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1.
Genes Immun ; 17(6): 321-7, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27307211

RESUMO

G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.


Assuntos
Artrite Reumatoide/genética , Quimiotaxia de Leucócito , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neutrófilos/metabolismo , Artrite Reumatoide/imunologia , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Interleucina-8/metabolismo , Leucopoese , Leucotrieno B4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Polimorfismo de Nucleotídeo Único , Tretinoína/metabolismo
2.
Sci Rep ; 6: 27054, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27270970

RESUMO

Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFß but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Mucosa Respiratória/metabolismo , Ativação Transcricional , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocina CXCL12/fisiologia , Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Linfotoxina-alfa/genética , Linfotoxina-alfa/metabolismo , Camundongos Endogâmicos C57BL , Mucina-1/genética , Mucina-1/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores CXCR4/metabolismo , Mucosa Respiratória/imunologia , Regulação para Cima
3.
Sci Rep ; 6: 23735, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27025979

RESUMO

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. Here, we show that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. We show that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or ß-tubulin in pins(-) mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. Our data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells.


Assuntos
Divisão Celular Assimétrica , Desdiferenciação Celular , Proteínas de Drosophila/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Chaperonas Moleculares/fisiologia , Animais , Encéfalo/citologia , Proteínas de Ciclo Celular , Linhagem Celular , Polaridade Celular , Proliferação de Células , Drosophila melanogaster/citologia , Homeostase , Larva/citologia , Células-Tronco Neurais
4.
Differentiation ; 89(5): 128-36, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26143356

RESUMO

Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis.


Assuntos
Polpa Dentária/citologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Células-Tronco Multipotentes/citologia , Osteogênese/fisiologia , Fator de Necrose Tumoral alfa , Adulto , Idoso , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Pessoa de Meia-Idade , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
5.
PLoS Comput Biol ; 9(12): e1003396, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24348237

RESUMO

Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/fisiologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Polaridade Celular , Saccharomyces cerevisiae/citologia
6.
Biochem Soc Trans ; 40(6): 1373-7, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23176483

RESUMO

Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.


Assuntos
Complexo de Golgi/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Complexo de Golgi/ultraestrutura , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Ligação Proteica , Transporte Proteico
7.
Biochem Soc Trans ; 40(6): 1383-8, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23176485

RESUMO

Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.


Assuntos
Melanossomas/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Membranas Intracelulares/enzimologia , Melanócitos/enzimologia , Transporte Proteico , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab27 de Ligação ao GTP
8.
Biochem Soc Trans ; 40(6): 1421-5, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23176492

RESUMO

Despite over two decades of research, the mechanism of Rab targeting to specific intracellular membranes is still not completely understood. Present evidence suggests that the original hypothesis that the message for targeting resides solely in the hypervariable C-terminus is incorrect, and a second mechanism involving a GDF [GDI (guanine-nucleotide-dissociation inhibitor) displacement factor] to disrupt stable Rab-GDI complexes has only been shown to apply in one case, despite the need for targeting over 60 human Rab proteins. Evidence for the involvement of Rab-effector interactions has only been presented for a few cases or in a very specific context. There is mounting evidence that GEFs (guanine-nucleotide-exchange factors) are essential for membrane targeting, although contributions from additional factors are likely to be of importance, at least in specific cases.


Assuntos
Membranas Intracelulares/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Modelos Biológicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas rab de Ligação ao GTP/química
9.
PLoS One ; 7(10): e48209, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23118954

RESUMO

Date hub proteins have 1 or 2 interaction interfaces but many interaction partners. This raises the question of whether all partner proteins compete for the interaction interface of the hub or if the cell carefully regulates aspects of this process? Here, we have used real-time rendering of protein interaction networks to analyse the interactions of all the 1 or 2 interface hubs of Saccharomyces cerevisiae during the cell cycle. By integrating previously determined structural and gene expression data, and visually hiding the nodes (proteins) and their edges (interactions) during their troughs of expression, we predict when interactions of hubs and their partners are likely to exist. This revealed that 20 out of all 36 one- or two- interface hubs in the yeast interactome fell within two main groups. The first was dynamic hubs with static partners, which can be considered as 'competitive hubs'. Their interaction partners will compete for the interaction interface of the hub and the success of any interaction will be dictated by the kinetics of interaction (abundance and affinity) and subcellular localisation. The second was static hubs with dynamic partners, which we term 'non-competitive hubs'. Regulatory mechanisms are finely tuned to lessen the presence and/or effects of competition between the interaction partners of the hub. It is possible that these regulatory processes may also be used by the cell for the regulation of other, non-cell cycle processes.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Modelos Biológicos , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Actinas/genética , Actinas/metabolismo , Actinas/fisiologia , Ligação Competitiva , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Cadeias Leves de Miosina/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcriptoma
10.
Pharmazie ; 67(3): 253-5, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22530308

RESUMO

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.


Assuntos
Neoplasias do Colo/secundário , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neoplasias da Próstata/secundário , Idoso , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Corantes Fluorescentes , Géis , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Hidrólise , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Análise em Microsséries , Pessoa de Meia-Idade , Tripsina/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
11.
J Clin Invest ; 122(4): 1503-18, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22406535

RESUMO

Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients.


