RESUMEN
Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.
Asunto(s)
Ácido Glicirrínico/farmacología , Dominios PDZ , Uniones Estrechas , Proteína de la Zonula Occludens-1 , Células CACO-2 , Humanos , Uniones Estrechas/química , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/antagonistas & inhibidores , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.
Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Proteínas de la Envoltura de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , COVID-19/patología , Interacciones Huésped-Patógeno , Humanos , Dominios PDZ , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SARS-CoV-2/aislamiento & purificación , Uniones Estrechas/metabolismoRESUMEN
Baicalein and scutellarein are bioactive flavonoids isolated from the traditional Chinese medicine Scutellaria baicalensis Georgi; however, there is a lack of effective strategies for producing baicalein and scutellarein. In this study, we developed a sequential self-assembly enzyme reactor involving two enzymes in the baicalein pathway with a pair of protein-peptide interactions in E. coli. These domains enabled us to optimize the stoichiometry of two baicalein biosynthetic enzymes recruited to be an enzymes complex. This strategy reduces the accumulation of intermediates and removes the pathway bottleneck. With this strategy, we successfully promoted the titer of baicalein by 6.6-fold (from 21.6 to 143.5 mg/L) and that of scutellarein by 1.4-fold (from 84.3 to 120.4 mg/L) in a flask fermentation, respectively. Furthermore, we first achieved the de novo biosynthesis of baicalein directly from glucose, and the strain was capable of producing 214.1 mg/L baicalein by fed-batch fermentation. This work provides novel insights for future optimization and large-scale fermentation of baicalein and scutellarein.
Asunto(s)
Apigenina/biosíntesis , Reactores Biológicos , Medicamentos Herbarios Chinos/metabolismo , Escherichia coli/metabolismo , Flavanonas/biosíntesis , Ingeniería Metabólica/métodos , Extractos Vegetales/biosíntesis , Técnicas de Cultivo Celular por Lotes/métodos , Escherichia coli/genética , Fermentación , Glucosa/metabolismo , Malonil Coenzima A/metabolismo , Microorganismos Modificados Genéticamente , Dominios PDZ , Fenilanina Amoníaco-Liasa/química , Fenilanina Amoníaco-Liasa/metabolismo , Scutellaria baicalensis , Sirolimus/metabolismoRESUMEN
Inhibition of PSD-95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide-based compounds that target the PDZ domains of PSD-95. In contrast, developing potent and drug-like small molecules against the PSD-95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD-95 PDZ1-2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter-test, and found four promising fragments. Three ligand efficient fragments were shown by 1 H,15 N HSQC NMR to bind in the small hydrophobic P0 pockets of PDZ1-2, and one of them underwent structure-activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1-2 of PSD-95 and disclose novel fragments that can serve as starting points for optimization towards small-molecule PDZ domain inhibitors.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos , Polarización de Fluorescencia , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Dominios PDZ/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein-protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein-which are pharmacologically active components of Chinese skullcap (Radix scutellariae)-with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein-but not baicalin-induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a "partial" epithelial-mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1-CLD interaction, activation of both transforming growth factor-ß (TGF-ß) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.
Asunto(s)
Flavanonas/administración & dosificación , Flavonoides/administración & dosificación , Dominios PDZ/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Proteína de la Zonula Occludens-1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células CACO-2 , Perros , Medicamentos Herbarios Chinos/administración & dosificación , Humanos , Células de Riñón Canino Madin Darby , Ratones , Dominios PDZ/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Unique intrinsic properties of peptides like low toxicity, high biological activity, and specificity make them attractive therapeutic agents. PDZ-binding peptide inhibitors have been demonstrated for curing of Alzheimer, Parkinson, Dementia, and other central nervous system ailments. In this article, we report the successful use of an integrated computational protocol to analyze the structural basis of how peptides bind to the shallow groove of the third PDZ domain (PDZ-3) from the postsynaptic density (PSD-95) protein. This protocol employs careful and precise computational techniques for design of new strategy for predicting novel and potent peptides against PDZ protein. We attempted to generate a pharmacophore model using crystal structure of peptide inhibitor bound to the PDZ-3. A highly specific and sensitive generated pharmacophore model was used for screening virtual database generated using different combination of amino acid substitutions as well as decoy peptide database for its sensitivity and specificity. Identified hit peptides were further analyzed by docking studies, and their stability analyzed using solvated molecular dynamics. Quantum Mechanics/Molecular Mechanics (QM/MM) interaction energy and GMX-PBSA scoring schemes were used for ranking of stable peptides. Computational approach applied here generated encouraging results for identifying peptides against PDZ interaction model. The workflow can be further exercised as a virtual screening technique for reducing the search space for candidate target peptides against PDZ domains.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Homólogo 4 de la Proteína Discs Large/química , Diseño de Fármacos , Modelos Moleculares , Dominios PDZ , Péptidos/química , Péptidos/farmacología , Sitios de Unión , Evaluación Preclínica de Medicamentos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Relación Estructura-Actividad CuantitativaRESUMEN
The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/ß-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2µM affinity and had a 0.186µM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural-kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.
Asunto(s)
Proteínas Dishevelled/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Modelos Químicos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Dishevelled/química , Modelos Moleculares , Terapia Molecular Dirigida/métodos , Dominios PDZ , Unión Proteica , Espectrometría de Fluorescencia/métodos , Vía de Señalización WntRESUMEN
Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.
Asunto(s)
Evaluación Preclínica de Medicamentos/normas , Proteínas Fluorescentes Verdes/metabolismo , Dominios PDZ/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Transcripción Genética/efectos de los fármacos , Secuencias de Aminoácidos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dobutamina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Etanolaminas/análisis , Etanolaminas/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Compuestos Heterocíclicos con 3 Anillos/análisis , Compuestos Heterocíclicos con 3 Anillos/farmacología , Vía de Señalización Hippo , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/análisis , Piridinas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Tiourea/análogos & derivados , Tiourea/análisis , Tiourea/farmacología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , ortoaminobenzoatos/análisis , ortoaminobenzoatos/farmacologíaRESUMEN
Virtual ligand screening is a powerful technique to identify potential hits of targets and to increase hit rates. Here, we describe how we used this technique combined with NMR (15)N HSQC experiments to obtain small molecules that bind to the PDZ domain of Dvl targeting the Wnt signaling pathway.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética/métodos , Bases de Datos Factuales , Ligandos , Dominios PDZ , Programas Informáticos , Vía de Señalización WntRESUMEN
Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.
Asunto(s)
Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Sitios de Unión , Barrera Hematoencefálica , Cristalografía por Rayos X , Homólogo 4 de la Proteína Discs Large , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Guanilato-Quinasas/antagonistas & inhibidores , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Discapacidades para el Aprendizaje/etiología , Discapacidades para el Aprendizaje/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Trastornos del Movimiento/etiología , Trastornos del Movimiento/prevención & control , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Resonancia Magnética Nuclear Biomolecular , Dominios PDZ/efectos de los fármacos , Péptidos/síntesis química , Péptidos/farmacología , Equilibrio Postural , Conformación Proteica , Trastornos de la Sensación/etiología , Trastornos de la Sensación/prevención & controlRESUMEN
PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.
Asunto(s)
Ubiquitina-Proteína Ligasas/química , Biología Computacional , Evaluación Preclínica de Medicamentos , Humanos , Dominios PDZ , Unión Proteica , Mapeo de Interacción de Proteínas , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C-terminus of guanylate-kinase-associated protein (GKAP). Here, small-molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high-affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein-protein interaction. Chemical synthesis via a hetero-Diels-Alder strategy is employed for hit optimization, and structure-activity relationship studies are performed. Best hits possess K(i) values in the 10 µM range, and binding to the PDZ domain is confirmed by ¹H,¹5N HSQCâ NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD-95, syntrophin, and DVL3 was studied using ¹H,¹5N HSQCâ NMR spectroscopy.
Asunto(s)
Proteínas Portadoras/química , Quinolinas/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Proteínas del Tejido Nervioso , Dominios PDZ/efectos de los fármacos , Péptidos/síntesis química , Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Quinolinas/síntesis química , Quinolinas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Protein fold recognition usually relies on a statistical model of each fold; each model is constructed from an ensemble of natural sequences belonging to that fold. A complementary strategy may be to employ sequence ensembles produced by computational protein design. Designed sequences can be more diverse than natural sequences, possibly avoiding some limitations of experimental databases. METHODOLOGY/PRINCIPAL FINDINGS: WE EXPLORE THIS STRATEGY FOR FOUR SCOP FAMILIES: Small Kunitz-type inhibitors (SKIs), Interleukin-8 chemokines, PDZ domains, and large Caspase catalytic subunits, represented by 43 structures. An automated procedure is used to redesign the 43 proteins. We use the experimental backbones as fixed templates in the folded state and a molecular mechanics model to compute the interaction energies between sidechain and backbone groups. Calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is used to scan the sequence and conformational space, yielding 200,000-300,000 sequences per backbone template. The results confirm and generalize our earlier study of SH2 and SH3 domains. The designed sequences ressemble moderately-distant, natural homologues of the initial templates; e.g., the SUPERFAMILY, profile Hidden-Markov Model library recognizes 85% of the low-energy sequences as native-like. Conversely, Position Specific Scoring Matrices derived from the sequences can be used to detect natural homologues within the SwissProt database: 60% of known PDZ domains are detected and around 90% of known SKIs and chemokines. Energy components and inter-residue correlations are analyzed and ways to improve the method are discussed. CONCLUSIONS/SIGNIFICANCE: For some families, designed sequences can be a useful complement to experimental ones for homologue searching. However, improved tools are needed to extract more information from the designed profiles before the method can be of general use.
Asunto(s)
Biología Computacional/métodos , Modelos Moleculares , Proteínas/química , Análisis de Secuencia de Proteína/métodos , Homología Estructural de Proteína , Aminoácidos/genética , Bases de Datos de Proteínas , Entropía , Mutación/genética , Dominios PDZ , Posición Específica de Matrices de Puntuación , Estabilidad Proteica , Estructura Secundaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
In this work, we describe how the Erbin PDZ domain interacts with Smad3, a transductor of the Transforming Growth Factor-beta (TGFbeta) pathway, via its MH2 domain. This interaction was described as important for TGFbeta signaling as it could potentially repress the transcriptional activity of the growth factor. In order to clarify our preliminary experimental observations pointing this interaction, we built a 3D model of the Erbin PDZ/Smad3 MH2 complex and checked its stability using molecular dynamics simulations. This model pointed out charged residues in Smad3 and Erbin which could be important for the interaction. By introducing point mutations of these residues within the proposed binding domains, we experimentally confirmed that arginine 279, glutamic acid 246 in Smad3 and glutamic acid 1321 in Erbin are important for the binding. These data suggest a possible novel interface of binding in the Erbin PDZ domain and reveal an unconventional mode of interaction for a PDZ domain and its ligand.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dominios PDZ , Proteína smad3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Arginina/genética , Arginina/metabolismo , Línea Celular , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas , Proteína smad3/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Despite similar molecular structures, the growth-related sodium/phosphate cotransporter NaPiIIc is regulated differently than the main NaPiIIa phosphate transporter. Using two-hybrid systems and immunoprecipitation, we identified several proteins that interact with NaPiIIc that might account for this differential regulation. NaPiIIc interacted with the PDZ domain-containing sodium-hydrogen exchange-regulating factor (NHERF) 1 and NHERF3 through novel binding motifs in its C terminus. NaPiIIc from brush-border membranes coprecipitated with both NHERF1 and NHERF3, with more NHERF3 co-precipitated in rats fed a low-phosphorus diet. NaPiIIc colocalizes with both NHERF1 and NHERF3 in brush-border membranes of rats fed either a low- or high-phosphorus diet. When mouse NaPiIIc was transfected into opossum kidney cells, it was localized mainly in apical microvilli and the trans-Golgi. Both confocal and total internal reflection microscopy show that NaPiIIc colocalizes with NHERF1 and NHERF3 in the apical microvilli, and this was not altered by truncation of the last three amino acids of NaPiIIc. Interactions of NaPiIIc with NHERF1 and NHERF3 were modulated by the membrane-associated 17 kDa protein (MAP17) similarly to NaPiIIa, but only the MAP17-NaPiIIc-NHERF3 complexes were internalized to the trans-Golgi. Our study shows that NaPiIIc interacts with a limited number of PDZ domain proteins, and the mechanisms and consequences of such interactions differ from those of NaPiIIa.
Asunto(s)
Corteza Renal/metabolismo , Dominios PDZ , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Animales , Dieta , Biblioteca de Genes , Inmunoprecipitación , Corteza Renal/química , Proteínas de la Membrana/metabolismo , Ratones , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fósforo/administración & dosificación , Mapeo de Interacción de Proteínas , Ratas , Intercambiadores de Sodio-Hidrógeno/análisis , Intercambiadores de Sodio-Hidrógeno/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/análisis , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Dominios PDZ/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Transferencia Resonante de Energía de Fluorescencia , Indicadores y Reactivos , Luz , Luminiscencia , Oxígeno/química , Oxígeno/efectos de la radiación , Ratas , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/efectos de la radiación , Proteínas Recombinantes/química , Estreptavidina/química , Especificidad por SustratoRESUMEN
The PDZ and LIM domain-containing protein family is encoded by a diverse group of genes whose phylogeny has currently not been analyzed. In mammals, ten genes are found that encode both a PDZ- and one or several LIM-domains. These genes are: ALP, RIL, Elfin (CLP36), Mystique, Enigma (LMP-1), Enigma homologue (ENH), ZASP (Cypher, Oracle), LMO7 and the two LIM domain kinases (LIMK1 and LIMK2). As conventional alignment and phylogenetic procedures of full-length sequences fell short of elucidating the evolutionary history of these genes, we started to analyze the PDZ and LIM domain sequences themselves. Using information from most sequenced eukaryotic lineages, our phylogenetic analysis is based on full-length cDNA-, EST-derived- and genomic- PDZ and LIM domain sequences of over 25 species, ranging from yeast to humans. Plant and protozoan homologs were not found. Our phylogenetic analysis identifies a number of domain duplication and rearrangement events, and shows a single convergent event during evolution of the PDZ/LIM family. Further, we describe the separation of the ALP and Enigma subfamilies in lower vertebrates and identify a novel consensus motif, which we call 'ALP-like motif' (AM). This motif is highly-conserved between ALP subfamily proteins of diverse organisms. We used here a combinatorial approach to define the relation of the PDZ and LIM domain encoding genes and to reconstruct their phylogeny. This analysis allowed us to classify the PDZ/LIM family and to suggest a meaningful model for the molecular evolution of the diverse gene architectures found in this multi-domain family.