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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.
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Vesículas Extracelulares , Melanoma , Humanos , Caveolina 1/metabolismo , Autofagossomos/metabolismo , Vesículas Extracelulares/metabolismo , Colesterol/metabolismoRESUMO
Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.
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Estágios do Ciclo de Vida/fisiologia , Malária/parasitologia , Microdomínios da Membrana/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Animais , Colesterol/química , Colesterol/metabolismo , Simulação por Computador , Detergentes/química , Modelos Animais de Doenças , Gametogênese/fisiologia , Humanos , Lipídeos/análise , Microdomínios da Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Proteômica , Esfingolipídeos/química , Esfingolipídeos/metabolismoRESUMO
The p66Shc gerontogene may affect healthspan by promoting fat accumulation. We assessed changes of p66Shc-mRNA in peripheral tissues in relation to maternal obesity and the moderating effects of resistance-training (RT) exercise in elderly frail women. Thirty-seven women participated in a 4-month RT program. Twenty were offspring of lean/normal weight mothers and 17 were offspring of overweight/obese mothers (OOM). P66Shc was assessed in peripheral blood mononuclear cells (PBMC) and in subcutaneous adipose tissue (SAT) before and after RT. Overall, OOM showed elevated p66Shc mRNA levels in the PBMC. Independently from maternal obesity, following RT there was a decrease in p66Shc expression in PBMC but not in SAT, particularly in subjects with a high body mass index. Results suggest that maternal obesity has long-term effects on the expression of genes involved in mitochondrial function and fat deposition and that RT modifies p66Shc expression in PBMC with greater effects in obese subjects.ClinicalTrials.gov ID: NCT01931540.
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Fragilidade/genética , Obesidade/genética , Treinamento Resistido/métodos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/sangue , Tecido Adiposo/metabolismo , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Mães , Projetos Piloto , Gravidez , RNA Mensageiro/sangueRESUMO
Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.
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Membrana Eritrocítica/química , Microdomínios da Membrana/química , Plasmodium falciparum/genética , Proteoma/genética , Proteínas de Protozoários/genética , Trofozoítos/metabolismo , Anticorpos/química , Centrifugação com Gradiente de Concentração , Colesterol/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Detergentes/química , Membrana Eritrocítica/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/química , Microdomínios da Membrana/parasitologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Anotação de Sequência Molecular , Fosfolipídeos/química , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Transporte Proteico , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/químicaRESUMO
Endocrine-disrupting compounds are chemicals that alter the normal functioning of the endocrine system of living organisms. They can be natural (N-EDCs) or synthetic compounds (S-EDCs). N-EDCs can belong to different groups, such as phytoestrogens (PEs), including flavonoids, or mycotoxins originating from plants or fungi, and cyanotoxins, derived from bacteria. Humans encounter these substances in their daily lives. The aim of this rapid review (RR) is to provide a fine mapping of N-EDCs and their toxicological effects on human health in terms of various medical conditions or adverse consequences. This work is based on an extensive literature search and follows a rigorous step-by-step approach (search strategy, analysis strategy and data extraction), to select eligible papers published between 2019 and 2023 in the PubMed database, and to define a set of aspects characterizing N-EDCs and the different human target systems. Of the N-EDCs identified in this RR, flavonoids are the most representative class. Male and female reproductive systems were the targets most affected by N-EDCs, followed by the endocrine, nervous, bone and cardiovascular systems. In addition, the perinatal, pubertal and pregnancy periods were found to be particularly susceptible to natural endocrine disruptors. Considering their current daily use, more toxicological research on N-EDCs is required.
