RESUMO
Caveolins are a family of transmembrane proteins located in caveolae, small lipid raft invaginations of the plasma membrane. The roles of caveolin-enriched lipid rafts are diverse, and include mechano-protection, lipid homeostasis, metabolism, transport, and cell signaling. Caveolin-1 (Cav-1) and other caveolins were described in endothelial cells and later in other cell types of the central nervous system (CNS), including neurons, astrocytes, oligodendrocytes, microglia, and pericytes. This pancellular presence of caveolins demands a better understanding of their functional roles in each cell type. In this review we describe the various functions of Cav-1 in the cells of normal and pathological brains. Several emerging preclinical findings suggest that Cav-1 could represent a potential therapeutic target in brain disorders.
Assuntos
Caveolinas , Sistema Nervoso Central , Humanos , Animais , Caveolinas/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Caveolina 1/metabolismo , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.
Assuntos
Cavéolas , Membrana Celular , Humanos , Cavéolas/metabolismo , Membrana Celular/metabolismo , Animais , Caveolinas/metabolismoRESUMO
We explored the potential of overcoming the dense interstitial barrier in pancreatic cancer treatment by enhancing the uptake of hydrophilic chemotherapeutic drugs. In this study, we synthesized the squalenoyl-chidamide prodrug (SQ-CHI), linking lipophilic squalene (SQ) with the hydrophilic antitumor drug chidamide (CHI) through a trypsin-responsive bond. Self-assembled nanoparticles with sigma receptor-bound aminoethyl anisamide (AEAA) modification, forming AEAA-PEG-SQ-CHI NPs (A-C NPs, size 116.6 ± 0.4 nm), and reference nanoparticles without AEAA modification, forming mPEG-SQ-CHI NPs (M-C NPs, size 88.3 ± 0.3 nm), were prepared. A-C NPs exhibited significantly higher in vitro CHI release (74.7 %) in 0.5 % trypsin medium compared to release (20.2 %) in medium without trypsin. In vitro cell uptake assays revealed 3.6 and 2.3times higher permeation of A-C NPs into tumorspheres of PSN-1/HPSC or CFPAC-1/HPSC, respectively, compared to M-C NPs. Following intraperitoneal administration to subcutaneous tumor-bearing nude mice, the A-C NPs group demonstrated significant anti-pancreatic cancer efficacy, inducing cancer cell apoptosis and inhibiting proliferation in vivo. Mechanistic studies revealed that AEAA surface modification on nanoparticles promoted intracellular uptake through caveolin-mediated endocytosis. This nanoparticle system presents a novel therapeutic approach for pancreatic cancer treatment, offering a delivery strategy to enhance efficacy through improved tumor permeation, trypsin-responsive drug release, and specific cell surface receptor-mediated intracellular uptake.
Assuntos
Aminopiridinas , Benzamidas , Nanopartículas , Neoplasias Pancreáticas , Pró-Fármacos , Animais , Camundongos , Caveolinas/uso terapêutico , Camundongos Nus , Tripsina , Nanopartículas/química , Pró-Fármacos/química , Neoplasias Pancreáticas/tratamento farmacológico , Linhagem Celular TumoralRESUMO
The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.
Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Camundongos , Humanos , Animais , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos Transgênicos , Caveolinas/metabolismo , Mamíferos/metabolismoRESUMO
Background Microalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. Methods We established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. Results In vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner (AU)
Antecedentes La microalbuminuria es un síntoma clínico común que se manifiesta en las fases tempranas de la enfermedad renal diabética (ERD), y también es característica del daño de las células endoteliales glomerulares (GEC). Existe evidencia creciente en cuanto a que la transcitosis de la albúmina a través de las GEC está estrechamente relacionada con la formación de albuminuria. Nuestros estudios previos reflejaron que angiopoyetina 2 (ANGPT2) puede inhibir la transcitosis de la albúmina a través de las células epiteliales tubulares renales activando la fosforilación de caveolina 1 (CAV1) durante la exposición a hiperglucemia (HG). El rol de ANGPT2 en la transcitosis de la albúmina a través de las GEC resulta incierto. Losartan reduce considerablemente la albuminuria, aunque no se ha esclarecido el mecanismo. Métodos Establecimos un modelo in vitro de transcitosis de la albúmina para investigar el cambio de dicho mecanismo a través de las células endoteliales glomerulares renales humanas (hrGEC) en condiciones de glucosa normal (GN), hiperglucemia (HG) e intervención de losartan. Realizamos breakdown de ANGPT2 y CAV1 para evaluar sus roles en la transcitosis de la albúmina a través de las hrGEC, y verificamos la relación entre ellas. Se establecieron modelos in vivo de ratones con ERD, tratados con diferentes dosis de losartan. Se utilizaron pruebas de inmunohistoquímica e inmunotransferencia para detectar la expresión de ANGPT2 y CAV1. Resultados In vitro, la transcitosis de la albúmina a través de hrGEC se incrementó considerablemente en condiciones de estimulación de la hiperglucemia, inhibiendo losartan este proceso. La expresión de ANGPT2 y CAV1 se incrementó en las hrGEC en condiciones de HG, reduciendo la intervención de losartan la expresión de ambas (AU)
Assuntos
Animais , Masculino , Camundongos , Diabetes Mellitus Experimental/metabolismo , Glomérulos Renais/metabolismo , Albuminas/metabolismo , Transcitose , Angiopoietinas/metabolismo , Camundongos Endogâmicos C57BL , Caveolinas/farmacologia , Losartan/farmacologia , Modelos AnimaisRESUMO
BACKGROUND AND OBJECTIVE: Studies have shown that Caveolin gene polymorphisms (CAV-1) are involved in chronic diseases, such as metabolic syndrome. Moreover, the dietary insulin index (DII) and dietary insulin load (DIL) have been shown to potentially elicit favorable effects on cardiovascular disease (CVD) risk. Therefore, this study sought to investigate the effect of DII DIL and CAV-1 interaction on CVD risk factors. METHODS: This cross-sectional study consisted of 333 overweight and obese women aged 18-48 years. Dietary intakes, DII, and DIL were evaluated using the 147-item food frequency questionnaire (FFQ). Serum profiles were measured by standard protocols. The CAV-1 rs 3,807,992 and anthropometric data were measured by the PCR-RFLP method and bioelectrical impedance analysis (BIA), respectively. Participants were also divided into three groups based on DII, DIL score, and rs3807992 genotype. RESULTS: This comparative cross-sectional study was conducted on 333 women classified as overweight or obese. Participants with A allele for the caveolin genotype and higher DII score showed significant interactions with high-density lipoprotein (HDL) (P for AA = 0.006 and P for AG = 0.019) and CRI-I (P for AA < 0.001 and P for AG = 0.024). In participants with AA genotype and greater DII score, interactions were observed in weight, systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol, CRI-II, fat-free mass (FFM), and skeletal muscle mass (SMM) (P < 0.079). Those with higher DIL scores and AA genotype had higher weight (P = 0.033), FFM (P = 0.022), and SMM (P = 0.024). In addition, DIL interactions for waist/hip ratio (WHR), waist circumference (WC), triglyceride (TG), CRI-I, and body fat mass (BFM) among individuals with AA genotype, while an HDL interaction was observed in individuals with AG and AA (P < 0.066). CONCLUSION: The findings of the present study indicate that people who carry the caveolin rs3807992 (A) allele and have greater DII and DIL scores are at higher risk for several cardiovascular disease and metabolic syndrome biomarkers. These results highlight that diet, gene variants, and their interaction, should be considered in the risk evaluation of developing CVD.
