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1.
J Biol Chem ; 290(13): 8360-72, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25694433

RESUMO

Integrins are heterodimeric α/ß extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. ß1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin ß1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin ß1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the ß1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin ß1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how ß1-containing integrins interact with and activate Abl family kinases.


Assuntos
Integrina beta1/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Ativação Enzimática , Células HEK293 , Humanos , Integrina beta1/química , Dados de Sequência Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/química
2.
J Neurosci ; 32(8): 2824-34, 2012 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-22357865

RESUMO

Integrins are heterodimeric extracellular matrix receptors that are essential for the proper development of the vertebrate nervous system. We report here that selective loss of integrin ß1 in excitatory neurons leads to reductions in the size and complexity of hippocampal dendritic arbors, hippocampal synapse loss, impaired hippocampus-dependent learning, and exaggerated psychomotor sensitivity to cocaine in mice. Our biochemical and genetic experiments demonstrate that the intracellular tail of integrin ß1 binds directly to Arg kinase and that this interaction stimulates activity of the Arg substrate p190RhoGAP, an inactivator of the RhoA GTPase. Moreover, genetic manipulations that reduce integrin ß1 signaling through Arg recapitulate the integrin ß1 knock-out phenotype in a gene dose-sensitive manner. Together, these results describe a novel integrin ß1-Arg-p190RhoGAP pathway that regulates dendritic arbor size, promotes synapse maintenance, supports proper hippocampal function, and mitigates the behavioral consequences of cocaine exposure.


Assuntos
Dendritos/metabolismo , Comportamento Exploratório/fisiologia , Integrina beta1/metabolismo , Neurônios/citologia , Transdução de Sinais/genética , Sinapses/fisiologia , alfa-Fetoproteínas/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Células Cultivadas , Cocaína/administração & dosagem , Dendritos/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Comportamento Exploratório/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/ultraestrutura , Imunoprecipitação , Integrina beta1/genética , Masculino , Camundongos , Camundongos Knockout , Mutação/fisiologia , Proteínas do Tecido Nervoso/deficiência , Neurônios/fisiologia , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Densidade Pós-Sináptica/genética , Densidade Pós-Sináptica/patologia , Densidade Pós-Sináptica/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/ultraestrutura , alfa-Fetoproteínas/genética , Domínios de Homologia de src/efeitos dos fármacos , Domínios de Homologia de src/fisiologia
3.
J Cell Sci ; 122(Pt 19): 3441-54, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19759284

RESUMO

The Abl-family non-receptor tyrosine kinases are essential regulators of the cytoskeleton. They transduce diverse extracellular cues into cytoskeletal rearrangements that have dramatic effects on cell motility and morphogenesis. Recent biochemical and genetic studies have revealed several mechanisms that Abl-family kinases use to mediate these effects. Abl-family kinases stimulate actin polymerization through the activation of cortactin, hematopoietic lineage cell-specific protein (HS1), WASp- and WAVE-family proteins, and Rac1. They also attenuate cell contractility by inhibiting RhoA and altering adhesion dynamics. These pathways impinge on several physiological processes, including development and maintenance of the nervous and immune systems, and epithelial morphogenesis. Elucidating how Abl-family kinases are regulated, and where and when they coordinate cytoskeletal changes, is essential for garnering a better understanding of these complex processes.


Assuntos
Movimento Celular , Morfogênese , Família Multigênica , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Transdução de Sinais
4.
Mol Biol Cell ; 18(10): 3860-72, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17652459

RESUMO

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.


Assuntos
Actomiosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibronectinas/metabolismo , Adesões Focais/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Adesão Celular , Movimento Celular , Fibroblastos/citologia , Fibroblastos/enzimologia , Camundongos , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/deficiência , Fibras de Estresse/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo
5.
ACS Med Chem Lett ; 11(6): 1205-1212, 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32551002

RESUMO

Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27-a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.

6.
Mol Biol Cell ; 17(11): 4827-36, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16971514

RESUMO

The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.


Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Integrinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Sítios de Ligação , Adesão Celular/fisiologia , Proteínas de Ligação a DNA/química , Fibroblastos/citologia , Genes Dominantes/genética , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Tirosina Quinases/deficiência , Ratos , Proteínas Repressoras/química , Proteínas rho de Ligação ao GTP/metabolismo
7.
J Neurosci ; 27(41): 10982-92, 2007 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-17928439

RESUMO

The RhoA (Rho) GTPase is a master regulator of dendrite morphogenesis. Rho activation in developing neurons slows dendrite branch dynamics, yielding smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes dendritic spine loss and dendritic regression, indicating that Rho can affect dendritic structure and function even after dendrites have developed. However, it is unclear whether and how endogenous Rho modulates dendrite and synapse morphology after dendrite arbor development has occurred. We demonstrate that a Rho inhibitory pathway involving the Arg tyrosine kinase and p190RhoGAP is essential for synapse and dendrite stability during late postnatal development. Hippocampal CA1 pyramidal dendrites develop normally in arg-/- mice, reaching their mature size by postnatal day 21 (P21). However, dendritic spines do not undergo the normal morphological maturation in these mice, leading to a loss of hippocampal synapses and dendritic branches by P42. Coincident with this synapse and dendrite loss, arg-/- mice exhibit progressive deficits in a hippocampus-dependent object recognition behavioral task. p190RhoGAP localizes to dendritic spines, and its activity is reduced in arg-/- hippocampus, leading to increased Rho activity. Although mutations in p190rhogap enhance dendritic regression resulting from decreased Arg levels, reducing gene dosage of the Rho effector ROCKII can suppress the dendritic regression observed in arg-/- mice. Together, these data indicate that signaling through Arg and p190RhoGAP acts late during synaptic refinement to promote dendritic spine maturation and synapse/dendrite stability by attenuating synaptic Rho activity.


Assuntos
Arginina/fisiologia , Espinhas Dendríticas/fisiologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/fisiologia , Hipocampo/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Arginina/deficiência , Arginina/genética , Dendritos/genética , Dendritos/fisiologia , Espinhas Dendríticas/genética , Proteínas Ativadoras de GTPase/genética , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/genética , Inibição Neural/genética , Sinapses/genética
8.
Antiviral Res ; 67(1): 10-7, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15950748

RESUMO

A novel non-chain terminating nucleoside analog anti-HIV inhibitor, KP-1212 has been designed to form base pairs with multiple bases that may lead to mutagenesis in the HIV-1 viral genome. After multiple replication cycles, the accumulation of mutations surpasses a crucial threshold beyond which the virus can no longer replicate. HIV-1 reverse transcriptase (RT) incorporates the KP-1212 monophosphate into the genome during viral replication after metabolic activation of the KP-1212 nucleoside to the triphosphate. The propensity for forming alternate base pairs with the KP-1212 nucleotide leads to mismatched nucleotides and the subsequent misincorporation is the basis for the inhibitory activity. The results showed that HIV-1 RT and human mitochondrial DNA polymerase (Pol gamma) incorporated KP-1212-TP with a significant level of efficiency, whereas mouse DNA polymerase beta (Pol beta) did not. Misincorporation studies suggest that both HIV-1 RT and Pol gamma may cause mutations at significantly high rates. These in vitro data confirm the mechanistic basis of KP-1212 as a viral mutagen but suggest that there may be a potential for toxicity to the mitochondria.


Assuntos
Fármacos Anti-HIV/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Transcriptase Reversa do HIV/metabolismo , Mutação , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Ligação Competitiva , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/efeitos dos fármacos , Humanos , Mitocôndrias/enzimologia , Inibidores da Síntese de Ácido Nucleico
9.
Chem Biol ; 21(11): 1463-75, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25457180

RESUMO

The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Histonas/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/toxicidade , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Histonas/química , Humanos , Cinética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Metilação , Camundongos , Camundongos Nus , Mutação , Peptídeos/análise , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Transplante Heterólogo
10.
Cancer Res ; 72(3): 779-89, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22180495

RESUMO

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Indóis/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Bevacizumab , Western Blotting , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Capecitabina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Erlotinib , Fluoruracila/administração & dosagem , Fluoruracila/análogos & derivados , Células HCT116 , Células HT29 , Humanos , Indóis/administração & dosagem , Indóis/farmacocinética , Irinotecano , Estimativa de Kaplan-Meier , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Quinazolinas/administração & dosagem , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Vemurafenib
11.
Cancer Res ; 72(4): 969-78, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22205714

RESUMO

A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Sulfonamidas/uso terapêutico , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imidazolidinas/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos SCID , Mutação , Fenilbutiratos/administração & dosagem , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/efeitos dos fármacos , Transfecção , Vemurafenib , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Arch Physiol Biochem ; 117(3): 165-76, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21671708

RESUMO

The intestine is an important metabolic organ that has gained attention in recent years for the newly identified role that it plays in the pathophysiology of various metabolic diseases including obesity, insulin resistance and diabetes. Recent insights regarding the role of enteroendocrine hormones, such as GIP, GLP-1, and PYY in metabolic diseases, as well as the emerging role of the gut microbial community and gastric bypass bariatric surgeries in modulating metabolic function and dysfunction have sparked a wave of interest in understanding the mechanisms involved, in an effort to identify new therapeutics and novel regulators of metabolism. This review summarizes the current evidence that the gastrointestinal tract has a key role in the development of obesity, inflammation, insulin resistance and diabetes and discusses the possible players that can be targeted for therapeutic intervention.


Assuntos
Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/fisiopatologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/fisiopatologia , Animais , Cirurgia Bariátrica , Diabetes Mellitus Tipo 2/fisiopatologia , Polipeptídeo Inibidor Gástrico/metabolismo , Hormônios Gastrointestinais/metabolismo , Trato Gastrointestinal/microbiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Inflamação/fisiopatologia , Resistência à Insulina/fisiologia , Metagenoma , Obesidade/fisiopatologia , Obesidade/cirurgia , Peptídeo YY/metabolismo
13.
J Biol Chem ; 278(27): 24651-7, 2003 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-12714598

RESUMO

The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers. This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel. Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation. Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features. Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity. Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47. I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis. However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost. Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol. G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type. These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces.


Assuntos
Bacillus thuringiensis/enzimologia , Fosfolipases Tipo C/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ativação Enzimática/genética , Corantes Fluorescentes , Modelos Moleculares , Mutação , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Fosfotransferases/genética , Fosfotransferases/metabolismo , Conformação Proteica , Triptofano , Fosfolipases Tipo C/genética
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