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Blood and airway eosinophilia represent markers for the endotype-driven treatment of allergic asthma. Little is known on mechanisms that link eosinophils and airway epithelial cells before and after these cells are infiltrated by eosinophils during allergic response. Given that innate immune mechanisms, mainly mediated by epithelial-derived cytokines (IL-33, IL-25, TSLP), induce eosinophil-maturing/attractive substances, we thought to evaluate the crosstalk between eosinophils and airway epithelial cells in the context of IL-33-mediated allergic inflammation. DUOX1 was previously described in clinically relevant aspects of allergic inflammation in a HDM -induced allergic asthma mice model, and in patients with chronic sinusitis or allergic asthma. Thus, we evaluated the involvement of HDM and eosinophils in the regulation of DUOX1 in airway epithelial cells. To recapitulate the lung environment present at the allergen challenge time in acute asthma, we set up an in vitro model based on murine bone marrow-derived eosinophils differentiated with IL-5 and then activated with IL-33 (EOs33) and TC1 or C57 airway epithelial cells. We found that treatment of epithelial cells with HDM induced an eosinophil-attractive environment and increased DUOX1 expression. Importantly, we found that the co-culture of airway epithelial cells with EOs33 or with conditioned medium from EOs33 enhanced the expression of DUOX1, which was further increased by combined stimulation (HDM plus EOs33). Our results suggest that lung recruited EOs once activated by IL-33 could be involved in a crosstalk loop with airway epithelial cells by DUOX1-mediated IL-33 secretion.
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A growing body of evidence suggests that regular consumption of natural products might promote healthy aging; however, their mechanisms of action are still unclear. Rosmarinic acid (RA) is a polyphenol holding anti-inflammatory, antioxidant and neuroprotective properties. The aim of this study was to characterise the efficacy of an oral administration of RA in promoting healthspan in a mouse model of physiological aging. Aged C57Bl/6 male and female (24-month-old) mice were either administered with RA (500 mg/Kg) or a vehicle in drinking bottles for 52 days while 3-month-old mice receiving the same treatment were used as controls. All subjects were assessed for cognitive abilities in the Morris water maze (MWM) and for emotionality in the elevated-plus maze test (EPM). Brain-derived Neurotrophic Factor (BDNF) protein levels were evaluated in the hippocampus. Since the interaction between metabolic signals and cerebral functions plays a pivotal role in the etiopathogenesis of cognitive decline, the glycaemic and lipid profiles of the mice were also assessed. RA enhanced learning and memory in 24-month-old mice, an effect that was associated to improved glucose homeostasis. By contrast, the lipid profile was disrupted in young adults. This effect was associated with worse glycaemic control in males and with reduced BDNF levels in females, suggesting powerful sex-dependent effects and raising a note of caution for RA administration in young healthy adult subjects.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Estresse Oxidativo , Masculino , Camundongos , Feminino , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cognição , Hipocampo/metabolismo , Camundongos Endogâmicos , Glucose/metabolismo , Lipídeos , Camundongos Endogâmicos C57BL , Ácido RosmarínicoRESUMO
Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.
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Caveolina 1/metabolismo , Melanoma/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Actinas/metabolismo , Caveolina 1/genética , Contagem de Células , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Melanoma/patologia , Fosforilação , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/genéticaRESUMO
A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.
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Amiloide/efeitos adversos , Apoptose/efeitos dos fármacos , Hipocampo/metabolismo , Microdomínios da Membrana/metabolismo , Neuritos/metabolismo , Amiloide/farmacologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Hipocampo/patologia , Microdomínios da Membrana/patologia , Camundongos , Células NIH 3T3 , Neuritos/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia , RatosRESUMO
Social isolation is a powerful stressor capable of affecting brain plasticity and function. In the case of breast cancer, previous data indicate that stressful experiences may contribute to a worse prognosis, activating neuroendocrine and metabolism pathways, although the mechanisms underlying these effects are still poorly understood. In this study, we tested the hypothesis that chronic isolation stress (IS) may boost hypothalamic-pituitary-adrenal (HPA) axis activity, leading to changes in the hypothalamic expression of genes modulating both mood and metabolism in an animal model of breast cancer. This centrally activated signaling cascade would, in turn, affect the mammary gland microenvironment specifically targeting fat metabolism, leading to accelerated tumor onset. MMTVNeuTg female mice (a model of breast cancer developing mammary hyperplasia at 5 months of age) were either group-housed (GH) or subjected to IS from weaning until 5 months of age. At this time, half of these subjects underwent acute restraint stress to assess corticosterone (CORT) levels, while the remaining subjects were characterized for their emotional profile in the forced swimming and saccharin preference tests. At the end of the procedures, all the mice were sacrificed to assess hypothalamic expression levels of Brain-derived neurotrophic factor (Bdnf), Neuropeptide Y (NpY), Agouti-Related Peptide (AgRP), and Serum/Glucocorticoid-Regulated Protein Kinase 1 (SgK1). Leptin and adiponectin expression levels, as well as the presence of brown adipose tissue (BAT), were assessed in mammary fat pads. The IS mice showed higher CORT levels following acute stress and decreased expression of NpY, AgRP, and SgK1, associated with greater behavioral despair in the forced swimming test. Furthermore, they were characterized by increased consumption of saccharin in a preference test, suggesting an enhanced hedonic profile. The IS mice also showed an earlier onset of breast lumps (assessed by palpation) accompanied by elevated levels of adipokines (leptin and adiponectin) and BAT in the mammary fat pads. Overall, these data point to IS as a pervasive stressor that is able to specifically target neuronal circuits, mastered by the hypothalamus, modulating mood, stress reactivity and energy homeostasis. The activation of such IS-driven machinery may hold main implications for the onset and maintenance of pro-tumorigenic environments.