Assuntos
Carcinoma de Células de Transição/secundário , Regulação Neoplásica da Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neoplasias Pulmonares/secundário , Macrófagos/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias da Bexiga Urinária/patologia , Versicanas/biossíntese , Animais , Carcinoma de Células de Transição/imunologia , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/prevenção & controle , Linhagem Celular Tumoral , Quimiocina CCL2/biossíntese , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Quimiocina CCL2/fisiologia , Ácido Clodrônico/farmacologia , Técnicas de Cocultura , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Inflamação , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Prognóstico , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Microambiente Tumoral , Células U937 , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/metabolismo , Versicanas/genética , Versicanas/fisiologia
13.
J Neurosci ; 31(32): 11553-62, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21832186

RESUMO

Proteins containing the G protein regulator (GPR) domain bind the major neural G protein Gα(o) in vitro. However, the biological functions of GPR proteins in neurons remain undefined, and based on the in vitro activities of GPR proteins it is unclear whether these proteins activate or inhibit G protein signaling in vivo. We found that the conserved GPR domain protein AGS-3 activates Gα(o) signaling in vivo to allow Caenorhabditis elegans to alter several behaviors after food deprivation, apparently so that the animals can more effectively seek food. AGS-3 undergoes a progressive change in its biochemical fractionation upon food deprivation, suggesting that effects of food deprivation are mediated by modifying this protein. We analyzed one C. elegans food-regulated behavior in depth; AGS-3 activates Gα(o) in the ASH chemosensory neurons to allow food-deprived animals to delay response to the aversive stimulus octanol. Genetic epistasis experiments show the following: (1) AGS-3 and the guanine nucleotide exchange factor RIC-8 act in ASH in a mutually dependent fashion to activate Gα(o); (2) this activation requires interaction of the GPR domains of AGS-3 with Gα(o); and (3) Gα(o)-GTP is ultimately the signaling molecule that acts in ASH to delay octanol response. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gα(o) signaling in vivo.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Privação de Alimentos/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas Nucleares/fisiologia , Ligação Proteica/genética , Transdução de Sinais/genética
14.
Mol Cell ; 43(4): 540-9, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21855794

RESUMO

Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, we find GLs 1 and 2 act as "decoys" that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Transdução de Sinais , Fuso Acromático/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Ciclo Celular , Polaridade Celular , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura
15.
Cancer Sci ; 102(8): 1476-85, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21518140

RESUMO

Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.


Assuntos
Gangliosídeo G(M3)/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Melanoma Experimental/patologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Camundongos , Antígenos de Histocompatibilidade Menor , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
16.
Biochem J ; 434(3): 445-57, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21171963

RESUMO

RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal ß-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fusão de Membrana , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/fisiologia , Citoplasma/metabolismo , Citosol/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
17.
J Proteome Res ; 9(11): 5668-76, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20858016

RESUMO

Ovarian cancer is a gynecological malignancy with the highest mortality. Chemoresistance is an important subject for the treatment of ovarian cancer, because obtaining significant drug resistance to the first line chemotherapy, paclitaxel, causes major therapeutic obstacles. It is essential to improve the survival rate of ovarian cancer patients by mining the biomarkers indicating the drug resistance and prognosis, and by further understanding underlying mechanisms of drug resistance. In the present study, we established paclitaxel-resistant subline (SKpac) from human epithelial ovarian cancer cell line, SKOV3, and performed comparative analysis of whole proteomes between paclitaxel-resistant SKpac sublines and paclitaxel-sensitive parental SKOV3 cells to identify differentially expressed proteins and useful biomarkers indicating chemoresistance. Proteins related to chemoresistant process were identified by two-dimensional gel electrophoresis (2DE) with mass spectrometry (MALDI-TOF and LC-MS/MS). Eighteen spots were differentially expressed and were identified in SKpac chemoresistant cells compared to SKOV3. The expressions of ALDH 1A1, annexin A1, hnRNP A2, and GDI 2 proteins were validated by Western blot, which was consistent with proteomic analysis. Among the selected proteins, downregulation of hnRNP A2 and GDI 2 was found to be the most significant finding in SKpac cells and chemoresistant ovarian cancer tissues. Our results suggest that hnRNP A2 and GDI 2 may represent potential biomarkers of the paclitaxel-resistant ovarian cancers for tailored cancer therapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Dissociação do Nucleotídeo Guanina/análise , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/análise , Neoplasias Ovarianas/fisiopatologia , Paclitaxel/farmacologia , Linhagem Celular Tumoral , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Proteômica/métodos
18.
Oncol Rep ; 24(2): 465-71, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20596634

RESUMO

Rho GDP dissociation inhibitors (RhoGDIs) are important regulators of the GTP hydrolase activity and biological functions of Rho GTPases. RhoGDI2 has been shown to be a metastasis suppressor in bladder cancer and several other cancers. However, the underlying mechanism, effector targets, and the cognate biological functions of RhoGDI2 are not fully understood. To investigate the possible role of RhoGDI2 in lung cancer tumorigenesis and metastasis, the expression pattern of RhoGDI2 in various lung cancer tissue samples and lung cancer-derived cell lines were profiled at both mRNA and protein levels. Furthermore, possible interplay between PI3K/Akt/mTOR pathway activation/inhibition and RhoGDI2 signalling is examined in lung cancer-related cell lines. Our results suggest that RhoGDI2 is likely to be involved in lung tumor malignancy and metastasis.


Assuntos
Carcinoma/patologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neoplasias Pulmonares/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inibidores de Dissociação do Nucleotídeo Guanina/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
19.
J Clin Invest ; 120(8): 2829-41, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20628200

RESUMO

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil-containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras-induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.


Assuntos
Transformação Celular Neoplásica , Genes ras , Neoplasias Pulmonares/etiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Animais , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Humanos , Camundongos , Células NIH 3T3 , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
20.
Nat Cell Biol ; 12(5): 477-83, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20400958

RESUMO

At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Homeostase , Humanos , Ligação Proteica , Dobramento de Proteína , Prenilação de Proteína , Estabilidade Proteica , Receptor Cross-Talk , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
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