RESUMEN
BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.
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Insuficiencia Cardíaca , Hipertensión , Células Madre Pluripotentes Inducidas , Humanos , Ratas , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Microtomografía por Rayos X , Células Madre Pluripotentes Inducidas/metabolismo , Hipertensión/complicaciones , Hipertensión/genética , Miocitos Cardíacos/metabolismo , Cardiomegalia , ARNRESUMEN
The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.
Asunto(s)
Acuaporina 2/metabolismo , Aurora Quinasa A/metabolismo , Túbulos Renales Colectores/metabolismo , Actinas/metabolismo , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Silenciador del Gen , Inmunohistoquímica , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Estructura Molecular , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , RatasRESUMEN
BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Hipertensión/genética , Mutación , Alelos , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Presión Arterial , Biomarcadores/sangre , Biomarcadores/orina , Braquidactilia/diagnóstico , Braquidactilia/genética , Sistemas CRISPR-Cas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Activación Enzimática , Marcación de Gen , Estudios de Asociación Genética/métodos , Genotipo , Inmunohistoquímica , Isoenzimas , Masculino , Linaje , Fenotipo , Radiografía , Ratas , Sistema Renina-Angiotensina/genéticaRESUMEN
Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking inâ vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Animales , Células Epiteliales/química , Células HeLa , Humanos , Luz , Proteínas Luminiscentes/química , Estructura Molecular , Procesos Fotoquímicos , Proteínas Recombinantes de Fusión/química , Pez CebraRESUMEN
BACKGROUND: Arginine-vasopressin (AVP) binding to vasopressin V2 receptors promotes redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane of renal collecting duct principal cells. This pathway fine-tunes renal water reabsorption and urinary concentration, and its perturbation is associated with diabetes insipidus. Previously, we identified the antimycotic drug fluconazole as a potential modulator of AQP2 localization. METHODS: We assessed the influence of fluconazole on AQP2 localization in vitro and in vivo as well as the drug's effects on AQP2 phosphorylation and RhoA (a small GTPase, which under resting conditions, maintains F-actin to block AQP2-bearing vesicles from reaching the plasma membrane). We also tested fluconazole's effects on water flow across epithelia of isolated mouse collecting ducts and on urine output in mice treated with tolvaptan, a VR2 blocker that causes a nephrogenic diabetes insipidus-like excessive loss of hypotonic urine. RESULTS: Fluconazole increased plasma membrane localization of AQP2 in principal cells independent of AVP. It also led to an increased AQP2 abundance associated with alterations in phosphorylation status and ubiquitination as well as inhibition of RhoA. In isolated mouse collecting ducts, fluconazole increased transepithelial water reabsorption. In mice, fluconazole increased collecting duct AQP2 plasma membrane localization and reduced urinary output. Fluconazole also reduced urinary output in tolvaptan-treated mice. CONCLUSIONS: Fluconazole promotes collecting duct AQP2 plasma membrane localization in the absence of AVP. Therefore, it might have utility in treating forms of diabetes insipidus (e.g., X-linked nephrogenic diabetes insipidus) in which the kidney responds inappropriately to AVP.
Asunto(s)
Acuaporina 2/metabolismo , Transporte Biológico/genética , Colforsina/farmacología , Diabetes Insípida Nefrogénica/tratamiento farmacológico , Fluconazol/farmacología , Proteína de Unión al GTP rhoA/efectos de los fármacos , Análisis de Varianza , Animales , Membrana Celular/metabolismo , Células Cultivadas , Diabetes Insípida Nefrogénica/metabolismo , Modelos Animales de Enfermedad , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación/genética , Distribución Aleatoria , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including Gâ protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamineâ D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100â fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.