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BACKGROUND: The NCAM or CD56 antigen is a cell surface glycoprotein belonging to the immunoglobulin super-family involved in cell-cell and cell-matrix adhesion. NCAM is also over-expressed in many tumour types and is considered a tumour associated antigen, even if its role and biological mechanisms implicated in tumour progression and metastasis have not yet to be elucidated. In particular, it is quite well documented the role of the interaction between the NCAM protein and the fibroblast growth factor receptor-1 in metastasis and invasion, especially in the ovarian cancer progression. OBJECTIVE: Here we describe the isolation and preliminary characterization of a novel human anti-NCAM single chain Fragment variable antibody able to specifically bind NCAM-expressing cells, including epithelial ovarian cancer cells. METHODS: The antibody was isolate by phage display selection and was characterized by ELISA, FACS analysis and SPR experiments. Interference in EOC migration was analyzed by scratch test. RESULTS: It binds a partially linear epitope lying in the membrane proximal region of two fibronectin-like domains with a dissociation constant of 3.43 × 10-8 M. Interestingly, it was shown to interfere with the NCAM-FGFR1 binding and to partially decrease migration of EOC cells. CONCLUSIONS: According to our knowledge, this is the first completely human antibody able to interfere with this newly individuated cancer mechanism.
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Bacteriófagos , Transdução de Sinais , Bacteriófagos/metabolismo , Humanos , Imunoglobulinas , Moléculas de Adesão de Célula Nervosa/metabolismo , Ligação ProteicaRESUMO
The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II-IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0-I, II and III-IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0-I) from late (III-IV) stages' CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III-IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV' FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.
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Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.
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Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Progressão da Doença , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Melanoma/patologia , Microscopia Confocal/métodos , Modelos Biológicos , Metástase Neoplásica , Prótons , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Espectrometria de Fluorescência/métodosRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.
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Astrócitos/metabolismo , Encéfalo/metabolismo , Cavéolas/metabolismo , Caveolina 1/metabolismo , Leucoencefalopatias/metabolismo , Proteínas de Membrana/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Encéfalo/fisiopatologia , Cavéolas/ultraestrutura , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Colesterol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Complexo de Proteínas Associadas Distrofina/metabolismo , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/fisiopatologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico/fisiologia , RatosRESUMO
Prion pathologies are fatal neurodegenerative diseases caused by the misfolding of the physiological Prion Protein (PrPC) into a ß-structure-rich isoform called PrPSc. To date, there is no available cure for prion diseases and just a few clinical trials have been carried out. The initial approach in the search of anti-prion agents had PrPSc as a target, but the existence of different prion strains arising from alternative conformations of PrPSc, limited the efficacy of the ligands to a straindependent ability. That has shifted research to PrPC ligands, which either act as chaperones, by stabilizing the native conformation, or inhibit its interaction with PrPSc. The role of transition-metal mediated oxidation processes in prion misfolding has also been investigated. Another promising approach is the indirect action via other cellular targets, like membrane domains or the Protein- Folding Activity of Ribosomes (PFAR). Also, new prion-specific high throughput screening techniques have been developed. However, so far no substance has been found to be able to extend satisfactorily survival time in animal models of prion diseases. This review describes the main features of the Structure-Activity Relationship (SAR) of the various chemical classes of anti-prion agents.
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Doenças Priônicas , Animais , Doenças Priônicas/tratamento farmacológico , Príons , Dobramento de Proteína , Relação Estrutura-AtividadeRESUMO
Caveolin-1 (Cav-1), a member of the caveolin family, regulates caveolae-associated signaling proteins, which are involved in many biological processes, including cancer development. Cav-1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav-1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav-1, and then utilized as a recipient for Cav-1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho (Ph)-Cav-1 on cell regulation was also tested by transducing the nonphosphorylatable Cav-1Y14A mutant. Wild-type Cav-1, but not mutated Cav-1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav-1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav-1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav-1 levels, the amount of microvesicles released in the culture medium and MMP-9 expression was also observed.
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Biomarcadores Tumorais/metabolismo , Caveolina 1/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Western Blotting , Caveolina 1/genética , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/patologia , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologiaRESUMO
MLC1 gene mutations have been associated with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurologic disorder in children. The MLC1 gene encodes a membrane protein (MLC1) with unknown function which is mainly expressed in astrocytes. Using a newly developed anti-human MLC1 polyclonal antibody, we have investigated the biochemical properties and localization of MLC1 in cultured astrocytes and brain tissue and searched for evidence of a relationship between MLC1 and proteins of the dystrophin-glycoprotein complex (DGC). Cultured astrocytes express two MLC1 components showing different solubilisation properties and subcellular distribution. Most importantly, we show that the membrane-associated component of MLC1 (60-64 kDa) localizes in astrocytic lipid rafts together with dystroglycan, syntrophin and caveolin-1, and co-fractionates with the DGC in whole rat brain tissue. In the human brain, MLC1 protein is expressed in astrocyte processes and ependymal cells, where it colocalizes with dystroglycan and syntrophin. These data indicate that the DGC may be involved in the organization and function of the MLC1 protein in astrocyte membranes.
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Astrócitos/metabolismo , Distroglicanas/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrocitoma/patologia , Encéfalo/citologia , Células Cultivadas , Proteínas Associadas à Distrofina/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Ratos , Frações Subcelulares/metabolismoRESUMO
Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (Lo) phase typical of the raft structure, namely "cholesterol-rich Lo phase," with a phase fully missing in Cav-1 KO that we named "caveolin-induced Lo phase." Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a "caveolin-induced Lo phase," as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources.
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Caveolina 1/metabolismo , Colesterol/metabolismo , Proteômica/métodos , Animais , Cavéolas/metabolismo , Humanos , Difração de Raios XRESUMO
Tamoxifen resistance is a major hurdle in the treatment of estrogen receptor (ER)-positive breast cancer. The mechanisms of tamoxifen resistance are not fully understood although several underlying molecular events have been suggested. Recently, we identified autoantibodies reacting with membrane-associated ERα (anti-ERα Abs) in sera of breast cancer patients, able to promote tumor growth. Here, we investigated whether anti-ERα Abs purified from sera of ER-positive breast cancer patients could contribute to tamoxifen resistance. Anti-ERα Abs inhibited tamoxifen-mediated effects on cell cycle and proliferation in MCF-7 cells. Moreover, anti-ERα Abs hampered the tamoxifen-mediated reduction of tumor growth in SCID mice xenografted with breast tumor. Notably, simvastatin-mediated disaggregation of lipid rafts, where membrane-associated ERα is embedded, restored tamoxifen sensitivity, preventing anti-ERα Abs effects. In conclusion, detection of serum anti-ERα Abs may help predict tamoxifen resistance and concur to appropriately inform therapeutic decisions concerning hormone therapy in ER-positive breast cancer patients.
Assuntos
Antineoplásicos Hormonais/imunologia , Autoanticorpos/sangue , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/imunologia , Receptor alfa de Estrogênio/imunologia , Tamoxifeno/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Hormonais/uso terapêutico , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.