RESUMO
AphB is a LysR-type transcriptional regulator (LTTR) that cooperates with a second transcriptional activator, AphA, at the tcpPH promoter to initiate expression of the virulence cascade in Vibrio cholerae. Because it is not yet known whether AphB responds to a natural ligand in V. cholerae that influences its ability to activate transcription, we used a computational approach to identify small molecules that influence its activity. In silico docking was used to identify potential ligands for AphB, and saturation transfer difference nuclear magnetic resonance was subsequently employed to access the validity of promising targets. We identified a small molecule, BP-15, that specifically binds the C-terminal regulatory domain of AphB and increases its activity. Interestingly, molecular docking predicts that BP-15 does not bind in the putative primary effector-binding pocket located at the interface of RD-I and RD-II as in other LTTRs, but rather at the dimerization interface. The information gained in this study helps us to further understand the mechanism by which transcriptional activation by AphB is regulated by suggesting that AphB has a secondary ligand binding site, as observed in other LTTRs. This study also lays the groundwork for the future design of inhibitory molecules to block the V. cholerae virulence cascade, thereby preventing the devastating symptoms of cholera infection.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Multimerização Proteica , Transativadores/química , Vibrio cholerae/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cólera/tratamento farmacológico , Cólera/genética , Ligantes , Domínios Proteicos , Estrutura Quaternária de Proteína , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Fatores de Transcrição/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.
Assuntos
Citoesqueleto de Actina/química , Proteínas dos Microfilamentos/química , Profilinas/química , Dobramento de Proteína , Citoesqueleto de Actina/genética , Motivos de Aminoácidos , Forminas , Humanos , Proteínas dos Microfilamentos/genética , Profilinas/genéticaRESUMO
The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.
Assuntos
Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Quinases Associadas a Fase S/química , Trocadores de Sódio-Hidrogênio/químicaRESUMO
The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals, the infection remains dormant and asymptomatic, but in immuno-suppressed patients, it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection nor for the treatment of PML. Here, we report the development of small-molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively, these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics.
Assuntos
Antivirais/química , Proteínas do Capsídeo/antagonistas & inibidores , Vírus JC/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/síntese química , Sítios de Ligação , Bioensaio , Células COS , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular Transformada , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Vírus JC/crescimento & desenvolvimento , Vírus JC/metabolismo , Simulação de Acoplamento Molecular , Neuroglia/efeitos dos fármacos , Neuroglia/virologia , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKß, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-κB pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44-111) present high thermal stability and cooperative melting and, most importantly, restore IKKß binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKKß(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.
Assuntos
Quinase I-kappa B/química , Engenharia de Proteínas , Sítios de Ligação , Cristalografia por Raios X , Humanos , Quinase I-kappa B/genética , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de ProteínaRESUMO
We have identified a series of small molecules that bind to the canonical peptide binding groove of the PDZ1 domain of NHERF1 and effectively compete with the association of the C-terminus of the parathyroid hormone 1 receptor (PTH1R). Employing nuclear magnetic resonance and molecular modeling, we characterize the mode of binding that involves the GYGF loop important for the association of the C-terminus of PTH1R. We demonstrate that the common core of the small molecules binds to the PDZ1 domain of NHERF1 and displaces a (15)N-labeled peptide corresponding to the C-terminus of PTH1R. The small size (molecular weight of 192) of this core scaffold makes it an excellent candidate for further elaboration in the development of an inhibitor for this important protein-protein interaction.
Assuntos
Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Fosfoproteínas/química , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Trocadores de Sódio-Hidrogênio/químicaRESUMO
We designed and characterized a soluble mimic of the parathyroid hormone (PTH) receptor (PTH1R) that incorporates the N-terminus and third extracellular loop of PTH1R, important for ligand binding. The engineered receptor (PTH1R-NE3) was conceived to enable easy production and the use of standard biochemical and biophysical assays for the screening of competitive antagonists of PTH. We show that PTH1R-NE3 is folded, thermodynamically stable and selectively binds PTH. We also demonstrate the utility of our mimic by identifying a small molecule that competes with PTH in our PTH1R-NE3-based fluorescence polarization assay. Antagonists to PTH1R, a transmembrane protein belonging to the class B G-protein coupled receptor family, may provide new therapeutic options for calcium metabolism diseases like humoral hypercalcemia of malignancy.
