RESUMEN
Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses â¼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Virus Sincitiales Respiratorios/inmunología , Vacunación/métodos , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Caveolina 1 , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/uso terapéutico , Cultivo Primario de Células , Virus Sincitiales Respiratorios/patogenicidad , Vacunas/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/metabolismo , Proteínas Virales de Fusión/fisiologíaRESUMEN
We have uncovered the existence of extracellular vesicle (EV)-mediated signaling between cell types within the adipose tissue (AT) proper. This phenomenon became evident in our attempts at generating an adipocyte-specific knockout of caveolin 1 (cav1) protein. Although we effectively ablated the CAV1 gene in adipocytes, cav1 protein remained abundant. With the use of newly generated mouse models, we show that neighboring endothelial cells (ECs) transfer cav1-containing EVs to adipocytes in vivo, which reciprocate by releasing EVs to ECs. AT-derived EVs contain proteins and lipids capable of modulating cellular signaling pathways. Furthermore, this mechanism facilitates transfer of plasma constituents from ECs to the adipocyte. The transfer event is physiologically regulated by fasting/refeeding and obesity, suggesting EVs participate in the tissue response to changes in the systemic nutrient state. This work offers new insights into the complex signaling mechanisms that exist among adipocytes, stromal vascular cells, and, potentially, distal organs.
Asunto(s)
Adipocitos/metabolismo , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Ayuno/metabolismo , Transducción de Señal , Animales , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular , Células Cultivadas , Endotelio Vascular/citología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-µ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell-induced autoimmunity by means of its role in plasma-membrane organization.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Caveolina 1/metabolismo , Tolerancia Inmunológica , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Caveolina 1/genética , Expresión Génica , Tolerancia Inmunológica/genética , Inmunoglobulina D/inmunología , Inmunoglobulina D/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Unión Proteica , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.
Asunto(s)
Proteínas Portadoras/inmunología , Células Endoteliales/inmunología , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Animales , Antígenos/inmunología , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Portadoras/genética , Caveolina 1/deficiencia , Caveolina 1/genética , Caveolina 1/inmunología , Células Endoteliales/citología , Endotelio Linfático/citología , Endotelio Linfático/inmunología , Femenino , Regulación de la Expresión Génica , Ganglios Linfáticos/citología , Vasos Linfáticos/citología , Vasos Linfáticos/inmunología , Linfocitos/citología , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Migración Transendotelial y Transepitelial/inmunologíaRESUMEN
Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
Asunto(s)
Caveolina 1 , Movimiento Celular , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Transporte Biológico , Cicatrización de Heridas/fisiología , Orgánulos/metabolismoRESUMEN
Zika virus (ZIKV) has gained notoriety in recent years because there are no targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveil that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches.
Asunto(s)
Caveolina 1 , ARN Viral , Replicación Viral , Infección por el Virus Zika , Virus Zika , Caveolina 1/metabolismo , Caveolina 1/genética , Virus Zika/fisiología , Virus Zika/metabolismo , Humanos , Infección por el Virus Zika/virología , Infección por el Virus Zika/metabolismo , ARN Viral/metabolismo , ARN Viral/genética , Animales , Interacciones Huésped-Patógeno , Chlorocebus aethiops , Células Vero , Células HEK293 , Internalización del Virus , Replicación de ARNRESUMEN
Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.
Asunto(s)
Proteínas Argonautas , Caveolina 1 , MicroARNs , Metástasis de la Neoplasia , Animales , Humanos , Ratones , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Caveolina 1/metabolismo , Caveolina 1/genética , Línea Celular Tumoral , Vesículas Extracelulares/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , MicroARNs/genética , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Sirtuina 2/metabolismo , Sirtuina 2/genéticaRESUMEN
Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.
Asunto(s)
Caveolina 1/metabolismo , Metástasis de la Neoplasia/patología , Neoplasias/patología , Animales , Movimiento Celular , Fibroblastos/patología , Humanos , Melanoma/patología , Ratones , Ratones NoqueadosRESUMEN
Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical-physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.
Asunto(s)
Vesículas Extracelulares , Melanoma , Humanos , Caveolina 1/metabolismo , Autofagosomas/metabolismo , Vesículas Extracelulares/metabolismo , Colesterol/metabolismoRESUMEN
Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA-MB-231 triple-negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non-phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho-associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA-MB-231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae-independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA-MB-231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1-ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild-type MDA-MB-231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK-dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy.
Asunto(s)
Caveolina 1 , Neoplasias de la Próstata , Masculino , Humanos , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Mecanotransducción Celular , Mitocondrias/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Mitocondriales/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.
Asunto(s)
Caveolina 1 , Endosomas , Caveolina 1/metabolismo , Endosomas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Endocitosis , Clatrina/metabolismoRESUMEN
BACKGROUND: In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) treated advanced atherosclerosis using a blocking antibody for IL-1ß (interleukin-1ß); this significantly reduced cardiovascular events. However, whether IL-1ß regulates early disease, particularly LDL transcytosis, remains unknown. METHODS: We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1ß. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1ß and LDL to visualize acute LDL deposition in the aortic arch. RESULTS: Exposure to picomolar concentrations of IL-1ß induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1ß increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1ß on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNA sequencing data to curate a list of Rab (Ras-associated binding) GTPases affected by IL-1ß, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1ß. This was phenocopied by depletion of the Rab27a effector JFC1 (synaptotagmin-like protein 1). In vivo, IL-1ß increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1ß-induced LDL accumulation in the aorta. CONCLUSIONS: IL-1ß induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1ß may contribute to the acceleration of early disease.
Asunto(s)
Vasos Coronarios , Células Endoteliales , Interleucina-1beta , Lipoproteínas LDL , Ratones Noqueados , Receptores de LDL , Transducción de Señal , Transcitosis , Proteínas de Unión al GTP rab , Interleucina-1beta/metabolismo , Animales , Humanos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Lipoproteínas LDL/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/efectos de los fármacos , Células Cultivadas , Ratones Endogámicos C57BL , Caveolina 1/metabolismo , Caveolina 1/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Modelos Animales de Enfermedad , Aorta Torácica/metabolismo , Aorta Torácica/efectos de los fármacos , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Masculino , RatonesRESUMEN
Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.
Asunto(s)
Caveolas , Proteínas de Unión al ARN , Caveolas/química , Caveolina 1/química , Células HEK293 , Humanos , Fosfatidilinositol 4,5-Difosfato/química , Dominios Proteicos , Transporte de Proteínas , Proteínas de Unión al ARN/química , Transducción de SeñalRESUMEN
Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitationtranscription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.
Asunto(s)
Canales de Calcio Tipo L , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Caveolas , Transcripción Genética , Remodelación Vascular , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Caveolas/metabolismo , Caveolina 1/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Acoplamiento Excitación-Contracción , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Neuronas/metabolismo , FosforilaciónRESUMEN
The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.
Asunto(s)
Homeostasis , Miocitos Cardíacos , Proteínas de Unión al ARN , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Miocitos Cardíacos/metabolismo , Ratones , Caveolina 1/metabolismo , Caveolina 1/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Miocardio/metabolismo , Regulación de la Expresión Génica , Adenosina/análogos & derivados , Adenosina/metabolismo , Ratones Noqueados , Biosíntesis de ProteínasRESUMEN
The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.
Asunto(s)
Caveolina 1 , Receptores Purinérgicos P2X7 , Animales , Ratones , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMEN
Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-ß signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSCs with the TGF-ß inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-ß signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.NEW & NOTEWORTHY A Na/K-ATPase α1 caveolin-binding motif regulates adipogenesis. Mutation of this binding motif in the mouse leads to reduced fat with increased extracellular matrix production and inflammation. RNA-seq analysis and pharmacological interventions in human iPSC-derived adipocytes revealed that TGF-ß signal, rather than Na/K-ATPase-mediated ion transport, is a key mediator of NKA regulation of adipogenesis.
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Adipocitos , Adipogénesis , Caveolina 1 , Células Madre Pluripotentes Inducidas , ATPasa Intercambiadora de Sodio-Potasio , Adipogénesis/genética , Animales , Caveolina 1/metabolismo , Caveolina 1/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Humanos , Ratones , Adipocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Diferenciación Celular , Masculino , Matriz Extracelular/metabolismo , Secuencias de Aminoácidos , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. METHODS: Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. RESULTS: Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. CONCLUSION: Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.
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Caveolina 1 , Proteína Quinasa C-alfa , Animales , Masculino , Ratones , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Ácidos y Sales Biliares/metabolismo , Caveolina 1/metabolismo , Colesterol/metabolismo , Hepatocitos/metabolismo , Ratones Endogámicos C57BL , Proteína Quinasa C-alfa/metabolismo , ARN Mensajero/metabolismo , HumanosRESUMEN
Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a disease-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with WT CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.
Asunto(s)
Caveolina 1 , Caveolinas , Enfermedad , Humanos , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Subunidades de Proteína/metabolismo , Enfermedad/genéticaRESUMEN
Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1, and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways, including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.NEW & NOTEWORTHY Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.