Assuntos
Doenças Cardiovasculares , Síndrome Metabólica , Feminino , Humanos , Insulina , Sobrepeso/genética , Caveolinas , Estudos Transversais , Fatores de Risco Cardiometabólico , Síndrome Metabólica/genética , Doenças Cardiovasculares/genética , Obesidade/genética , DietaRESUMO
Micro(nano)plastics are prevalent in the environment, and prolonged exposure to them represents a threat to human health. The goal of this study is to assess the health risk of long-term exposure to nanoplastics (NPs) at environmental concentrations on the intestinal mechanical and immune barrier in mice. In this study, mice were provided drinking water containing polystyrene NPs (PS-NPs; 0.1, 1, and 10 mg·L-1) for 32 consecutive weeks. The levels of endocytosis proteins caveolin and clathrin and of tight junctional proteins claudin-1, occludin, and ZO-1, and morphological changes, proportion of lymphocytes B in MLNs and lymphocytes T in IELs and LPLs were determined by immunohistochemistry, hematoxylin-eosin, and flow cytometry assays in the intestinal tissues of mice at 28 weeks. The activities or concentrations of ROS, SOD, MDA, and GSH-Px and inflammatory factors (IL-1ß, IL-6, and TNF-α) in the intestinal tissues of mice were measured by ELISA at 12, 16, 20, 24, and 32 weeks. Compared with the control group, oral ingested PS-NPs entered the intestinal tissues of mice and upregulated expression levels of the clathrin and caveolin. The intestinal tissue structure of mice in the PS-NPs (1 and 10 mg·L-1) exposure groups showed significant abnormalities, such as villus erosion, decreased of crypts numbers and large infiltration of inflammatory cells. Exposure to 0.1 mg·L-1 PS-NPs decreased occludin protein levels, but not claudin-1 and ZO-1 levels. The levels of these three tight junction proteins decreased significantly in the 1 and 10 mg·L-1 PS-NPs exposed groups. Exposure to PS-NPs led to a significant time- and dose-dependent increase in ROS and MDA levels, and concurrently decreased GSH-Px and SOD contents. Exposure to PS-NPs increased the proportion of B cells in MLNs, and decreased the proportion of CD8+ T cells in IELs and LPLs. The levels of pro-inflammatory cytokines IL-6, TNF-α and IL-1ß were markedly elevated after PS-NPs exposure. Long-term PS-NPs exposure impaired intestinal mechanical and immune barrier, and indicate a potentially significant threat to human health.
Assuntos
Nanopartículas , Poliestirenos , Humanos , Poliestirenos/toxicidade , Microplásticos , Linfócitos T CD8-Positivos , Interleucina-6 , Ocludina , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa , Caveolinas , Clatrina , Superóxido DismutaseRESUMO
In the mid-1950s, a groundbreaking discovery revealed the fascinating presence of caveolae, referred to as flask-shaped invaginations of the plasma membrane, sparking renewed excitement in the field of cell biology. Caveolae are small, flask-shaped invaginations in the cell membrane that play crucial roles in diverse cellular processes, including endocytosis, lipid homeostasis, and signal transduction. The structural stability and functionality of these specialized membrane microdomains are attributed to the coordinated activity of scaffolding proteins, including caveolins and cavins. While caveolae and caveolins have been long appreciated for their integral roles in cellular physiology, the accumulating scientific evidence throughout the years reaffirms their association with a broad spectrum of human disorders. This review article aims to offer a thorough account of the historical advancements in caveolae research, spanning from their initial discovery to the recognition of caveolin family proteins and their intricate contributions to cellular functions. Furthermore, it will examine the consequences of a dysfunctional caveolar network in the development of human diseases.
Assuntos
Cavéolas , Caveolinas , Humanos , Cavéolas/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de SinaisRESUMO
Lung cancer is one of the most frequently diagnosed cancers worldwide. Due to its late diagnosis, it remains the leading cause of cancer-related deaths. Despite it is mostly associated to tobacco smoking, recent data suggested that genetic factors are of the highest importance. In this context, different processes meaningful for the development and progression of lung cancer such endocytosis, protein secretion and signal transduction, are controlled by membrane rafts. These highly ordered membrane domains contain proteins such as caveolins and flotillins, which were traditionally considered scaffold proteins but have currently been given a preponderant role in lung cancer. Here, we summarize current knowledge regarding the involvement of caveolins and flotillins in lung cancer from a molecular point of view.
Assuntos
Caveolinas , Neoplasias Pulmonares , Humanos , Caveolinas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microdomínios da MembranaRESUMO
Caveolins form complexes of various sizes that deform membranes into polyhedral shapes. However, the recent structure of the 8S complex was disk-like with a flat membrane-binding surface. How can a flat complex deform membranes into nonplanar structures? Molecular dynamics simulations revealed that the 8S complex rapidly takes the form of a suction cup. Simulations on implicit membrane vesicles determined that binding is stronger when E140 gets protonated. In that case, the complex binds much more strongly to 5- and 10-nm-radius vesicles. A concave membrane-binding surface readily explains the membrane-deforming ability of caveolins by direct scaffolding. We propose that the 8S complex sits at the vertices of the caveolar polyhedra, rather than at the center of the polyhedral faces.