Asunto(s)
Antagonistas de los Receptores de Dopamina D2/química , Imidazoles/química , Indoles/química , Receptores de Dopamina D2/análisis , Animales , Antagonistas de los Receptores de Dopamina D2/síntesis química , Antagonistas de los Receptores de Dopamina D2/efectos de la radiación , Células HEK293 , Humanos , Imidazoles/síntesis química , Imidazoles/efectos de la radiación , Indoles/síntesis química , Indoles/efectos de la radiación , Ligandos , Simulación del Acoplamiento Molecular , Ensayo de Unión Radioligante , Ratas , Receptores de Dopamina D2/efectos de la radiación , Espiperona/química , Relación Estructura-Actividad , Porcinos , Espectrometría de Masas en Tándem , Rayos UltravioletaRESUMEN
Encapsulation of diagnostic and therapeutic compounds in transporters improves their delivery to the point of need. An even more efficient treatment of diseases can be achieved using carriers with targeting or protecting moieties. In the present work, we investigated micellar and liposomal nanocarriers modified with fluorescein, peptides, and polymers that are covalently bound to fatty acids or phospholipids to ensure a self-driven incorporation into the micelles or liposomes. First, we characterized the photophysics of the fluorescent probes in the absence and in the presence of nanocarriers. Changes in the fluorescence decay time, quantum yield, and intensity of a fluorescein-labeled fatty acid (fluorescein-labeled palmitic acid [fPA]) and a fluorescein-labeled lipopeptide (P2fA2) were found. By exploiting these changes, we investigated a lipopeptide (P2A2 as an uptake-mediating unit) in combination with different nanocarriers (micelles and liposomes) and determined the corresponding association constant Kass values, which were found to be very high. In addition, the mobility of fPA was exploited using fluorescence correlation spectroscopy (FCS) and fluorescence depolarization (FD) experiments to characterize the nanocarriers. Cellular uptake experiments with mouse brain endothelial cells provided information on the uptake behavior of liposomes modified by uptake-mediating P2A2 and revealed differences in the uptake behavior between pH-sensitive and pH-insensitive liposomes.
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Células Endoteliales/metabolismo , Fluoresceína/química , Lipopéptidos , Nanoestructuras/química , Ácido Palmítico/química , Animales , Línea Celular , Células Endoteliales/citología , Concentración de Iones de Hidrógeno , Lipopéptidos/química , Lipopéptidos/farmacocinética , Lipopéptidos/farmacología , Liposomas , RatonesRESUMEN
In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.
Asunto(s)
Acuaporina 2/metabolismo , Benzodioxoles/farmacología , Benzofuranos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/fisiología , Fluconazol/farmacología , Aparato de Golgi/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Microscopía Fluorescente , Fosforilación , Transporte de Proteínas/efectos de los fármacos , RatasRESUMEN
Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antineoplásicos/química , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cetuximab , Humanos , Inmunotoxinas/química , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Saponinas/química , Saporinas , Trastuzumab , Triterpenos/químicaRESUMEN
GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.
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GTP Fosfohidrolasas/química , Proteínas de la Membrana/química , Modelos Moleculares , Filogenia , Conformación Proteica , Estructura Terciaria de Proteína , Calorimetría , Cromatografía Líquida de Alta Presión , Cristalización , Dimerización , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Hidrólisis , Células Jurkat , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis , Nucleótidos/metabolismo , UltracentrifugaciónRESUMEN
We have developed and characterized efficient caged compounds of the neurotransmitter octopamine. For derivatization, we introduced [6-bromo-8-(diethylaminomethyl)-7-hydroxycoumarin-4-yl]methoxycarbonyl (DBHCMOC) and {6-bromo-7-hydroxy-8-[(piperazin-1-yl)methyl]coumarin-4-yl}methoxycarbonyl (PBHCMOC) moieties as novel photo-removable protecting groups. The caged compounds were functionally inactive when applied to heterologously expressed octopamine receptors (AmOctα1R). Upon irradiation with UV-visible or IR light, bioactive octopamine was released and evoked Ca2+ signals in AmOctα1R-expressing cells. The pronounced water solubility of compounds 2-4 in particular holds great promise for these substances as excellent phototriggers of this important neurotransmitter.
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Cumarinas/química , Octopamina/química , Receptores de Amina Biogénica/metabolismo , Animales , Abejas/metabolismo , Señalización del Calcio , Dióxido de Carbono/química , Células HEK293 , Humanos , Rayos Infrarrojos , Octopamina/síntesis química , Fotólisis , Receptores de Amina Biogénica/genética , Solubilidad , Rayos UltravioletaRESUMEN
The paracellular passage of ions and small molecules across epithelia is controlled by tight junctions, complex meshworks of claudin polymers that form tight seals between neighboring cells. How the nanoscale architecture of tight junction meshworks enables paracellular passage of specific ions or small molecules without compromising barrier function is unknown. Here we combine super-resolution stimulated emission depletion microscopy in live and fixed cells and tissues, multivariate classification of super-resolution images and fluorescence resonance energy transfer to reveal the nanoscale organization of tight junctions formed by mammalian claudins. We show that only a subset of claudins can assemble into characteristic homotypic meshworks, whereas tight junctions formed by multiple claudins display nanoscale organization principles of intermixing, integration, induction, segregation, and exclusion of strand assemblies. Interestingly, channel-forming claudins are spatially segregated from barrier-forming claudins via determinants mainly encoded in their extracellular domains also known to harbor mutations leading to human diseases. Electrophysiological analysis of claudins in epithelial cells suggests that nanoscale segregation of distinct channel-forming claudins enables barrier function combined with specific paracellular ion flux across tight junctions.
Asunto(s)
Claudinas , Uniones Estrechas , Animales , Claudinas/genética , Células Epiteliales , Epitelio , Humanos , Iones , MamíferosRESUMEN
Rhodamine fluorophores are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, show improved photophysical parameters (i.e. brightness, lifetimes) and reduced chemical bleaching. We explore this finding for SNAP- and Halo-tag labelling in live cells, and highlight enhanced properties in several applications, such as fluorescence activated cell sorting, fluorescence lifetime microscopy, stimulated emission depletion nanoscopy and single-molecule Förster-resonance energy transfer. We finally extend this idea to other dye families and envision deuteration as a generalizable concept to improve existing and to develop new chemical biology probes.
RESUMEN
The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615, and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology.
RESUMEN
A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Transducción de Señal/fisiología , Proteínas de Anclaje a la Quinasa A/genética , Secuencias de Aminoácidos/fisiología , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Unión Proteica/fisiología , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Membrane-bound human angiotensin-converting enzyme (ACE) has been reported to initiate intracellular signaling after interaction with substrates or inhibitors. Somatic ACE is known to contain two distinct, extracellular catalytic centers. We analyzed the signal transduction mechanisms in cells transfected with different forms of murine ACE (mACE) and investigated whether the two domains are similarly involved in these processes. For this purpose, CHO cells were stably transfected with mACE or with its domain-selective mutants. In addition to these modified cellular models, human umbilical vein endothelial cells were used in this study. Signal transduction molecules such as JNK and c-Jun were analyzed after activation of cells with several ACE substrates and inhibitors. ACE-targeting compounds such as substrates, inhibitors, or even the ACE product angiotensin-II induce in mACE-expressing cells a signal transduction response. These processes are also evoked by partially inactivated forms of mACE and finally result in an enhanced cyclooxygenase-2 transcription. Surprisingly, the membrane-bound ACE activity is also influenced by ACE-targeted interventions. Our data suggest that the two catalytic domains of mACE do not function independently but that the signal transduction is influenced by negative cooperativity of the two catalytic domains. This study underlines that ACE indeed has receptor-like properties which occur in a species-specific manner.
Asunto(s)
Dominio Catalítico , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Transducción de Señal , Regulación Alostérica , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Biocatálisis , Células CHO , Dominio Catalítico/genética , Células Cultivadas , Cricetinae , Cricetulus , Células Endoteliales/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Mutación , Peptidil-Dipeptidasa A/genética , TransfecciónRESUMEN
Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Acuaporina 2/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Ligasas/metabolismo , Ratones , Ubiquitina/metabolismoRESUMEN
The heptapeptide angiotensin (Ang)-(1-7) is part of the beneficial arm of the renin-angiotensin system. Ang-(1-7) has cardiovascular protective effects, stimulates regeneration, and opposes the often detrimental effects of AngII. We recently identified the G protein-coupled receptors Mas and MrgD as receptors for the heptapeptide. Ala1-Ang-(1-7) (Alamandine), a decarboxylated form of Ang-(1-7), has similar vasorelaxant effects, but has been described as only stimulating MrgD. Therefore, this study aimed to characterise the consequences of the lack of the carboxyl group in amino acid 1 on intracellular signalling and to identify the receptor fingerprint for Ala1-Ang-(1-7). In primary endothelial and mesangial cells, Ala1-Ang-(1-7) elevated cAMP concentration. Dose response curves generated with Ang-(1-7) and Ala1-Ang-(1-7) significantly differed from each other, with a much lower EC50 and a bell-shape curve for Ala1-Ang-(1-7). We provided pharmacological proof that both, Mas and MrgD, are functional receptors for Ala1-Ang-(1-7). Consequently, in primary mesangial cells with genetic deficiency in both receptors, the heptapeptide failed to increase cAMP concentration. As we previously described for Ang-(1-7), the Ala1-Ang-(1-7)-mediated cAMP increase in Mas/MrgD-transfected HEK293 cells and primary cells was blocked by the AT2 receptor blocker, PD123319. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, electrostatic potential calculations, and an involvement of Galpha i for higher concentrations of Ala1-Ang-(1-7). Our results identify Ala1-Ang-(1-7) as a peptide with specific pharmacodynamic properties and builds the basis for the design of more potent and efficient Ang-(1-7) analogues for therapeutic intervention in a rapidly growing number of diseases.
Asunto(s)
Angiotensina I/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Descarboxilación , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones Noqueados , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renales Colectores/citología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Membrana Celular/efectos de los fármacos , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR) expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1. RESULTS: We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG) and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 +/- 92 fmol/mg) were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X) or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation. CONCLUSION: We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.