Assuntos
Engenharia de Proteínas/métodos , Receptor Tipo 1 de Hormônio Paratireóideo/química , Proteínas Recombinantes de Fusão/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
RESUMO
The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine-glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3'UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 Variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/farmacologia , Inflamação/imunologia , Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Transportador de Glucose Tipo 1/biossíntese , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Macrófagos/imunologia , Camundongos , Peptídeos/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Células U937 , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
We propose a new molecular dynamics (MD) protocol to identify the binding site of a guest within a host. The method utilizes a four spatial (4D) dimension representation of the ligand allowing for rapid and efficient sampling within the receptor. We applied the method to two different model receptors characterized by diverse structural features of the binding site and different ligand binding affinities. The Abl kinase domain is comprised of a deep binding pocket and displays high affinity for the two chosen ligands examined here. The PDZ1 domain of PSD-95 has a shallow binding pocket that accommodates a peptide ligand involving far fewer interactions and a micromolar affinity. To ensure completely unbiased searching, the ligands were placed in the direct center of the protein receptors, away from the binding site, at the start of the 4D MD protocol. In both cases, the ligands were successfully docked into the binding site as identified in the published structures. The 4D MD protocol is able to overcome local energy barriers in locating the lowest energy binding pocket and will aid in the discovery of guest binding pockets in the absence of a priori knowledge of the site of interaction.
Assuntos
Sítios de Ligação/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Domínios PDZ/genética , Sítios de Ligação/efeitos dos fármacos , Simulação por Computador , Cristalografia por Raios X , Proteína 4 Homóloga a Disks-Large , Descoberta de Drogas/métodos , Transferência de Energia , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Oncogênicas v-abl/efeitos dos fármacos , Proteínas Oncogênicas v-abl/genética , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The dimerization of the G protein-coupled receptors for endothelin-1 (ET-1), endothelin A receptor (ETA) and endolethin B receptor (ETB), is well established. However, the signaling consequences of the homodimerization and heterodimerization of ETA and ETB is not well understood. Here, we demonstrate that peptides derived from the C-termini of these receptors regulate the signaling capacity of ET-1. The C-termini of the ETA and ETB receptors are believed to consist of three α-helices, which may serve as points of interaction between the receptors. The third α-helix in the C-terminus is of particular interest because of its amphipathic nature. In a cell line expressing only the ETA receptor, expression of residues Y430-S442, representing the third helix of the ETB C-terminus, leads to a dramatic increase in the signaling induced by ET-1. In contrast, in a cell line containing only ETB , Y430-S442 has an antagonistic effect, slightly reducing the ET-1 induced signal. Computational docking results suggest that the α-helical ETB -derived peptide binds to the second and third intracellular loops of the ETA receptor consistent with the alteration of its signaling capacity. Our results described here provide important insight into ETA /ETB receptor interactions and possibly a new approach to regulate specific G protein-coupled receptor signal transmission.
Assuntos
Endotelina-1 , Receptor de Endotelina A , Receptor de Endotelina B , Transdução de Sinais/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Endotelina-1/química , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptor de Endotelina A/química , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismoRESUMO
The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1-34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of (15) N-enriched human parathyroid hormone, (15) N PTH(1-34), generated via a plasmid vector that gives reasonable yield, low-cost protease cleavage (leaving the native N-terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS.