Assuntos
Caveolinas , Proteínas de Membrana , Caveolinas/análise , Caveolinas/metabolismo , Proteínas de Membrana/química , Caveolina 1/metabolismo , Membranas/metabolismo , Membrana Celular/metabolismoRESUMO
Efficient transfection remains a challenge for gene delivery in both cell biological scientific research and gene therapeutic fields. Existing transfection strategies rarely pay attention to altering the endocytosis pathway of nanocarriers for transfection efficiency improvement. In this work, we innovatively postulated that calcium phosphate nanoparticles coated with glycosaminoglycan could be internalized by cells mainly through caveolin-mediated endocytosis pathway allowing genes to bypass lysosome route, and hence enhance the transfection efficiency. To achieve this, we developed calcium phosphate nanoparticles (CP-ALN-CS) coated with chondroitin sulfate (CS) and alendronate (ALN) in a modular manner. The CP-ALN-CS had a hydrodynamic size of 131.0 ± 8.7 nm and exhibited favorable dispersity, stability, and resistance to nuclease degradation. Unlike conventional calcium phosphate and PEI-based transfection, CP-ALN-CS exhibited efficient cellular uptake with co-localization in Golgi apparatus and endoplasmic reticulum. Through bypassing the lysosome involved cellular uptake route, CP-ALN-CS can effectively protect genes from degradation and relieve cytotoxicity. After loading plasmid DNA, CP-ALN-CS showed extraordinary transfection efficiency in HEK 293T cells, outperforming the PEI which is considered as the gold standard. The current work provides a novel and facile approach to improve gene transfection efficiency and is valuable for the design of next-generation in vitro transfection reagents.
Assuntos
Sulfatos de Condroitina , Nanopartículas , Transfecção , Plasmídeos , Endocitose , Fosfatos de Cálcio , Caveolinas/genéticaRESUMO
Hepatocellular carcinoma (HCC) is largely associated with aberrant activation of Wnt/ß-catenin signaling. Nevertheless, how membrane lipid composition is altered in HCC cells with abnormal Wnt signaling remains elusive. Here, by exploiting comprehensive lipidome profiling, we unravel the membrane lipid composition of six different HCC cell lines with mutations in components of Wnt/ß-catenin signaling, leading to differences in their endogenous signaling activity. Among the differentially regulated lipids are diacylglycerol (DAG) and ceramide, which were downregulated at the membrane of HCC cells after Wnt3a treatment. DAG and ceramide enhanced Wnt/ß-catenin signaling by inducing caveolin-mediated endocytosis of the canonical Wnt-receptor complex, while their depletion suppressed the signaling activity along with a reduction of caveolin-mediated endocytosis in SNU475 and HepG2 cells. Moreover, depletion of DAG and ceramide significantly impeded the proliferation, tumor growth, and in vivo migration capacity of SNU475 and HepG2 cells. This study, by pioneering plasma membrane lipidome profiling in HCC cells, exhibits the remarkable potential of lipids to correct dysregulated signaling pathways in cancer and stop abnormal tumor growth.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , beta Catenina/metabolismo , Carcinoma Hepatocelular/patologia , Caveolinas , Linhagem Celular Tumoral , Proliferação de Células/genética , Ceramidas , Diglicerídeos , Lipidômica , Neoplasias Hepáticas/patologia , Via de Sinalização WntRESUMO
Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.
Assuntos
Caveolinas , Proteostase , Caveolinas/metabolismo , Caveolina 1/metabolismo , Proteínas de Membrana/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismoRESUMO
Caveolae are plasma membrane invaginations with a distinct lipid composition. Membrane lipids cooperate with the structural components of caveolae to generate a metastable surface domain. Recent studies have provided insights into the structure of essential caveolar components and how lipids are crucial for the formation, dynamics, and disassembly of caveolae. They also suggest new models for how caveolins, major structural components of caveolae, insert into membranes and interact with lipids.
Assuntos
Cavéolas , Lipídeos de Membrana , Cavéolas/química , Cavéolas/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Caveolinas/análise , Caveolinas/metabolismo , EndocitoseRESUMO
GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.
