Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
2.
Nature ; 524(7565): 315-21, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26245379

RESUMO

Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.


Assuntos
Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfinanos/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 2/química , Receptores Opioides mu/agonistas , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Relação Estrutura-Atividade
3.
FEBS J ; 291(7): 1506-1529, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38145505

RESUMO

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.


Assuntos
Ácido Caínico , Receptores de Ácido Caínico , Tiazinas , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/agonistas , Ácido Caínico/farmacologia , Óxidos S-Cíclicos , Zinco/metabolismo
4.
J Biol Chem ; 287(15): 12293-308, 2012 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-22303009

RESUMO

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the ß(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.


Assuntos
Proteínas de Transporte/fisiologia , Endocitose , Proteínas Nucleares/fisiologia , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de AMPA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , proteínas de unión al GTP Rab7
5.
Proc Natl Acad Sci U S A ; 107(1): 413-8, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20018661

RESUMO

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.


Assuntos
Carbamatos/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Cinamatos/metabolismo , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Domínios PDZ , Animais , Sítios de Ligação , Células COS , Carbamatos/química , Proteínas de Transporte/química , Proteínas de Transporte/genética , Chlorocebus aethiops , Cinamatos/química , Proteínas do Citoesqueleto , Hipocampo/citologia , Humanos , Modelos Moleculares , Estrutura Molecular , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Biochemistry ; 51(2): 586-96, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22129425

RESUMO

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Metabolismo dos Lipídeos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Domínios PDZ , Proteína Quinase C-alfa/metabolismo , Serina , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas do Citoesqueleto , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/genética , Fosforilação , Transporte Proteico
7.
Org Biomol Chem ; 8(19): 4281-8, 2010 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-20668766

RESUMO

Recently, we described the first small-molecule inhibitor, (E)-ethyl 2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with Calpha-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction. Herein, we explore structure-activity relationships of 1 by introducing subtle modifications of the acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration around the double bond of 1 and analogues was settled by a combination of X-ray crystallography, NMR and density functional theory calculations. Thereby, docking studies were used to correlate biological affinities with structural considerations for ligand-protein interactions. The most potent analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a lead in further studies of PICK1 inhibition.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Domínios PDZ , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas de Transporte/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Relação Estrutura-Atividade
8.
Biochim Biophys Acta ; 1764(4): 671-6, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16488199

RESUMO

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.


Assuntos
Ascomicetos/enzimologia , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Fungos Mitospóricos/enzimologia , Ascomicetos/genética , Clonagem Molecular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Fungos Mitospóricos/genética , Dados de Sequência Molecular , Peso Molecular , Filogenia , Temperatura
9.
J Am Soc Mass Spectrom ; 26(5): 808-817, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25740347

RESUMO

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., ß2-adrenergic receptor [ß2AR], µ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-ß-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of ß2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of ß2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in ß2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.


Assuntos
Bicamadas Lipídicas/química , Modelos Moleculares , Receptor PAR-1/química , Receptores Adrenérgicos beta 2/química , Receptores Opioides mu/química , Sequência de Aminoácidos , Detergentes/química , Medição da Troca de Deutério , Estudos de Viabilidade , Humanos , Cinética , Bicamadas Lipídicas/metabolismo , Maltose/análogos & derivados , Maltose/química , Micelas , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Espectrometria de Massas por Ionização por Electrospray
10.
Structure ; 23(7): 1258-1270, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26073603

RESUMO

PICK1 is a neuronal scaffolding protein containing a PDZ domain and an auto-inhibited BAR domain. BAR domains are membrane-sculpting protein modules generating membrane curvature and promoting membrane fission. Previous data suggest that BAR domains are organized in lattice-like arrangements when stabilizing membranes but little is known about structural organization of BAR domains in solution. Through a small-angle X-ray scattering (SAXS) analysis, we determine the structure of dimeric and tetrameric complexes of PICK1 in solution. SAXS and biochemical data reveal a strong propensity of PICK1 to form higher-order structures, and SAXS analysis suggests an offset, parallel mode of BAR-BAR oligomerization. Furthermore, unlike accessory domains in other BAR domain proteins, the positioning of the PDZ domains is flexible, enabling PICK1 to perform long-range, dynamic scaffolding of membrane-associated proteins. Together with functional data, these structural findings are compatible with a model in which oligomerization governs auto-inhibition of BAR domain function.


Assuntos
Proteínas de Transporte/química , Proteínas Nucleares/química , Animais , Células COS , Cálcio/química , Chlorocebus aethiops , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Soluções , Difração de Raios X
11.
Comb Chem High Throughput Screen ; 14(7): 590-600, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21534917

RESUMO

PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters (Z'≈0.7 and Z≈0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 µM to ~193 µM. Binding to the PICK1 PDZ domain was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 µM) in the FP assay, and was according to fluorescence resonance energy transfer (FRET) experiments capable of inhibiting the interaction between the C-terminus of the GluR2 subunit of the AMPA-type glutamate receptor and PICK1 in live cells. Additional experiments suggested that CSC-03 most likely is an irreversible inhibitor but with specificity for PICK1 since it did not bind three different PDZ domains of PSD-95. Summarized, our data suggest that FP based screening assays might be a widely applicable tool in the search for small molecule inhibitors of PDZ domain interactions.


Assuntos
Ensaios de Triagem em Larga Escala , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Peso Molecular , Proteínas Nucleares/antagonistas & inibidores , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA