation-induced changes in the proteome were investigated after different time intervals (1, 7 and 14 d) using ICPL technology. Key changes identified by proteomics and bioinformatics were validated by immunoblotting and ELISA. RESULTS: The radiation-induced alteration of the endothelial proteome was characterized by sustained perturbation of Rho GDP-dissociation inhibitor (RhoGDI) and nitric oxide (NO) signalling pathways. At later time-points, this was accompanied by reduced proteasome activity, enhanced protein carbonylation indicating augmented oxidative stress, and senescence. CONCLUSIONS: These molecular changes are indicative of long-term premature endothelial dysfunction and provide a mechanistic framework to the epidemiological data showing increased risk of cardiovascular disease at 0.5 Gy.


Assuntos
Células Endoteliais/fisiologia , Células Endoteliais/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Óxido Nítrico/metabolismo , Proteoma/metabolismo , Transdução de Sinais/efeitos da radiação , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Células Cultivadas , Senescência Celular/fisiologia , Senescência Celular/efeitos da radiação , Regulação da Expressão Gênica/fisiologia , Humanos , Doses de Radiação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Raios X
13.
Am J Physiol Cell Physiol ; 312(4): C527-C536, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148498

RESUMO

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.


Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Ativação Plaquetária/fisiologia , Adesividade Plaquetária/fisiologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Células Cultivadas , Hemostasia/fisiologia , Humanos , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismo
14.
Mol Cell Biol ; 37(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28069739

RESUMO

Signaling by the small GTPase Cdc42 governs a diverse set of cellular processes that contribute to tissue morphogenesis. Since these processes often require highly localized signaling, Cdc42 activity must be clustered in order to prevent ectopic signaling. During cell polarization, apical Cdc42 signaling directs the positioning of the nascent apical membrane. However, the molecular mechanisms that drive Cdc42 clustering during polarity establishment are largely unknown. Here, we demonstrate that during cell polarization localized Cdc42 signaling is enabled via activity-dependent control of Cdc42 mobility. By performing photoconversion experiments, we show that inactive Cdc42-GDP is 30-fold more mobile than active Cdc42-GTP. This switch in apical mobility originates from a dual mechanism involving RhoGDI-mediated membrane dissociation of Cdc42-GDP and Tuba-mediated immobilization of Cdc42-GTP. Interference with either mechanism affects Cdc42 clustering and as a consequence impairs Cdc42-mediated apical membrane clustering. We therefore identify a molecular network, comprised of Cdc42, the guanine nucleotide exchange factor (GEF) Tuba, and RhoGDI, that enables differential diffusion of inactive and active Cdc42 and is required to establish localized Cdc42 signaling during enterocyte polarization.


Assuntos
Polaridade Celular , Enterócitos/citologia , Enterócitos/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Microvilosidades/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
15.
Free Radic Biol Med ; 103: 57-68, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27974245

RESUMO

Reactive oxygen species (ROS) produced by many kinds of stimuli are essential for cellular signaling including cell proliferation. The dysregulation of ROS, therefore, is related to a variety of diseases including cancer. However, it was not clearly elucidated how ROS regulate cell proliferation and tumorigenesis. In this study, we investigated a mechanism by which the oxidation of RhoA GTPase regulates nuclear factor-κB (NF-κB) and cell proliferation. Hydrogen peroxide activated NF-κB and RhoA GTPase, but did not activate RhoA C16/20A mutant, an oxidation-resistant form. Remarkably, the oxidation of RhoA reduced its affinity towards RhoGDI, leading to the dissociation of RhoA-RhoGDI complex. Si-Vav2, a guanine nucleotide exchange factor (GEF), inhibited RhoA activation upon hydrogen peroxide. The oxidized RhoA (oxRhoA)-GTP was readily bound to IκB kinase γ (IKKγ), whereas oxidized RhoGDI did not bind to IKKγ. The oxRhoA-GTP bound to IKKγ activated IKKß, leading to IκB phosphorylation and degradation, consequently NF-κB activation. Hydrogen peroxide induced cell proliferation, but RhoA C16/20A mutant suppressed cell proliferation and tumorigenesis. Conclusively, RhoA oxidation at Cys16/20 is critically involved in cell proliferation and tumorigenesis through NF-κB activation in response to ROS.


Assuntos
Proliferação de Células , NF-kappa B/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Feminino , Células HEK293 , Células HT29 , Humanos , Peróxido de Hidrogênio/farmacologia , Quinase I-kappa B/metabolismo , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Oxirredução , Ligação Proteica , Proteínas Proto-Oncogênicas c-vav/metabolismo , Células RAW 264.7 , Transdução de Sinais , Carga Tumoral , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo
16.
Mol Cell Endocrinol ; 436: 23-32, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27424144

RESUMO

Nerve growth factor (NGF) is a protein required for neuronal development that also has regulatory functions in non-neuronal cells. Both NGF and its membrane receptors trkA and p75(NTR) are expressed by islet ß-cells. In this study we dynamically profiled NGF secretion from islets and used selective trkA and p75(NTR) inhibitors to identify the role of endogenous NGF in ß-cell stimulus-secretion coupling. NGF secretion from mouse islets was transient and did not accompany the sustained second phase of glucose-induced insulin secretion. Despite being present in human islets, NGF was not released at sufficient levels to be quantified. Inhibition of NGF signaling through trkA and p75(NTR) increased basal insulin secretion from both human and mouse islets, but impaired glucose-stimulated insulin secretion. These data support a role for islet NGF in fine-tuning insulin secretion, to both maintain a low basal level of insulin output and contribute to the biphasic secretory response to glucose.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fator de Crescimento Neural/metabolismo , Animais , Feminino , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos ICR , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo
18.
Plant Physiol ; 170(2): 841-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26662604

RESUMO

Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Transdução de Sinais , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Homeostase , Mutação , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/fisiologia , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética
19.
Elife ; 4: e11692, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26646181

RESUMO

Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75(NTR)) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75(NTR) DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75(NTR) DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Receptores de Fator de Crescimento Neural/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/química
20.
J Proteome Res ; 14(11): 4674-86, 2015 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-26420666

RESUMO

Recent epidemiological data indicate that radiation doses as low as those used in computer tomography may result in long-term neurocognitive side effects. The aim of this study was to elucidate long-term molecular alterations related to memory formation in the brain after low and moderate doses of γ radiation. Female C57BL/6J mice were irradiated on postnatal day 10 with total body doses of 0.1, 0.5, or 2.0 Gy; the control group was sham-irradiated. The proteome analysis of hippocampus, cortex, and synaptosomes isolated from these brain regions indicated changes in ephrin-related, RhoGDI, and axonal guidance signaling. Immunoblotting and miRNA-quantification demonstrated an imbalance in the synapse morphology-related Rac1-Cofilin pathway and long-term potentiation-related cAMP response element-binding protein (CREB) signaling. Proteome profiling also showed impaired oxidative phosphorylation, especially in the synaptic mitochondria. This was accompanied by an early (4 weeks) reduction of mitochondrial respiration capacity in the hippocampus. Although the respiratory capacity was restored by 24 weeks, the number of deregulated mitochondrial complex proteins was increased at this time. All observed changes were significant at doses of 0.5 and 2.0 Gy but not at 0.1 Gy. This study strongly suggests that ionizing radiation at the neonatal state triggers persistent proteomic alterations associated with synaptic impairment.


Assuntos
Córtex Cerebral/efeitos da radiação , Raios gama/efeitos adversos , Hipocampo/efeitos da radiação , Potenciação de Longa Duração/efeitos da radiação , Proteoma/genética , Transmissão Sináptica/efeitos da radiação , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/efeitos da radiação , Axônios/ultraestrutura , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Efrinas/genética , Efrinas/metabolismo , Feminino , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fosforilação Oxidativa/efeitos da radiação , Proteoma/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/efeitos da radiação , Irradiação Corporal Total , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo
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