Assuntos
Arrestina , Receptores Acoplados a Proteínas G , Humanos , Arrestina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo , Dinaminas/metabolismo , Endocitose/fisiologia , Clatrina/metabolismo , Caveolinas/metabolismo , Receptores de Peptídeos/metabolismoRESUMO
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.
Assuntos
Caveolina 1 , Caveolinas , Doença , Humanos , Cavéolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Subunidades Proteicas/metabolismo , Doença/genéticaRESUMO
Background and Objectives: The saphenous vein is one of the most common used grafts (SVG) for surgical revascularization. The mechanism of the SVGs occlusion is still unknown. Surgical preparation techniques have an important role in the early and late graft occlusion. Our study analyzed the influence of the three different surgical techniques on the histological and immunohistochemical characteristics of the vein grafts. Methods: Between June 2019 and December 2020, 83 patients who underwent surgical revascularization were prospectively randomly assigned to one of the three groups, according to saphenous vein graft harvesting (conventional (CVH), no-touch (NT) and endoscopic (EVH)) technique. The vein graft samples were sent on the histological (hematoxylin-eosin staining) and immunohistochemical (CD31, Factor VIII, Caveolin and eNOS) examinations. Results: The CVH, NT, and EVH groups included 27 patients (mean age 67.66 ± 5.6), 31 patients (mean age 66.5 ± 7.4) and 25 patients (mean age 66 ± 5.5), respectively. Hematoxylin-eosin staining revealed a lower grade of microstructural vein damage in the NT group (2, IQR 1-2) in comparison with CVH and EVH (3, IQR 2-4), (4, IQR 2-4) respectively (p < 0.001). Immunohistochemical examination revealed a high grade of staining in the NT group compared to the CVH and EVH group (CD 31 antibody p = 0.02, FVIII, p < 0.001, Caveolin, p = 0.001, and eNOS, p = 0.003). Conclusion: The best preservation of the structural vein integrity was in the NT group, while the lowest rate of leg wound complication was in the EVH group. These facts increase the interest in developing and implementing the endoscopic no-touch technique.
Assuntos
Ponte de Artéria Coronária , Veia Safena , Idoso , Humanos , Pessoa de Meia-Idade , Caveolinas/análise , Ponte de Artéria Coronária/métodos , Endoscopia , Veia Safena/química , Veia Safena/patologia , Veia Safena/transplante , Grau de Desobstrução VascularRESUMO
Bovine herpesvirus-1 (BoHV-1) can infect all breeds of cattle and cause severe respiratory organs and genital tract diseases. However, the mechanism of BoHV-1 entering the cells remains unclear. In this study, we explored the mechanism of BoHV-1 entering MDBK cells. We found that the entry of BoHV-1 was blocked by NH4Cl and bafilomycin A1, indicating that BoHV-1 entry is dependent on the acidic environment of endosome. Specific inhibitor dynasore and small interfering RNA (siRNA) knockdown of dynamin-2 inhibited BoHV-1 entry, showing that dynamin is required in BoHV-1 entry. The results of specific inhibitor, siRNA knockdown and co-localization indicating clathrin- and caveolin- mediated endocytosis play a role in BoHV-1 entry. BoHV-1 infection was not affected by EIPA which is a specific inhibitor of macropinocytosis. In addition, we found that BoHV-1 triggered PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways to induce clathrin-mediated and caveolin-mediated endocytosis at the early stage of BoHV-1 infection. BoHV-1 binding was sufficient to activate the endocytic signaling pathways and promote viral entry. These two signaling pathways were activated by transfection of viral gD protein, and were inhibited by deletion of viral gD protein and the siRNA knockdown of cellular receptor nectin-1. The results of co-localization indicating the entered BoHV-1 is traced to late endosomes via early endosomes. Our results suggested the interaction of viral gD protein and cellular receptor nectin-1 triggered the PI3K-Akt-NF-κB and Ras-p38 MAPK signaling pathways and induced clathrin-mediated and caveolin-mediated endocytosis to promote BoHV-1 entry into MDBK cells at the early stage of BoHV-1 infection.
Assuntos
Herpesvirus Bovino 1 , Internalização do Vírus , Bovinos , Animais , NF-kappa B , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Herpesvirus Bovino 1/fisiologia , Clatrina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno , Nectinas , Linhagem Celular , Endocitose , Caveolinas , RNA Interferente PequenoRESUMO
In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide-sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs.