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The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.
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Nanopartículas , Neoplasias , Citosol , Humanos , Liposomas/química , Nanopartículas/químicaRESUMEN
Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.
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Troponina C/metabolismo , Troponina T/metabolismo , Sitios de Unión , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Femenino , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Contracción Miocárdica , Miocardio/metabolismo , Miofibrillas/fisiología , Resonancia Magnética Nuclear Biomolecular , Linaje , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Troponina C/química , Troponina T/química , Troponina T/genéticaRESUMEN
Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.
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Calcio/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Miofibrillas/metabolismo , Troponina C/metabolismo , Algoritmos , Animales , Señalización del Calcio , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Cardiomiopatía Hipertrófica/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Contracción Isométrica , Cinética , Masculino , Ratones , Modelos Moleculares , Modelos Teóricos , Mutación , Miocardio/química , Miofibrillas/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Troponina C/química , Troponina C/genéticaRESUMEN
Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition.
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Lípidos , Proteolisis , Humanos , Proteolisis/efectos de los fármacos , Lípidos/química , Nanopartículas/química , Animales , Citosol/metabolismo , Sistemas de Liberación de Medicamentos , Proteínas Recombinantes/metabolismo , Ratones , LiposomasRESUMEN
Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information.
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Fenómenos Fisiológicos Celulares , Proteínas , Microscopía FluorescenteRESUMEN
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
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Escherichia coli , Optogenética , Optogenética/métodos , Retroalimentación , Escherichia coli/genética , Algoritmos , Análisis EspectralRESUMEN
Several vaccines against COVID-19 use a recombinant SARS-CoV-2 receptor-binding domain (RBD) as antigen, making the purification of this protein a key step in their production. In this work, citrate-coated magnetic iron oxide nanoparticles were evaluated as nano adsorbents in the first step (capture) of the purification of recombinant RBD. The nanoparticles were isolated through coprecipitation and subsequently coated with sodium citrate. The citrate-coated nanoparticles exhibited a diameter of 10 ± 2 nm, a hydrodynamic diameter of 160 ± 3 nm, and contained 1.9 wt% of citrate. The presence of citrate on the nanoparticles' surface was confirmed through FT-IR spectra and thermogravimetric analysis. The crystallite size (10.1 nm) and the lattice parameter (8.3646 Å) were determined by X-ray diffraction. In parallel, RBD-containing supernatant extracted from cell culture was exchanged through ultrafiltration and diafiltration into the adsorption buffer. The magnetic capture was then optimized using different concentrations of nanoparticles in the purified supernatant, and we found 40 mg mL-1 to be optimal. The ideal amount of nanoparticles was assessed by varying the RBD concentration in the supernatant (between 0.113 mg mL-1 and 0.98 mg mL-1), which resulted in good capture yields (between 83 ± 5% and 94 ± 4%). The improvement of RBD purity after desorption was demonstrated by SDS-PAGE and RP-HPLC. Furthermore, the magnetic capture was scaled up 100 times, and the desorption was subjected to chromatographic purifications. The obtained products recognized anti-RBD antibodies and bound the ACE2 receptor, proving their functionality after the developed procedure.
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COVID-19 , SARS-CoV-2 , Humanos , Vacunas contra la COVID-19 , Ácido Cítrico , Espectroscopía Infrarroja por Transformada de Fourier , CitratosRESUMEN
Extrusion three-dimensional (3D) bioprinting is a promising technology with many applications in the biomedical and tissue engineering fields. One of the key limitations for the widespread use of this technology is the narrow window of printability that results from the need to have bioinks with rheological properties that allow the extrusion of continuous filaments while maintaining high cell viability within the materials during and after printing. In this work, we use Carbopol (CBP) as rheology modifier for extrusion printing of biomaterials that are typically nonextrudable or present low printability. We show that low concentrations of CBP can introduce the desired rheological properties for a wide range of formulations, allowing the use of polymers with different cross-linking mechanisms and the introduction of additives and cells. To explore the opportunities and limitations of CBP as a rheology modifier, we used ink formulations based on poly(ethylene glycol)diacrylate with extrusion 3D printing to produce soft, yet stable, hydrogels with tunable mechanical properties. Cell-laden constructs made with such inks presented high viability for cells seeded on top of cross-linked materials and cells incorporated within the bioink during printing, showing that the materials are noncytotoxic and the printed structures do not degrade for up to 14 days. To our knowledge, this is the first report of the use of CBP-containing bioinks to 3D-print complex cell-laden structures that are stable for days and present high cell viability. The use of CBP to obtain highly printable inks can accelerate the evolution of extrusion 3D bioprinting by guaranteeing the required rheological properties and expanding the number of materials that can be successfully printed. This will allow researchers to develop and optimize new bioinks focusing on the biochemical, cellular, and mechanical requirements of the targeted applications rather than the rheology needed to achieve good printability.
