RESUMO
R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Motivos de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Trombospondinas/químicaRESUMO
The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.
Assuntos
Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Catálise , Cristalografia por Raios X , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Humanos , Hidrólise , Cinética , Modelos Moleculares , Mutação/genética , Diester Fosfórico Hidrolases/genética , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.
Assuntos
Deinococcus/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/química , Proteínas de Bactérias/análise , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Bases de Dados de Proteínas , Expressão Gênica , Cloreto de Magnésio , Oligopeptídeos , Fragmentos de Peptídeos/análise , Peptídeos/genética , RNA Bacteriano/análise , RNA Ribossômico/análise , Proteínas Recombinantes de Fusão , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor alpha at a resolution of 2.0 A. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERalpha/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents.
Assuntos
Receptor alfa de Estrogênio/química , Pirrolidinas/química , Tetra-Hidronaftalenos/química , Cristalografia por Raios X , Receptor alfa de Estrogênio/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirrolidinas/metabolismo , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Tetra-Hidronaftalenos/metabolismoRESUMO
Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant to the anticipated binding interaction for the fragments 1-methyl-1H-benzotriazole 5, 3-methyl-benzo[d]isoxazole 3, and 3-methyl-[1,2,4]triazolo[4,3-a]pyridine 4, pyridine 1, and 1,3-dimethyl-1,3-dihydro-benzoimidazol-2-one 2 are reported. The design considerations and synthesis of p38 inhibitors based on these H-bond acceptor fragments is detailed. Comparative evaluation of the pyridine-, benzimidazolone-, benzotriazole-, and triazolopyridine-based inhibitors shows the triazoles 20 and 25 to be significantly more potent experimentally than the benzimidazolone after which they were modeled. An X-ray crystal structure of 25 bound to the active site shows that the triazole group serves as the H-bond acceptor but unexpectedly as a dual acceptor, inducing movement of the crossover connection of p38alpha. The computed descriptors for the hydrophobic and pi-pi interaction capacities were the most useful in ranking potency.
Assuntos
Benzimidazóis/química , Piridinas/química , Triazóis/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química , Benzimidazóis/síntese química , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Relação Quantitativa Estrutura-Atividade , Eletricidade Estática , Triazóis/síntese químicaRESUMO
The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.
Assuntos
Citocinas/farmacologia , Células Caliciformes/efeitos dos fármacos , Pulmão/patologia , Receptor Notch2/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-13/farmacologia , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Pulmão/metabolismo , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Current antiangiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor endothelial marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer. Furthermore, antibodies developed against the TEM8 extracellular domain blocked anthrax intoxication, inhibited tumor-induced angiogenesis, displayed broad antitumor activity, and augmented the activity of clinically approved anticancer agents without added toxicity. Thus, TEM8 targeting may allow selective inhibition of pathological angiogenesis.
Assuntos
Proteínas de Neoplasias/fisiologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Receptores de Superfície Celular/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina G/farmacologia , Imunoglobulina G/toxicidade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neovascularização Patológica/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/imunologia , Transplante Heterólogo , Cicatrização/genéticaRESUMO
High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.
Assuntos
Histidina/metabolismo , Oligopeptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/biossíntese , Serina/metabolismo , Acetilação , Fosfatase Alcalina/química , Animais , Aurora Quinases , Baculoviridae/genética , Domínio Catalítico/genética , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Quinase 2 de Adesão Focal/biossíntese , Quinase 2 de Adesão Focal/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Gluconatos/metabolismo , Humanos , Luz , Peso Molecular , Ácido Okadáico/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Espalhamento de Radiação , Spodoptera , Trombina/químicaRESUMO
Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants. On average, antagonist ligands (such as tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists (such as estriol and beta-estradiol). This finding is consistent with these antagonists binding to an altered conformation of the receptor. The biosensor assay also could identify subtle differences in how the same ligand interacted with two different isoforms of the receptor (alpha and beta). The biosensor's ability to determine kinetic rate constants for small molecule/protein interactions provides unique opportunities to understand the mechanisms associated with complex formation as well as new information to drive the optimization of drug candidates.
Assuntos
Receptores de Estrogênio/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos Monoclonais/metabolismo , Compostos Benzidrílicos , Desidroepiandrosterona/metabolismo , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Cinética , Ligantes , Fenóis/metabolismo , Receptores de Estrogênio/química , Proteínas Recombinantes/químicaRESUMO
In this communication, we wish to describe the discovery of a novel series of 6-azauracil-based thyromimetics that possess up to 100-fold selectivities for binding and functional activation of the beta(1)-isoform of the thyroid receptor family. Structure-activity relationship studies on the 3,5- and 3'-positions provided compounds with enhanced TR beta affinity and selectivity. Key binding interactions between the 6-azauracil moiety and the receptor have been determined through of X-ray crystallographic analysis.