RESUMEN
Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine.
RESUMEN
Myocardial fibroblast activation coupled with extracellular matrix production is a pathological signature of myocardial fibrosis and is governed mainly by transforming growth factor TGFß-Smad2/3 signaling. Targeting the ubiquitous TGFß leads to cellular homeostasis deregulation with adverse consequences. We previously showed the anti-fibrotic effects upon downregulation of 90-kDa heat shock protein (Hsp90), a chaperone that associates to the TGFß signaling cascade. In the present study, we use a fluorescent-labeled Hsp90 protein inhibitor (CTPR390-488) with specific Hsp90 binding properties to reduce myocardial pro-fibrotic events in vitro and in vivo. The mechanism of action involves the disruption of TGFßRI-Hsp90 complex, resulting in a decrease in TGFß signaling and reduction in extracellular matrix collagen. In vivo, decreased myocardial collagen deposition was observed upon CTPR390-488 treatment in a pro-fibrotic mouse model. This is the first study demonstrating the ability of an engineered Hsp90 protein inhibitor to block collagen expression, reduce the motility of myocardial TGFß-activated fibroblasts and ameliorate angiotensin-II induced cardiac myocardial fibrosis in vivo.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Miocardio/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Fibrosis , Técnica del Anticuerpo Fluorescente , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Ratones , Ratones Noqueados , Microscopía Confocal , Modelos Moleculares , Miocardio/patología , Péptidos/química , Péptidos/farmacología , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. RESULTS: The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. CONCLUSIONS: Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.
Asunto(s)
Fosfatos de Inositol/aislamiento & purificación , Fosfatos de Inositol/farmacología , Insulina/farmacología , Lathyrus/química , Polisacáridos/aislamiento & purificación , Polisacáridos/farmacología , Animales , División Celular/efectos de los fármacos , Línea Celular , Embrión de Pollo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ácidos Grasos/análisis , Ganglios/citología , Ganglios/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/aislamiento & purificación , Glicosilfosfatidilinositoles/farmacología , Hidrólisis , Técnicas In Vitro , Fosfatos de Inositol/química , Hígado/efectos de los fármacos , Hígado/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Polisacáridos/química , Ratas , Semillas/químicaRESUMEN
Diacylglycerol increased the hydrolytic activity of phosphatidylinositol-specific phospholipase C on large unilamellar vesicles containing 5-40% phosphatidylinositol. Moreover, diacylglycerol increased the rate and extent of vesicle fusion (contents mixing) induced by the enzyme. Kinetic studies of intervesicular lipid mixing revealed that fusion was limited by the frequency of contacts involving two diacylglycerol-rich domains.
Asunto(s)
Diglicéridos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana/fisiología , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/metabolismo , Bacillus cereus/enzimología , Liposomas/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa CRESUMEN
Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.
Asunto(s)
Membrana Dobles de Lípidos/química , Fusión de Membrana , Fosfolipasas de Tipo C/química , Colesterol/química , Diglicéridos/química , Colorantes Fluorescentes , Liposomas/química , Modelos Químicos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Fosfolípidos/química , Espectrometría de FluorescenciaRESUMEN
Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D.
Asunto(s)
Gangliósidos/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Liposomas/síntesis química , Liposomas/metabolismo , Hígado/metabolismo , Hígado/fisiología , Animales , Hidrólisis , Técnicas In Vitro , Neuraminidasa/metabolismo , Permeabilidad , Fosfolipasa D/metabolismo , Ratas , Factores de Tiempo , alfa-Galactosidasa/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI) purified from rat liver lipids was incorporated into lipid bilayers of defined compositions, in the form of large unilamellar vesicles. The GPI concentration in the bilayers was kept constant at 25 mole%, whereas the remaining lipids being phosphatidylcholine, phosphastidylethanolamine, sphingomyelin and/or cholesterol were varied. The resulting liposomes consisted of spherical vesicles, approximately 100 nm in diameter, that could keep their aqueous contents separated from the extravesicular medium. When these liposomes were treated with either Bacillus cereus phosphatidylinositol-phospholipase C, Trypanosoma brucei GPI-phospholipase C, or bovine serum GPI-phospholipase D, GPI was hydrolyzed at different rates, depending on the enzyme and the bilayer lipid composition. These observations open the way to biophysical and biochemical studies of enzymic GPI cleavage under defined conditions. Extensive GPI hydrolysis was observed in certain cases that could allow the use of these systems for the preparation of inositol phosphoglycans, proposed second messengers of a wide variety of hormones, cytokines and growth factors.