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Bioimpresión , Polímeros , Bioimpresión/métodos , Materiales Biocompatibles/química , Ingeniería de Tejidos/métodos , Impresión Tridimensional , Reología , Hidrogeles/química , Tinta , Andamios del Tejido/químicaRESUMEN
Carbonation of epoxidized linseed oil (CELO) containing five-membered cyclic carbonate (CC5) groups has been optimized to 95% by reacting epoxidized linseed oil (ELO) with carbon dioxide (CO2) and tetrabutylammonium bromide (TBAB) as catalysts. The effect of reaction variables (temperature, CO2 pressure, and catalyst concentration) on the reaction parameters (conversion, carbonation and selectivity) in an autoclave system was investigated. The reactions were monitored, and the products were characterized by Fourier Transform Infrared Spectroscopy (FT-IR), carbon-13 nuclear magnetic resonance (13C-NMR) and proton nuclear magnetic resonance (1H-NMR) spectroscopies. The results showed that when carrying out the reaction at high temperature (from 90 °C to 120 °C) and CO2 pressure (60-120 psi), the reaction's conversion improves; however, the selectivity of the reaction decreases due to the promotion of side reactions. Regarding the catalyst, increasing the TBAB concentration from 2.0 to 5.0 w/w% favors selectivity. The presence of a secondary mechanism is based on the formation of a carboxylate ion, which was formed due to the interaction of CO2 with the catalyst and was demonstrated through 13C-NMR and FT-IR. The combination of these factors makes it possible to obtain the largest conversion (96%), carbonation (95%), and selectivity (99%) values reported until now, which are obtained at low temperature (90 °C), low pressure (60 psi) and high catalyst concentration (5.0% TBAB).
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Pheromones have been shown to induce sexually dimorphic responses in LH secretion. Here we asked whether the sexually dimorphic population of kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) could relay sexually dimorphic information from the olfactory systems to the GnRH system. Furthermore, we analyzed the effects of aromatase mutation (ArKO) and thus the role of estradiol on RP3V kisspeptin neuronal numbers and on the response of these kisspeptin neurons to same- versus opposite-sex urinary pheromones. Exposure to male but not female urinary odors induced Fos protein in kisspeptin neurons in the RP3V of female wildtype (WT) mice, suggesting that these kisspeptin neurons may be part of the neural circuitry that relays information from the olfactory brain to the GnRH system in a sexually dimorphic manner. Male pheromones induced Fos in kisspeptin neurons in ArKO females, albeit significantly less compared to WT females. The sexual differentiation of kisspeptin neuronal number was lost in ArKO mice, i.e. the number of kisspeptin-immunoreactive neurons in the RP3V of ArKO females was as low as in male mice, whereas male ArKO mice had somewhat increased numbers of kisspeptin neurons. These results suggest that the sex difference in kisspeptin neuronal number in WT mice reflects an organizational action of estradiol in females. By contrast, the ability of male urinary pheromones to activate kisspeptin neurons in WT females may not depend on the organizational action of estradiol since ArKO females still showed some Fos/kisspeptin co-activation.
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Aromatasa/genética , Mutación/fisiología , Neuronas/fisiología , Atractivos Sexuales/farmacología , Diferenciación Sexual/genética , Proteínas Supresoras de Tumor/fisiología , Animales , Recuento de Células , Interpretación Estadística de Datos , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Inmunohistoquímica , Kisspeptinas , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Odorantes , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/fisiología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Atractivos Sexuales/orina , Caracteres Sexuales , Olfato/genética , Olfato/fisiología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Sex differences in gonadal function are driven by either cyclical (females) or tonic (males) hypothalamic GnRH1 release and, subsequently, gonadotrophin (LH and FSH) secretion from the pituitary. This sex difference seems to depend on the perinatal actions of gonadal hormones on the hypothalamus. We used alpha-fetoprotein (AFP) knockout mice (Afp(-/-)) to study the mechanisms by which estrogens affect the sexual differentiation of the GnRH1 system. Afp(-/-) mice lack the protective actions of AFP against estrogens circulating during embryonic development, leading to infertility probably due to a hypothalamic dysfunction. Therefore, we first determined whether Afp(-/-) females are capable of showing a steroid-induced preovulatory LH surge by FOS/GnRH1 immunohistochemistry and RIA of plasma LH levels. Because the KISS1/GPR54 system is a key upstream regulator of the GnRH1 system as well as being sexually dimorphic, we also analyzed whether Kisspeptin-10 neurons were activated in Afp(-/-) mice after treatment with estradiol and progesterone. We found that the GnRH1 and Kisspeptin-10 neuronal systems are defeminized in Afp(-/-) females because they did not show either steroid-induced LH surges or significant FOS/GnRH1 double labeling. Furthermore, Kisspeptin-10 immunoreactivity and neural activation, measured by the number of double-labeled FOS/Kisspeptin-10 cells, were lower in Afp(-/-) females, suggesting a down-regulation of GnRH1 function. Thus, the sex difference in the ability to show preovulatory LH surges depends on the prenatal actions of estrogens in the male hypothalamus and, thus, is lost in Afp(-/-) females because they lack AFP to protect them against the defeminizing effects of estrogens during prenatal development.
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Estrógenos/farmacología , Feminización/sangre , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Luteinizante/sangre , Ovulación/sangre , Efectos Tardíos de la Exposición Prenatal/sangre , Proteínas/metabolismo , alfa-Fetoproteínas/genética , Animales , Regulación hacia Abajo , Femenino , Kisspeptinas , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Proteínas Oncogénicas v-fos/metabolismo , Embarazo , Caracteres SexualesRESUMEN
The eIMRT project is producing new remote computational tools for helping radiotherapists to plan and deliver treatments. The first available tool will be the IMRT treatment verification using Monte Carlo, which is a computational expensive problem that can be executed remotely on a GRID. In this paper, the current implementation of this process using GRID and SOA technologies is presented, describing the remote execution environment and the client.
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Informática Médica , Método de Montecarlo , Radioterapia de Intensidad Modulada , Humanos , Diseño de Software , EspañaRESUMEN
Mutations in TNNC1-the gene encoding cardiac troponin C (cTnC)-that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca2+-regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca2+], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca2+]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband.
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Defects of either anosmin-1 or fibroblast growth factor receptor 1 (FGFR1) are known to underlie hereditary Kallmann's syndrome (KS), a human disorder of olfactory and gonadotropin-releasing hormone (GnRH) neuronal ontogeny. Here, we report a functional interaction between anosmin-1 and the FGFR1-FGF2-heparan sulfate complex, leading to amplified responses in the FGFR1 signaling pathway. In human embryonic GnRH olfactory neuroblasts, wild-type anosmin-1, but not proteins with loss-of-function KS mutations, induces neurite outgrowth and cytoskeletal rearrangements through FGFR1-dependent mechanisms involving p42/44 and p38 mitogen-activated protein kinases and Cdc42/Rac1 activation. Furthermore, anosmin-1 enhances FGF2 signaling specifically through FGFR1 IIIc in heterologous BaF3 lymphoid cells in a heparan sulfate-dependent manner. Our study provides compelling evidence for anosmin-1 as an isoform-specific co-ligand modulator of FGFR signaling that amplifies and specifies FGFR1 signaling responses during human nervous system development and defines a mechanism underlying the link between autosomal and X-linked KS.