Assuntos
Endopeptidases/química , Hormônio Paratireóideo/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Escherichia coli , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/química , Hormônio Paratireóideo/isolamento & purificação , Estrutura Secundária de Proteína , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The BK polyoma virus is a leading cause of chronic post kidney transplantation rejection. One target for therapeutic intervention is the initial association of the BK virus with the host cell. We hypothesize that the rate of BKV infection can be curbed by competitively preventing viral binding to cells. The X-ray structures of homologous viruses complexed with N-terminal glycoproteins suggest that the BC and HI loops of the viral coat are determinant for binding and thereby infection of the host cell. The large size of the viral coat precludes it from common biophysical and small molecule screening studies. Hence, we sought to develop a smaller protein template incorporating the identified binding loops of the BK viral coat in a manner that adequately mimics the binding characteristics of the BK virus coat protein to cells. Such a mimic may serve as a tool for the identification of inhibitors of BK viral progression. Herein, we report the design and characterization of a reduced-size and soluble template derived from a four-helix protein-TM1526 of Thermatoga maritima archaea bacteria-which maintains the topological display of the BC and HI loops as found in the viral coat protein, VP1, of BKV. We demonstrate that the GT1b and GD1b sialogangliosides, which bind to the VP1 of BKV, also associate with our BKV template. Employing a GFP-tagged template, we show host cell association that is dose dependent and that can be reduced by neuraminidase treatment. These data demonstrate that the BKV template mimics the host cell binding observed for the wild-type virus coat protein VP1.
Assuntos
Vírus BK/patogenicidade , Proteínas do Capsídeo/metabolismo , Nefropatias/virologia , Mimetismo Molecular , Sequência de Aminoácidos , Animais , Vírus BK/metabolismo , Proteínas do Capsídeo/química , Chlorocebus aethiops , Citometria de Fluxo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Células Vero , Difração de Raios XRESUMO
Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/química , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Calmodulina/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Engenharia de Proteínas/métodos , Sítios de Ligação , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Estrutura Secundária de ProteínaRESUMO
The Keap1/Nrf2/ARE pathway controls a network of cytoprotective genes that defend against the damaging effects of oxidative and electrophilic stress, and inflammation. Induction of this pathway is a highly effective strategy in combating the risk of cancer and chronic degenerative diseases, including atherosclerosis and neurodegeneration. An acetylenic tricyclic bis(cyano enone) bearing two highly electrophilic Michael acceptors is an extremely potent inducer in cells and in vivo. We demonstrate spectroscopically that both cyano enone functions of the tricyclic molecule react with cysteine residues of Keap1 and activate transcription of cytoprotective genes. Novel monocyclic cyano enones, representing fragments of rings A and C of the tricyclic compound, reveal that the contribution to inducer potency of the ring C Michael acceptor is much greater than that of ring A, and that potency is further enhanced by spatial proximity of an acetylenic function. Critically, the simultaneous presence of two cyano enone functions in rings A and C within a rigid three-ring system results in exceptionally high inducer potency. Detailed understanding of the structural elements that contribute to the reactivity with the protein sensor Keap1 and to high potency of induction is essential for the development of specific and selective lead compounds as clinically relevant chemoprotective agents.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Modelos Químicos , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse OxidativoRESUMO
In addition to the heptahelical transmembrane domain shared by all G protein-coupled receptors (GPCRs), many class A GPCRs adopt a helical domain, termed helix 8, in the membrane-proximal region of the C terminus. We investigated the role of residues in the hydrophobic and hydrophilic faces of amphiphilic helix 8 of human cannabinoid receptor 1 (CB1). To differentiate between a role for specific residues and global features, we made two key mutants: one involving replacement of the highly hydrophobic groups, Leu404, Phe408, and Phe412, all with alanine and the second involving substitution of the basic residues, Lys402, Arg405, and Arg409, all with the neutral glutamine. The former showed a very low B(max) based on binding isotherms, a minimal E(max) based on GTPgammaS binding analysis, and defective localization relative to the wild-type CB1 receptor as revealed by confocal microscopy. However, the latter mutant and the wild-type receptors were indistinguishable. Circular dichroism spectroscopy of purified peptides with corresponding sequences indicated that the highly hydrophobic residues are critical for maintaining a strong helical structure in detergent, whereas the positively charged residues are not. Further investigation of mutant receptors revealed that CB1 localization requires a threshold level of hydrophobicity but not specific amino acids. Moreover, mutant receptors carrying two- to six-residue insertions amino-terminal to helix 8 revealed a graded decrease in B(max) values. Our results identify the key helix 8 components (including hydrophobicity of specific residues, structure, and location relative to TM7) determinant for receptor localization leading to robust ligand binding and G protein activation.
Assuntos
Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Dicroísmo Circular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
A growing body of evidence indicates that enhanced AMPA-mediated glutamate transmission in the core of the nucleus accumbens is critically involved in cocaine priming-induced reinstatement of drug seeking, an animal model of relapse. However, the extent to which increased glutamate transmission in the other major subregion of the nucleus accumbens, the shell, contributes to the reinstatement of cocaine seeking remains unclear. In the present experiments, administration of the AMPA/kainate receptor antagonist CNQX (0, 0.03, or 0.3 mug) into either the core or the shell of the nucleus accumbens before a systemic cocaine priming injection (10 mg/kg, i.p.) dose-dependently attenuated the reinstatement of drug seeking. Cocaine priming-induced reinstatement of cocaine seeking also was associated with increases in GluR2-pSer880 in the nucleus accumbens shell. The phosphorylation of GluR2 by PKC at Ser880 plays an important role in the trafficking of GluR2-containing AMPA receptors from the plasma membrane. The current results showed that administration of a cell-permeable peptide that disrupts GluR2 trafficking (Pep2-EVKI) into either the accumbens core or shell attenuated cocaine-induced reinstatement of drug seeking. Together, these findings indicate that changes in AMPA receptor-mediated glutamate transmission in both the nucleus accumbens core and shell are necessary for the reinstatement of drug seeking induced by a priming injection of cocaine. The present results also demonstrate that the reinstatement of cocaine seeking is associated with increases in the phosphorylation-dependent trafficking of GluR2-containing AMPA receptors in the nucleus accumbens.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/patologia , Transtornos Relacionados ao Uso de Cocaína/psicologia , Núcleo Accumbens/metabolismo , Receptores de AMPA/metabolismo , Reforço Psicológico , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Cocaína/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Preferências Alimentares , Masculino , Núcleo Accumbens/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Esquema de Reforço , Autoadministração , Serina/metabolismoRESUMO
The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.
Assuntos
Receptor CB1 de Canabinoide/metabolismo , Sequência de Aminoácidos , Benzofuranos/farmacologia , Linhagem Celular , Cicloexanóis/farmacologia , Humanos , Ligantes , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ensaio Radioligante , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/química , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
Binding of hormones to their cognate G protein-coupled receptors (GPCRs) induces conformational shifts within the receptor based on evidence from a few hormone-receptor systems. Employing an engineered disulfide bond formation strategy and guided by a previously established model of the PTH-PTH receptor (PTHR)1 bimolecular complex, we set out to document and characterize the nature of agonist-induced changes in this family B GPCR. A mutant PTHR1 was generated which incorporates a Factor Xa cleavage site in the third intracellular loop. Treatment with Factor Xa fragments the receptor. However, if a new disulfide bond was formed before exposure to the enzyme, the fragments remain held together. A set of double cysteine-containing mutants were designed to probe the internal relative movements of transmembrane (TM) helices 2 and TM7. PTH enhanced formation of disulfide bonds in the K240C/F447C and A242C/F447C mutants. For the F238C/F447C mutant, a disulfide bond is formed in the basal state, but is disrupted by interaction with PTH. For the D241C/F447C PTHR1 construct, no disulfide bond formation was observed in either the basal or hormone-bound state. These findings demonstrate that the conformation of PTHR1 is altered from the basal state when PTH is bound. Novel information regarding spatial proximities between TM2 and TM7 of PTHR1 and the nature of relative movements between the two transmembrane regions was revealed. The data confirm and extend the experimentally derived model of the PTH-PTHR1 complex and provide insights at a new level of detail into the early events in PTHR1 activation by PTH.
Assuntos
Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/química , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Chlorocebus aethiops , Simulação por Computador , Cisteína/genética , Cisteína/metabolismo , Fator Xa/metabolismo , Modelos Moleculares , Mutação , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , TransfecçãoRESUMO
NEMO is an essential component in the activation of the canonical NF-κB pathway and exerts its function by recruiting the IκB kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-κB mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKKß-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKKß-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKKß-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes.