RESUMEN

Bioactive peptides, derived from short protein fragments, are recognized for their neuroprotective properties and potential therapeutic applications in treating central nervous system (CNS) diseases. However, a significant challenge for these peptides is their ability to penetrate the blood-brain barrier (BBB). EVSGPGYSPN (EV-10) peptide, a walnut-derived peptide, has demonstrated promising neuroprotective effects in vivo. This study aimed to investigate the transportability of EV-10 across the BBB, explore its capacity to penetrate this barrier, and elucidate the regulatory mechanisms underlying peptide-induced cellular internalization and transport pathways within the BBB. The results indicated that at a concentration of 100 µM and osmotic time of 4 h, the apparent permeability coefficient of EV-10 was Papp = 8.52166 ± 0.58 × 10-6 cm/s. The penetration efficiency of EV-10 was influenced by time, concentration, and temperature. Utilizing Western blot analysis, immunofluorescence, and flow cytometry, in conjunction with the caveolin (Cav)-specific inhibitor M-ß-CD, we confirmed that EV-10 undergoes transcellular transport through a Cav-dependent endocytosis pathway. Notably, the tight junction proteins ZO-1, occludin, and claudin-5 were not disrupted by EV-10. Throughout its transport, EV-10 was localized within the mitochondria, Golgi apparatus, endoplasmic reticulum, lysosomes, endosomes, and cell membranes. Moreover, Cav-1 overexpression facilitated the release of EV-10 from lysosomes. Evidence of EV-10 accumulation was observed in mouse brains using brain slice scans. This study is the first to demonstrate that Cav-1 can facilitate the targeted delivery of walnut-derived peptide to the brain, laying a foundation for the development of functional foods aimed at CNS disease intervention.

Asunto(s)

Barrera Hematoencefálica , Juglans , Péptidos , Juglans/química , Juglans/metabolismo , Barrera Hematoencefálica/metabolismo , Animales , Ratones , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Transporte Biológico , Caveolinas/metabolismo , Caveolinas/química , Humanos , Endocitosis , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Nueces/química , Nueces/metabolismo

RESUMEN

SLC38A6 (SNAT6) is the only known member of the SLC38 family that is expressed exclusively in the excitatory neurons of the brain. It has been described as an orphan transporter with an unknown substrate profile, therefore very little is known about SNAT6. In this study, we addressed the substrate specificity, mechanisms for internalization of SNAT6, and the regulatory role of SNAT6 with specific insights into the glutamate-glutamine cycle. We used tritium-labeled amino acids in order to demonstrate that SNAT6 is functioning as a glutamine and glutamate transporter. SNAT6 revealed seven predicted transmembrane segments in a homology model and was localized to caveolin rich sites at the plasma membrane. SNAT6 has high degree of specificity for glutamine and glutamate. Presence of these substrates enables formation of SNAT6-caveolin complexes that aids in sodium dependent trafficking of SNAT6 off the plasma membrane. To further understand its mode of action, several potential interacting partners of SNAT6 were identified using bioinformatics. Among them where CTP synthase 2 (CTPs2), phosphate activated glutaminase (Pag), and glutamate metabotropic receptor 2 (Grm2). Co-expression analysis, immunolabeling with co-localization analysis and proximity ligation assays of these three proteins with SNAT6 were performed to investigate possible interactions. SNAT6 can cycle between cytoplasm and plasma membrane depending on availability of substrates and interact with Pag, synaptophysin, CTPs2, and Grm2. Our data suggest a potential role of SNAT6 in glutamine uptake at the pre-synaptic terminal of excitatory neurons. We propose here a mechanistic model of SNAT6 trafficking that once internalized influences the glutamate-glutamine cycle in presence of its potential interacting partners.

Asunto(s)

Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Caveolinas/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Caveolinas/química , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Ratones , Modelos Biológicos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/genética , Transducción de Señal , Sodio/metabolismo , Relación Estructura-Actividad

RESUMEN

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 µm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Asunto(s)

Caveolinas/química , Composición de Medicamentos/métodos , Inhaladores de Polvo Seco/métodos , Excipientes , Péptidos/administración & dosificación , Polvos/administración & dosificación , Dominios Proteicos , Fibrosis Pulmonar/tratamiento farmacológico , Administración por Inhalación , Aerosoles , Animales , Química Farmacéutica/métodos , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos/métodos , Estabilidad de Medicamentos , Femenino , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Polvos/química , Resultado del Tratamiento

RESUMEN

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.

Asunto(s)

Caveolinas/química , Secuencia de Aminoácidos , Animales , Humanos , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-ß-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.

Asunto(s)

Calcio/metabolismo , Caveolinas/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Caveolinas/química , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar

RESUMEN

Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae.

Asunto(s)

Caveolas/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caveolas/química , Caveolinas/química , Caveolinas/metabolismo , Membrana Celular/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Unión a Fosfato , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilserinas/química , Multimerización de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Transporte Vesicular

RESUMEN

Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.

Asunto(s)

Caveolas/química , Caveolas/metabolismo , Caveolinas/química , Caveolinas/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Caveolas/ultraestructura , Cristalografía por Rayos X , Citoplasma/química , Citoplasma/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Transducción de Señal/fisiología , Pez Cebra/metabolismo

RESUMEN

Vascular integrity or the maintenance of blood vessel continuity is a fundamental process regulated by endothelial cell-cell junctions. Defects in endothelial barrier function are an initiating factor in several disease processes including tumor angiogenesis and metastasis. The glycosaminoglycan, hyaluronan (HA), maintains vascular integrity through specific mechanisms including HA-binding protein signaling in caveolin-enriched microdomains, a subset of lipid rafts. Certain disease states, including cancer, increase enzymatic hyaluronidase activity and reactive oxygen species generation, which break down high molecular weight HA (HMW-HA) to low molecular weight fragments (LMW-HA). LMW-HA can activate specific HA-binding proteins during tumor progression to promote disruption of endothelial cell-cell contacts. In contrast, exogenous administration of HMW-HA promotes enhancement of vascular integrity. This review focuses on the roles of HA in regulating angiogenic and metastatic processes based on its size and the HA-binding proteins present. Further, potential therapeutic applications of HMW-HA in treating cancer are discussed.

Asunto(s)

Endotelio/metabolismo , Regulación Neoplásica de la Expresión Génica , Ácido Hialurónico/química , Neoplasias/metabolismo , Animales , Antineoplásicos/química , Caveolinas/química , Progresión de la Enfermedad , Glicosaminoglicanos/química , Humanos , Receptores de Hialuranos , Microdominios de Membrana/química , Peso Molecular , Metástasis de la Neoplasia , Neovascularización Patológica , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno , Transducción de Señal/fisiología

RESUMEN

BACKGROUND: SG2NA is a member of the striatin sub-family of WD-40 repeat proteins. Striatin family members have been associated with diverse physiological functions. SG2NA has also been shown to have roles in cell cycle progression, signal transduction etc. They have been known to interact with a number of proteins including Caveolin and Calmodulin and also propagate the formation of a multimeric protein unit called striatin-interacting phosphatase and kinase. As a pre-requisite for such interaction ability, these proteins are known to be unstable and primarily disordered in their arrangement. Earlier we had identified that it has multiple isoforms (namely 35, 78, 87 kDa based on its molecular weight) which are generated by alternative splicing. However, detailed structural information of SG2NA is still eluding the researchers. RESULTS: This study was aimed towards three-dimensional molecular modeling and characterization of SG2NA protein and its isoforms. One structure out of five was selected for each variant having the least value for C score. Out of these, m35 kDa with a C score value of -3.21 was the most poorly determined structure in comparison to m78 kDa and m87 kDa variants with C scores of -1.16 and -1.97 respectively. Further evaluation resulted in about 61.6% residues of m35 kDa, 76.6% residues of m78 kDa and 72.1% residues of m87 kDa falling in the favorable regions of Ramchandran Plot. Molecular dynamics simulations were also carried out to obtain biologically relevant structural models and compared with previous atomic coordinates. N-terminal region of all variants was found to be highly disordered. CONCLUSION: This study provides first-hand detailed information to understand the structural conformation of SG2NA protein variants (m35 kDa, m78 kDa and m87 kDa). The WD-40 repeat domain was found to constitute antiparallel strands of ß-sheets arranged circularly. This study elucidates the crucial structural features of SG2NA proteins which are involved in various protein-protein interactions and also reveals the extent of disorder present in the SG2NA structure crucial for excessive interaction and multimeric protein complexes. The study also potentiates the role of computational approaches for preliminary examination of unknown proteins in the absence of experimental information.

Asunto(s)

Autoantígenos/química , Proteínas de Unión a Calmodulina/química , Calmodulina/química , Caveolinas/química , Bases de Datos de Proteínas , Humanos , Simulación de Dinámica Molecular , Peso Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Estabilidad Proteica , Estructura Secundaria de Proteína

RESUMEN

AIM: This study is designed to test the hypothesis that tenofovir-loaded (an anti-HIV microbicide) chitosan-thioglycolic acid-conjugated (CS-TGA) nanoparticles (NPs) exhibit superior biophysical properties for mucoadhesion compared with those of native CS NPs. MATERIALS & METHODS: The NPs are prepared by ionotropic gelation. The particle mean diameter, encapsulation efficiency and release profile are analyzed by dynamic light scattering and UV spectroscopy, respectively. The cytotoxicity, cellular uptake and uptake mechanism are assessed on VK2/E6E7 and End1/E6E7 cell lines by colorimetry/fluorimetry, and percentage mucoadhesion is assessed using porcine vaginal tissue. RESULTS: The mean diameter of the optimal NP formulations ranges from 240 to 252 nm, with a maximal encapsulation efficiency of 22.60%. Tenofovir release from CS and CS-TGA NPs follows first-order and Higuchi models, respectively. Both NPs are noncytotoxic in 48 h. The cellular uptake, which is time dependent, mainly occurs via the caveolin-mediated pathway. The percentage of mucoadhesion of CS-TGA NPs is fivefold higher than that of CS NPs, and reached up to 65% after 2 h. CONCLUSION: Collectively, CS-TGA NPs exhibit superior biophysical properties and can potentially maximize the retention time of a topical microbicide, such as tenofovir, intended for the prevention of HIV transmission.

Asunto(s)

Adenina/análogos & derivados , Quitosano/química , Infecciones por VIH/tratamiento farmacológico , Nanopartículas/química , Organofosfonatos/administración & dosificación , Compuestos de Sulfhidrilo/química , Tioglicolatos/química , Adenina/administración & dosificación , Animales , Fármacos Anti-VIH/administración & dosificación , Caveolinas/química , Línea Celular , Sistemas de Liberación de Medicamentos , Fluorometría , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Nanomedicina , Porcinos , Tenofovir

Asunto(s)

Caveolas/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Animales , Caveolas/química , Caveolinas/química , Membrana Celular/química , Humanos

RESUMEN

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

Asunto(s)

Caveolas/fisiología , Membrana Celular/química , Membrana Celular/metabolismo , Animales , Caveolas/química , Caveolas/metabolismo , Caveolinas/química , Caveolinas/genética , Caveolinas/metabolismo , Caveolinas/fisiología , Citoprotección/genética , Citoprotección/fisiología , Endocitosis/genética , Endocitosis/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Modelos Biológicos , Transducción de Señal/genética , Transducción de Señal/fisiología

RESUMEN

Caveolins are coat proteins of caveolae, small flask-shaped pits of the plasma membranes of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules. Caveolin-protein interactions are commonly considered to occur between a ∼20 amino acid region within caveolin, the caveolin scaffolding domain (CSD), and an aromatic-rich caveolin binding motif (CBM) on the binding partner (фXфXXXXф, фXXXXфXXф or фXфXXXXфXXф, where ф is an aromatic and X an unspecified amino acid). The CBM resembles a typical linear motif--a short, simple sequence independently evolved many times in different proteins for a specific function. Here we exploit recent improvements in bioinformatics tools and in our understanding of linear motifs to critically examine the role of CBMs in caveolin interactions. We find that sequences conforming to the CBM occur in 30% of human proteins, but find no evidence for their statistical enrichment in the caveolin interactome. Furthermore, sequence- and structure-based considerations suggest that CBMs do not have characteristics commonly associated with true interaction motifs. Analysis of the relative solvent accessible area of putative CBMs shows that the majority of their aromatic residues are buried within the protein and are thus unlikely to interact directly with caveolin, but may instead be important for protein structural stability. Together, these findings suggest that the canonical CBM may not be a common characteristic of caveolin-target interactions and that interfaces between caveolin and targets may be more structurally diverse than presently appreciated.

Asunto(s)

Aminoácidos Aromáticos , Caveolinas/química , Caveolinas/metabolismo , Biología Computacional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteoma/metabolismo , Solventes/química

Asunto(s)

Caveolas/metabolismo , Caveolas/ultraestructura , Caveolinas/metabolismo , Membrana Celular/metabolismo , Animales , Caveolas/química , Caveolinas/química , Caveolinas/genética , Membrana Celular/ultraestructura , Cordados/metabolismo , Humanos , Invertebrados/citología , Invertebrados/metabolismo , Especificidad de Órganos , Transducción de Señal

RESUMEN

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1ß and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Asunto(s)

Apoptosis/fisiología , Caveolinas/metabolismo , Cerebelo/citología , Citocromo-B(5) Reductasa/metabolismo , Microdominios de Membrana/metabolismo , Neuronas/fisiología , Estrés Oxidativo/fisiología , Animales , Caveolinas/química , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/fisiología , Activación Enzimática , Microdominios de Membrana/química , Neuronas/citología , Neuronas/metabolismo , Transporte de Proteínas , Proteómica/métodos , Ratas , Ratas Wistar , Superóxidos/análisis , Superóxidos/metabolismo

RESUMEN

Immunophilins belong to a highly conserved family of proteins with cis-trans peptidyl-prolyl isomerase activity, generally classified by their ability to selectively bind specific immunosuppressive drugs, thereby regulating their activity. Immunophilins include Cyclophilins (CyPs), which are specific targets of the immunosuppressant drug cyclosporin A (CsA); FKBPs (FK506-binding proteins), that are sensitive to both FK506 (tacrolimus) and rapamycin (sirolimus); and FCBPs which are sensitive to CsA and FK506. Immunophilins are expressed in multiple human tissues, including brain, heart, kidney, liver and lung and regulate functions as diverse as intracellular calcium signaling, protein transport, protein folding and gene transcription. In particular, immunophilins play key functional roles in the cardiovascular system, where they can associate with proteins such as ryanodine and IP3 receptors (RyR and IP3R), calcineurin, and mitochondrial permeability transition pore (MPTP) and Heat-shock proteins-caveolin-cholesterol complex and regulate their function. The biological importance of immunophilins is further revealed by the pathophysiology, as they have been implicated in several cardiovascular diseases, including vascular stenosis, atherosclerosis, heart failure and arrhythmias. This review summarizes some of the most recent studies on immunophilins and focuses on their roles in the mechanisms underlying the cardiovascular disease.

Asunto(s)

Enfermedades Cardiovasculares/complicaciones , Inmunofilinas/metabolismo , Inmunosupresores/uso terapéutico , Animales , Señalización del Calcio , Cardiotónicos/metabolismo , Cardiotónicos/uso terapéutico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Caveolinas/química , Caveolinas/metabolismo , Colesterol/química , Colesterol/metabolismo , Humanos , Inmunofilinas/genética , Inmunosupresores/metabolismo

RESUMEN

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

Asunto(s)

Caveolinas/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos/toxicidad , Triglicéridos/biosíntesis , Aminas/química , Aminas/metabolismo , Caveolina 1/química , Caveolina 1/metabolismo , Caveolina 3/química , Caveolina 3/metabolismo , Caveolinas/química , Línea Celular , Relación Dosis-Respuesta a Droga , Espacio Extracelular/metabolismo , Fluoresceínas/metabolismo , Regulación de la Expresión Génica , Movimiento , Fosfatidiletanolaminas/metabolismo

RESUMEN

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-beta-cyclodextrin (MbetaCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged mu-opioid receptors (HEK FLAG-mu) or delta-opioid receptors (HEK FLAG-delta) with MbetaCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MbetaCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing mu-opioid receptors. The effects of MbetaCD may be due to uncoupling of the mu receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-mu cells, MbetaCD treatment of HEK FLAG-delta cells had no effect on acute inhibition or sensitization of adenylyl cyclase by delta-opioid agonists. The differential responses of mu- and delta-opioid agonists to cholesterol depletion suggest that mu-opioid receptors are more dependent on cholesterol for efficient signaling than delta receptors and can be partly explained by localization of mu- but not delta-opioid receptors in cholesterol- and caveolin-enriched membrane domains.

Asunto(s)

Adenilil Ciclasas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Transducción de Señal , Caveolinas/química , Línea Celular , Línea Celular Tumoral , Toxina del Cólera/química , Colesterol/química , Citoesqueleto/metabolismo , Humanos , Modelos Biológicos , Estructura Terciaria de Proteína , Receptores de Transferrina/metabolismo

RESUMEN

Caveolins, essential scaffold proteins of caveolae, have emerged as versatile molecules that can influence numerous signaling pathways and disease processes. This brief review is focused on the role of caveolins in the physiology and pathology of musculoskeletal system and will discuss the key features of caveolins as related to differentiation and diseases of bone and muscle cells.

Asunto(s)

Caveolinas/fisiología , Enfermedades Musculoesqueléticas/metabolismo , Animales , Antineoplásicos/farmacocinética , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Huesos/metabolismo , Huesos/patología , Caveolas/fisiología , Caveolas/ultraestructura , Caveolinas/química , Caveolinas/deficiencia , Caveolinas/genética , Diferenciación Celular , Resistencia a Múltiples Medicamentos/fisiología , Resistencia a Antineoplásicos/fisiología , Humanos , Ratones , Ratones Noqueados , Células Musculares/metabolismo , Desarrollo de Músculos/fisiología , Neoplasias de los Músculos/tratamiento farmacológico , Neoplasias de los Músculos/metabolismo , Neoplasias de los Músculos/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculoesqueléticas/patología , Osteoblastos/metabolismo , Osteoblastos/patología , Osteogénesis/fisiología

RESUMEN

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.

Asunto(s)

Caveolas/metabolismo , Integrina alfa2beta1/metabolismo , Microdominios de Membrana/química , Quinasas p21 Activadas/metabolismo , Amilorida/farmacología , Antígenos Transformadores de Poliomavirus/metabolismo , Caveolinas/química , Línea Celular Tumoral , Clatrina/metabolismo , Enterovirus Humano B/metabolismo , Humanos , Microscopía Confocal/métodos , Modelos Biológicos , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismo