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1.
Biochem Biophys Res Commun ; 565: 85-90, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34102474

RESUMO

GTP-bound forms of Ras proteins (Ras•GTP) assume two interconverting conformations, "inactive" state 1 and "active" state 2. Our previous study on the crystal structure of the state 1 conformation of H-Ras in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp) indicated that state 1 is stabilized by intramolecular hydrogen-bonding interactions formed by Gln61. Since Ras are constitutively activated by substitution mutations of Gln61, here we determine crystal structures of the state 1 conformation of H-Ras•GppNHp carrying representative mutations Q61L and Q61H to observe the effect of the mutations. The results show that these mutations alter the mode of hydrogen-bonding interactions of the residue 61 with Switch II residues and induce conformational destabilization of the neighboring regions. In particular, Q61L mutation results in acquirement of state 2-like structural features. Moreover, the mutations are likely to impair an intramolecular structural communication between Switch I and Switch II. Molecular dynamics simulations starting from these structures support the above observations. These findings may give a new insight into the molecular mechanism underlying the aberrant activation of the Gln61 mutants.


Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Cristalografia por Raios X , Guanosina Trifosfato/genética , Humanos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética
2.
Respir Res ; 20(1): 9, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30634975

RESUMO

BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/metabolismo , Quimiocina CXCL5/biossíntese , Neutrófilos/metabolismo , Fosfoinositídeo Fosfolipase C/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Células Cultivadas , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Distribuição Aleatória
3.
Biochemistry ; 57(36): 5350-5358, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30141910

RESUMO

The ras oncogene products (H-Ras, K-Ras, and N-Ras) have been regarded as some of the most promising targets for anticancer drug discovery because their activating mutations are frequently found in human cancers. Nonetheless, molecular targeted therapy for them is currently unavailable. Here, we report the discovery of a small-molecule compound carrying a naphthalene ring, named KBFM123, which binds to the GTP-bound form of H-Ras. The solution structure of its complex with the guanosine 5'-(ß,γ-imide) triphosphate-bound form of H-RasT35S (H-RasT35S·GppNHp) indicates that the naphthalene ring of KBFM123 interacts directly with a hydrophobic pocket located between switch I and switch II and allosterically inhibits the effector interaction by inducing conformational changes in switch I and its flanking region in strand ß2, which are directly involved in recognition of the effector molecules, including c-Raf-1. In particular, Asp38 of H-Ras, a crucial residue for the interaction with c-Raf-1 via the formation of a salt bridge with Arg89 of the Ras-binding domain (RBD) of c-Raf-1, shows a drastic conformational change: its side chain orients toward the opposite direction. Consistent with these results, KBFM123 exhibits an activity to inhibit, albeit weakly, the association of H-RasG12V·GppNHp with the c-Raf-1 RBD. The binding of the naphthalene ring to the hydrophobic pocket of H-RasT35S·GppNHp is further supported by nuclear magnetic resonance analyses showing that two other naphthalene-containing compounds with distinct structures also exhibit similar binding properties with KBFM123. These results indicate that the naphthalene ring could become a promising scaffold for the development of Ras inhibitors.


Assuntos
Guanosina Trifosfato/metabolismo , Naftalenos/química , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Catálise , Domínio Catalítico , Descoberta de Drogas , Humanos , Modelos Moleculares , Conformação Proteica
4.
Cancer Lett ; 410: 82-91, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28951129

RESUMO

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-rasG12Vgene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of RasG12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-rasG12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes.


Assuntos
Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pancreáticas/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos , Camundongos Nus , Mutação , Células NIH 3T3 , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Mol Graph Model ; 77: 51-63, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28837923

RESUMO

The state transitions of solvated H-Ras protein with GTP were theoretically analyzed through molecular dynamics (MD) simulations. To accelerate the structural changes associated with the locations of two switch regions (I and II), the Parallel Cascade Selection MD (PaCS-MD) method was employed in this study. The interconversions between the State 1 and State 2 were thus studied in atomic details, leading to a reasonable agreement with experimental observations and consequent scenarios concerning the transition mechanism that would be essential for the development of Ras inhibitors as anti-cancer agents. Furthermore, the state-transition-based local network entropy (SNE) was calculated for the transition process from State 1 to State 2, by which the temporal evolution of information entropy associated with the dynamical behavior of hydrogen bond network composed of hydration water molecules was described. The calculated results of SNE thus proved to provide a good indicator to detect the dynamical state transition of solvated Ras protein system (and probably more general systems) from a viewpoint of nonequilibrium statistical thermodynamics.


Assuntos
Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Termodinâmica , Entropia , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Água/química
6.
FEBS Lett ; 591(16): 2470-2481, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28730604

RESUMO

Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.


Assuntos
Guanosina Trifosfato/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas ras/química , Proteínas ras/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica
7.
J Biol Chem ; 291(24): 12586-12600, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27053111

RESUMO

Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon.


Assuntos
Células Epiteliais/metabolismo , NF-kappa B/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Células CACO-2 , Colite/genética , Colite/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Citoplasma/enzimologia , Humanos , Proteínas I-kappa B/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , Fosfoinositídeo Fosfolipase C/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
8.
J Biochem ; 158(2): 91-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26100833

RESUMO

Despite the importance of ras as driver genes in many cancers, clinically effective anti-cancer drugs targeting their products, Ras, have been unavailable so far, which was in part ascribable to the apparently 'undruggable' nature of their tertiary structures. Nonetheless, recent studies in academia and industry have identified novel surface pockets accepting small-molecule ligands in both their active GTP-bound and inactive GDP-bound forms (Ras•GTP and Ras•GDP, respectively), which has led to a surge of investigations into the discovery of Ras-specific inhibitors particularly by utilizing their structural information for structure-based drug design (SBDD). We have been developing Ras inhibitors by SBDD targeting a novel conformation of Ras•GTP called state 1, possessing 'druggable' surface pockets, which emerges from the conformational dynamics. In this article, we will survey Ras functions from the structural biological point of view and summarize the current status of the development of Ras inhibitors including our own.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas ras/antagonistas & inibidores , Animais , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/química , Proteínas ras/metabolismo
9.
PLoS One ; 9(9): e108373, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25269075

RESUMO

BACKGROUND: Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma. METHODS: We prepared a mouse model of allergic asthma using PLCε+/+ mice and PLCεΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells. RESULTS: After OVA challenge, the OVA-immunized PLCεΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCεΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCεΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency. CONCLUSIONS: PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.


Assuntos
Asma/enzimologia , Brônquios/enzimologia , Hiper-Reatividade Brônquica/enzimologia , Células Epiteliais/enzimologia , Fosfoinositídeo Fosfolipase C/imunologia , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Brônquios/imunologia , Brônquios/patologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Knockout , Ovalbumina , Fosfoinositídeo Fosfolipase C/deficiência , Fosfoinositídeo Fosfolipase C/genética , Cultura Primária de Células , Mucosa Respiratória/enzimologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais , Equilíbrio Th1-Th2/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Células Th2/patologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/imunologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/imunologia
10.
Proc Natl Acad Sci U S A ; 110(20): 8182-7, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23630290

RESUMO

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.


Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Biologia Computacional/métodos , Glutationa Transferase/metabolismo , Guanosina Trifosfato/química , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Mutação , Células NIH 3T3 , Transplante de Neoplasias , Ligação Proteica , Conformação Proteica , Transdução de Sinais
11.
Cell Signal ; 25(6): 1361-71, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23499910

RESUMO

The small GTPase Rac1 plays a pivotal role in insulin-stimulated glucose uptake in skeletal muscle, which is mediated by GLUT4 translocation to the plasma membrane. However, regulatory mechanisms for Rac1 and its role in the signaling pathway composed of phosphoinositide 3-kinase and the serine/threonine kinase Akt remain obscure. Here, we investigate the role of Akt in the regulation of Rac1 in myocytes. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt inhibitor IV. Insulin-induced Rac1 activation, on the other hand, was completely inhibited by this inhibitor. Constitutively activated phosphoinositide 3-kinase induced Rac1 activation and GLUT4 translocation. This GLUT4 translocation was almost completely suppressed by Rac1 knockdown. Furthermore, constitutively activated phosphoinositide 3-kinase-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt2 knockdown. Finally, insulin-induced Rac1 activation was indeed inhibited by Akt2 knockdown. Together, these results reveal a novel regulatory mechanism involving Akt2 for insulin-dependent Rac1 activation.


Assuntos
Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética
12.
Enzymes ; 34 Pt. B: 1-23, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-25034098

RESUMO

Ras proteins, particularly their active GTP-bound forms (Ras·GTP), were thought "undruggable" owing to the absence of apparent drug-accepting pockets in their crystal structures. Only recently, such pockets have been found in the crystal structures representing a novel Ras·GTP conformation. We have conducted an in silico docking screen targeting a pocket in the crystal structure of M-Ras(P40D)·GTP and obtained Kobe0065, which, along with its analogue Kobe2602, inhibits binding of H-Ras·GTP to c-Raf-1. They inhibit the growth of H-rasG12V-transformed NIH3T3 cells, which are accompanied by downregulation of not only MEK/ERK but also Akt, RalA, and Sos, indicating the blockade of interaction with multiple effectors. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma carrying K-rasG12V. The nuclear magnetic resonance structure of a complex of the compound with H-Ras(T35S)·GTP confirms its insertion into the surface pocket. Thus, these compounds may serve as a novel scaffold for the development of Ras inhibitors with higher potency and specificity.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Neoplasias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Animais , Descoberta de Drogas , Humanos , Neoplasias/metabolismo , Bibliotecas de Moléculas Pequenas
13.
FEBS Lett ; 586(12): 1715-8, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22584058

RESUMO

GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.


Assuntos
Guanosina Trifosfato/química , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras)/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/genética
14.
J Biol Chem ; 286(45): 39644-53, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-21930707

RESUMO

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and (31)P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5'-(ß, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone (1)H,(15)N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.


Assuntos
Guanosina Trifosfato/química , Proteínas Proto-Oncogênicas p21(ras)/química , Substituição de Aminoácidos , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-Atividade
15.
Biochem Biophys Res Commun ; 414(1): 106-11, 2011 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-21951843

RESUMO

Phospholipase Cε (PLCε) is a unique class of PLC regulated by both Ras family small GTPases and heterotrimeric G proteins. We previously showed by using mice bearing its null or transgenic allele that PLCε plays a crucial role in various forms of skin inflammation through upregulation of proinflammatory cytokine production from keratinocytes. However, molecular mechanisms how PLCε augments cytokine production were largely unknown. We show here using cultured human keratinocyte PHK16-0b cells that induction of the expression of chemokine (C-C motif) ligand 2 (CCL2) following stimulation with tumor necrosis factor (TNF)α, which primarily depends on the activation of the NF-κB pathway, is abrogated by small interfering RNA-mediated knockdown of PLCε. Enforced expression of PLCε causes substantial CCL2 expression and cooperates with low level TNFα stimulation to induce marked overexpression of CCL2, both of which are only partially blocked by pharmacological inhibition of the NF-κB signaling. However, PLCε knockdown exhibits no effect on both the NF-κB-cis-element-mediated transcription per se and the post-translational modifications of NF-κB implicated in transcriptional regulation, suggesting that PLCε constitutes a yet unknown signaling pathway distinct from the NF-κB pathway. This pathway can cooperate with the NF-κB pathway to achieve a synergistic TNFα-stimulated CCL2 induction in keratinocytes.


Assuntos
Quimiocina CCL2/biossíntese , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/efeitos dos fármacos , Camundongos , Fosfoinositídeo Fosfolipase C/genética , Fator de Necrose Tumoral alfa/farmacologia
16.
J Biol Chem ; 286(17): 15403-12, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21388959

RESUMO

GTP-bound forms of Ras family small GTPases exhibit dynamic equilibrium between two interconverting conformations, "inactive" state 1 and "active" state 2. A great variation exists in their state distribution; H-Ras mainly adopts state 2, whereas M-Ras predominantly adopts state 1. Our previous studies based on comparison of crystal structures representing state 1 and state 2 revealed the importance of the hydrogen-bonding interactions of two flexible effector-interacting regions, switch I and switch II, with the γ-phosphate of GTP in establishing state 2 conformation. However, failure to obtain both state structures from a single protein hampered further analysis of state transition mechanisms. Here, we succeed in solving two crystal structures corresponding to state 1 and state 2 from a single Ras polypeptide, M-RasD41E, carrying an H-Ras-type substitution in residue 41, immediately preceding switch I, in complex with guanosine 5'-(ß,γ-imido)triphosphate. Comparison among the two structures and other state 1 and state 2 structures of H-Ras/M-Ras reveal two new structural features playing critical roles in state dynamics; interaction of residues 31/41 (H-Ras/M-Ras) with residues 29/39 and 30/40, which induces a conformational change of switch I favoring its interaction with the γ-phosphate, and the hydrogen-bonding interaction of switch II with its neighboring α-helix, α3-helix, which induces a conformational change of switch II favoring its interaction with the γ-phosphate. The importance of the latter interaction is proved by mutational analyses of the residues involved in hydrogen bonding. These results define the two novel functional regions playing critical roles during state transition.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Homologia Estrutural de Proteína , Proteínas ras
17.
Lab Invest ; 91(5): 711-8, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21321537

RESUMO

Phospholipase C (PLC) ɛ is a phosphoinositide-specific PLC regulated by small GTPases including Ras and Rap. We previously demonstrated that PLCɛ has an important role in the development of phorbol ester-induced skin inflammation. In this study, we investigated the role of PLCɛ in ultraviolet (UV) B-induced acute inflammatory reactions in the skin. Wild-type (PLCɛ+/+) and PLCɛ gene knockout (PLCɛ⁻/⁻) mice were irradiated with a single dose of UVB at 1, 2.5, and 10 kJ/m² on the dorsal area of the skin, and inflammatory reactions in the skin were histologically evaluated up to 168 h after irradiation. In PLCɛ+/+ mice, irradiation with 1 and 2.5 kJ/m² UVB resulted in dose-dependent neutrophil infiltration in the epidermis at 24 and 48 h after irradiation. When mice were irradiated with 10 kJ/m² of UVB, most mice developed skin ulcers by 48 h and these ulcers became more severe at 168 h. In PLCɛ⁻/⁻ mice, UVB (1 or 2.5 kJ/m²)-induced neutrophil infiltration was markedly suppressed compared with PLCɛ+/+ mice. The suppression of neutrophil infiltration in PLCɛ⁻/⁻ mice was accompanied by attenuation of UVB-induced production of CXCL1/keratinocyte-derived chemokine (KC), a potent chemokine for neutrophils, in the whole skin. Cultured epidermal keratinocytes and dermal fibroblasts produced CXCL1/KC in a PLCɛ-dependent manner after UVB irradiation, and the UVB-induced upregulation of CXCL1/KC in these cells was significantly abolished by a PLC inhibitor. Furthermore, UVB-induced epidermal thickening was noticeably reduced in the skin of PLCɛ⁻/⁻ mice. These results indicate that PLCɛ has a crucial role in UVB-induced acute inflammatory reactions such as neutrophil infiltration and epidermal thickening by at least in part regulating the expression of CXCL1/KC in skin cells such as keratinocytes and fibroblasts.


Assuntos
Quimiocina CXCL1/genética , Dermatite/etiologia , Regulação da Expressão Gênica/fisiologia , Neutrófilos/imunologia , Fosfoinositídeo Fosfolipase C/fisiologia , Raios Ultravioleta , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Dermatite/imunologia , Ensaio de Imunoadsorção Enzimática , Camundongos , Fosfoinositídeo Fosfolipase C/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Eur J Immunol ; 41(1): 202-13, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21182091

RESUMO

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases. We showed previously using PLCε-deficient mice that PLCε plays a critical role in activation of cytokine production in non-immune skin cells in a variety of inflammatory reactions. For further investigation of its role in inflammation, we created transgenic mice overexpressing PLCε in epidermal keratinocytes. The resulting transgenic mice spontaneously developed skin inflammation as characterized by formation of adherent silvery scales, excessive growth of keratinocytes, and aberrant infiltration of immune cells such as T cells and DC. Development of the skin symptoms correlated well with increased expression of factors implicated in human inflammatory skin diseases, such as IL-23, in keratinocytes, and with the accumulation of CD4(+) T cells producing IL-22, a potent inducer of keratinocyte proliferation. Intradermal injection of a blocking antibody against IL-23 as well as treatment with the immunosuppressant FK506 reversed these skin phenotypes, which was accompanied by suppression of the IL-22-producing T-cell infiltration. These results reveal a crucial role of PLCε in the development of skin inflammation and suggest a mechanism in which PLCε induces the production of cytokines including IL-23 from keratinocytes, leading to the activation of IL-22-producing T cells.


Assuntos
Citocinas/imunologia , Dermatite/imunologia , Queratinócitos/imunologia , Fosfoinositídeo Fosfolipase C/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Dermatite/enzimologia , Dermatite/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Interleucina-23/análise , Interleucina-23/antagonistas & inibidores , Interleucina-23/imunologia , Interleucinas/análise , Interleucinas/imunologia , Queratinócitos/enzimologia , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoinositídeo Fosfolipase C/análise , Fosfoinositídeo Fosfolipase C/metabolismo , Tacrolimo/farmacologia , Regulação para Cima , Interleucina 22
19.
Carcinogenesis ; 31(10): 1897-902, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20688835

RESUMO

Phospholipase C (PLC) ε is a phosphoinositide-specific PLC regulated by small guanosine triphosphatases including Ras and Rap. Our previous studies revealed that PLCε gene-knockout (PLCε(-/-)) mice exhibit marked resistance to tumor formation in two-stage skin chemical carcinogenesis using 7,12-dimethylbenz(a)anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate as a promoter. In this model, PLCε functions in tumor promotion through augmentation of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. In this study, we have further assessed the role of PLCε in tumorigenesis using a mouse model of ultraviolet (UV) B-induced skin tumor development. We irradiated PLCε(+/+), PLCε(+/-) or PLCε(-/-) mice with doses of UVB increasing from 1 to 10 kJ/m(2) three times a week for a total of 25 weeks and observed tumor formation for up to 50 weeks. In sharp contrast to the results from the two-stage chemical carcinogenesis study, PLCε(-/-) mice developed a large number of neoplasms including malignant tumors, whereas PLCε(+/+) and PLCε(+/-) mice developed a relatively small number of benign tumors. However, UVB-induced skin inflammation was greatly suppressed in PLCε(-/-) mice, as observed with 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, implying that PLCε's role in the suppression of UVB-induced tumorigenesis is not mediated by inflammation. Studies of the tumor initiation stage revealed that UVB-induced cell death in the skin was markedly suppressed in PLCε(-/-)mice. Our findings identify a novel function for PLCε as a critical molecule regulating UVB-induced cell death and suggest that resistance to UVB-induced cell death conferred by the absence of PLCε is closely related to the higher incidence of skin tumor formation.


Assuntos
Apoptose , Neoplasias Induzidas por Radiação/etiologia , Fosfoinositídeo Fosfolipase C/fisiologia , Neoplasias Cutâneas/etiologia , Raios Ultravioleta/efeitos adversos , Animais , Interleucina-10/biossíntese , Interleucina-1beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Induzidas por Radiação/patologia , RNA Mensageiro/análise , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol , Proteína X Associada a bcl-2/fisiologia
20.
J Biol Chem ; 285(29): 22696-705, 2010 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-20479006

RESUMO

Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the (31)P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.


Assuntos
Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas ras
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