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Proteínas de la Matriz Extracelular/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Heparitina Sulfato/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Vías Olfatorias/citología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Embrión de Mamíferos/metabolismo , Activación Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Humanos , Inmunohistoquímica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuronas/metabolismo , Vías Olfatorias/embriología , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Defective function of anosmin-1, the protein encoded by KAL-1, underlies X-linked Kallmann's syndrome (X-KS), a human hereditary developmental disorder. Anosmin-1 appears to play a role in neurite outgrowth and axon branching, although molecular mechanisms of its action are still unknown. Anosmin-1 contains a WAP (whey acidic protein-like) domain and four contiguous FnIII (fibronectin-like type III) repeats; its WAP domain shows similarity to known serine protease inhibitors, whereas the FnIII domains contain HS (heparan sulphate)-binding sequences. To investigate the functional role of these domains, we have generated both wild-type and mutant recombinant anosmin-1 proteins using a Drosophila S2 cell expression system. Here we present the first biochemical evidence demonstrating the high-binding affinity between HS and anosmin-1, as measured by SPR (surface plasmon resonance) (K(d)=2 nM). The FnIII domains, particularly the first, are essential for dose-dependent HS binding and HS-mediated cell surface association. Furthermore, we have identified uPA (urokinase-type plasminogen activator) as an anosmin-1 interactant. Anosmin-1 significantly enhances the amidolytic activity of uPA in vitro; and anosmin-1-HS-uPA co-operation induces cell proliferation in the PC-3 prostate carcinoma cell line. Both the HS interaction and an intact WAP domain are required for the mitogenic activity of anosmin-1. These effects appear to be mediated by a direct protein interaction between anosmin-1 and uPA, since anosmin-1-uPA could be co-immunoprecipitated from PC-3 cell lysates, and their direct binding with high affinity (K(d)=6.91 nM) was demonstrated by SPR. We thus propose that anosmin-1 may modulate the catalytic activity of uPA and its signalling pathway, whereas HS determines cell surface localization of the anosmin-1-uPA complex.
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Cromosomas Humanos X , Proteínas de la Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Síndrome de Kallmann , Proteínas del Tejido Nervioso/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Bovinos , Línea Celular , Proliferación Celular , Cromosomas Humanos X/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Vectores Genéticos/genética , Heparina/metabolismo , Humanos , Síndrome de Kallmann/genética , Cinética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
BACKGROUND: Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. METHODS AND RESULTS: The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V(+/-); homozygote=KI-TnC-A8V(+/+)) were characterized by echocardiography and pressure-volume studies. Three-month-old KI-TnC-A8V(+/+) mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V(+/-) mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography-mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈ 21%) into the KI-TnC-A8V(+/-) cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca(2+) and contractile transients were recorded in intact KI-TnC-A8V(+/-) and KI-TnC-A8V(+/+) cardiomyocytes. Ca(2+) sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V(+/+)>KI-TnC-A8V(+/-)>wild-type, whereas KI-TnC-A8V(+/+) relaxed more slowly on flash photolysis of diazo-2. CONCLUSIONS: The TNNC1-A8V mutant increases the Ca(2+)-binding affinity of the thin filament and elicits changes in Ca(2+) homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.
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Cardiomiopatía Hipertrófica/genética , Predisposición Genética a la Enfermedad , Troponina C/genética , Adulto , Animales , Calcio/metabolismo , Cardiomiopatía Hipertrófica/diagnóstico por imagen , Técnicas de Sustitución del Gen , Corazón , Humanos , Masculino , Ratones , Mutación , Contracción Miocárdica , Miocardio/patología , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos , Sarcómeros , UltrasonografíaRESUMEN
The distribution of the cells expressing three prepro-gonadotrophin-releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH-II (cGnRH-II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross-reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH-associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken-II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP-immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP-ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP-ir fibers. The seabream GAP-ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP-ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP-ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken-II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP-ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species.
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Lubina/metabolismo , Química Encefálica , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/análisis , Hipófisis/química , Animales , Pollos , Femenino , Hormona Liberadora de Gonadotropina/inmunología , Inmunohistoquímica , Masculino , Precursores de Proteínas/análisis , Precursores de Proteínas/inmunología , Salmón , Dorada , Especificidad de la EspecieRESUMEN
In this study, we have analyzed the ontogenic expression of three gonadotrophin-releasing hormones (GnRH) systems expressed in the brain of a perciform fish, the European sea bass, using in situ hybridization. The riboprobes used correspond to the GnRH-associated peptide (GAP) coding regions of the three prepro-GnRH cDNAs cloned from the same species: prepro-salmon GnRH, prepro-seabream GnRH and prepro-chicken GnRH II. On day 4 after hatching, the first prepro-chicken GnRH-II mRNA-expressing cells appeared in the germinal zone of the third ventricle. They increased in number and size from 10 to 21 days, reaching at day 30 their adult final position, within the synencephalic area, at the transitional zone between the diencephalon and the mesencephalon. First prepro-salmon GnRH mRNA-expressing cells became evident on day 7 arising from the olfactory placode and migrating towards the olfactory nerve. On day 10, this cell group reached the olfactory bulb, being evident in the ventral telencephalon and preoptic area from days 15 and 45, respectively. Weakly labeled prepro-seabream GnRH mRNA-expressing cells were first detected at 30 days in the olfactory area and ventral telencephalon. On day 45, prepro-seabream GnRH mRNA-expressing cells were also present in the preoptic region reaching the ventrolateral hypothalamus on day 60. The results obtained in sea bass indicate that sGnRH and sbGnRH cells have a common origin in an olfactory primordium suggesting that both forms might arise from a duplication of a single ancestral gene, while cGnRH-II cells develop from a synencephalic primordium.
Asunto(s)
Lubina/genética , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Hormona Liberadora de Gonadotropina/biosíntesis , Precursores de Proteínas/biosíntesis , ARN Mensajero/biosíntesis , Animales , Lubina/embriología , Lubina/crecimiento & desarrollo , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Pollos , Hormona Liberadora de Gonadotropina/genética , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Salmón , DoradaRESUMEN
The knowledge of the roles and origins of different gonadotrophin-releasing hormone (GnRH) systems could greatly contribute to improve the understanding of mechanisms involved in the physiological control of early development, puberty and spawning. Thus, in this study, we have analyzed the distribution of the cells expressing salmon GnRH, seabream GnRH and chicken GnRH-II forms in the brain and pituitary of developing sea bass using specific antibodies to their corresponding GnRH-associated peptides. The first prepro-chicken GnRH-II-immunoreactive cells arose in the germinal zone of the third ventricle at 4 days after hatching, increasing their number from days 10 to 30, in which they adopted their adult position. The prepro-chicken GnRH-II-immunoreactive fibers became conspicuous in the first week and from day 26 they reached almost all brain areas, especially the hindbrain, being never detected in the pituitary. First prepro-salmon GnRH-immunoreactive cells were detected in the olfactory placode at day 7 after hatching and reached the olfactory bulbs at day 10. Migrating prepro-salmon GnRH cells arrived at the ventral telencephalon at day 15, and became apparent in the preoptic area from day 45. The prepro-salmon GnRH innervation was more evident in the forebrain and increased notably between 10 and 30 days, at which fibers already extended from the olfactory bulbs to the medulla. A few prepro-salmon GnRH-immunoreactive fibers were observed in the pituitary from day 30. The prepro-seabream GnRH-immunoreactive cells were first detected at day 26 in the rostral olfactory bulbs. On day 30, prepro-seabream GnRH-immunoreactive cells were also present in the ventral telencephalon, reaching the preoptic area and the hypothalamus at 45 and 60 days, respectively. The prepro-seabream GnRH innervation appeared restricted to the ventral forebrain, increasing notably during the sixth week, when fibers also reached the pituitary. A significant prepro-seabream GnRH innervation was not detected in the pituitary until day 60.