Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 208
Filtrar
Mais filtros










Filtros aplicados

Base de dados
Intervalo de ano de publicação
1.
FEBS Lett ; 592(23): 3835-3864, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29802621

RESUMO

Gangliosides (GGs) are sialic acid-containing glycosphingolipids (GSLs) and major membrane components enriched on cellular surfaces. Biosynthesis of mammalian GGs starts at the cytosolic leaflet of endoplasmic reticulum (ER) membranes with the formation of their hydrophobic ceramide anchors. After intracellular ceramide transfer to Golgi and trans-Golgi network (TGN) membranes, anabolism of GGs, as well as of other GSLs, is catalyzed by membrane-spanning glycosyltransferases (GTs) along the secretory pathway. Combined activity of only a few promiscuous GTs allows for the formation of cell-type-specific glycolipid patterns. Following an exocytotic vesicle flow to the cellular plasma membranes, GGs can be modified by metabolic reactions at or near the cellular surface. For degradation, GGs are endocytosed to reach late endosomes and lysosomes. Whereas membrane-spanning enzymes of the secretory pathway catalyze GSL and GG formation, a cooperation of soluble glycosidases, lipases and lipid-binding cofactors, namely the sphingolipid activator proteins (SAPs), act as the main players of GG and GSL catabolism at intralysosomal luminal vesicles (ILVs).


Assuntos
Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Gangliosídeos/metabolismo , Lisossomos/metabolismo , Animais , Sequência de Carboidratos , Gangliosídeos/química , Glicosiltransferases/metabolismo , Humanos , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo
2.
Biochimie ; 130: 146-151, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27157270

RESUMO

Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases.


Assuntos
Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Esfingolipidoses/metabolismo , Esfingolipídeos/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Humanos , Modelos Biológicos
4.
Eur Rev Med Pharmacol Sci ; 19(20): 3955-60, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26531285

RESUMO

OBJECTIVE: To observe the one-year neurologic prognostic outcome of newborns with moderate and severe hypoxic-ischemic encephalopathy (HIE) who received recombinant human erythropoietin (rhuEPO) combined with exogenous monosialotetrahexosylganglioside (GM1) treatment to provide new guidelines for clinical treatment. PATIENTS AND METHODS: Seventy-six newborns with moderate and severe HIE were selected from February 2011 to February 2014 in our hospital. This study received the informed consent of our hospital's Ethics Committee and the newborns' guardians. The newborns were divided to an observation group (n = 34 cases) and a control group (n = 42 cases). All newborns underwent hypothermia and conventional treatment for their conditions. The control group received GMl treatment and observation group received rhuEPO combined with GMl treatment. The curative differences and neural behavior from these two groups were compared. RESULTS: The excellent, efficient proportion and total effective rate of the newborns from the observation group were higher than the control group. The death rate, cerebral palsy and the invalid ratio of the newborns from the observation group were lower than that of the control group. Awareness, muscle tension, primitive reflex and increased intracranial pressure recovery time of the newborns in the observation group were less than those of the control group. The Neonatal Behavior Neurological Assessment (NBNA) score of both groups after the treatment of 7, 14 and 28 days were significantly higher and increased with time (p < 0.05). The MDI, PDI and DQ score of newborns from the two groups all increased after treatment of 3, 6 and 12 months than those of before, which increased with time (p < 0.05). CONCLUSIONS: The rhuEPO + GMl treatment in newborns with HIE improves short-term clinical effects and long-term neurological symptoms.


Assuntos
Eritropoetina/administração & dosagem , Hipóxia-Isquemia Encefálica/diagnóstico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Proteínas Recombinantes/administração & dosagem , Índice de Gravidade de Doença , Proteínas Ativadoras de Esfingolipídeos/administração & dosagem , Paralisia Cerebral/diagnóstico , Paralisia Cerebral/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Recém-Nascido , Masculino , Exame Neurológico/métodos , Prognóstico , Resultado do Tratamento
5.
Biochim Biophys Acta ; 1841(5): 799-810, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24184515

RESUMO

Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates, including cytotoxic lysosphingolipids, such as galactosylsphingosine and glucosylsphingosine, and lead to a sphingolipidosis. Analysis of patients with prosaposin deficiency revealed the accumulation of intra-endolysosmal vesicles and membrane structures (IM). Feeding of prosaposin reverses the storage, suggesting inner membrane structures as platforms of sphingolipid degradation. Water soluble enzymes can hardly attack sphingolipids embedded in the membrane of inner endolysosomal vesicles. The degradation of sphingolipids with few sugar residues therefore requires the help of the SAPs, and is strongly stimulated by anionic membrane lipids. IMs are rich in anionic bis(monoacylglycero)phosphate (BMP). This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.


Assuntos
Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Esfingolipídeos/metabolismo , Animais , Humanos , Proteínas Ativadoras de Esfingolipídeos/metabolismo
6.
J Cell Sci ; 126(Pt 22): 5293-304, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24046456

RESUMO

Cadherins are essential in many fundamental processes and assemble at regions of cell-cell contact in large macromolecular complexes named adherens junctions. We have identified flotillin 1 and 2 as new partners of the cadherin complexes. We show that flotillins are localised at cell-cell junctions (CCJs) in a cadherin-dependent manner. Flotillins and cadherins are constitutively associated at the plasma membrane and their colocalisation at CCJ increases with CCJ maturation. Using three-dimensional structured illumination super-resolution microscopy, we found that cadherin and flotillin complexes are associated with F-actin bundles at CCJs. The knockdown of flotillins dramatically affected N- and E-cadherin recruitment at CCJs in mesenchymal and epithelial cell types and perturbed CCJ integrity and functionality. Moreover, we determined that flotillins are required for cadherin association with GM1-containing plasma membrane microdomains. This allows p120 catenin binding to the cadherin complex and its stabilization at CCJs. Altogether, these data demonstrate that flotillin microdomains are required for cadherin stabilization at CCJs and for the formation of functional CCJs.


Assuntos
Caderinas/metabolismo , Junções Intercelulares/genética , Proteínas de Membrana/metabolismo , Caderinas/genética , Cateninas/metabolismo , Membrana Celular/metabolismo , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Junções Intercelulares/metabolismo , Células MCF-7 , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Proteínas Ativadoras de Esfingolipídeos/metabolismo
7.
Cell Immunol ; 284(1-2): 9-19, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23916875

RESUMO

Hepatitis B is considered to be a worldwide public health problem. An immunosuppressor microenvironment has been proposed to contribute to viral persistence during chronic disease. Understanding the intracellular signaling cascade in T-cells from HBV-infected patients, will contribute to unravel the mechanisms that control the development of immune response during hepatitis B. We analyze lipid rafts formation and early activation signals in chronic HBV infected patients, compared to naturally immune subjects (NIS). Patients show: (1) diminished GM1 clustering, (2) A deficient lipid rafts recruitment of CD3ζ/ZAP-70/Grb2, and (3) these proteins do not merge with GM1 within the lipid rafts. Finally, immunoprecipitation assays proved that ZAP-70 does not associate to CD3ζ. These results show for the first time, defects regarding early key events in T-cell activation, in chronically infected HBV patients, which may contribute not only to understand HBV immune tolerance, but to reveal new potential therapeutic targets to control the infection.


Assuntos
Complexo CD3/imunologia , Proteína Adaptadora GRB2/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Microdomínios da Membrana/imunologia , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/imunologia , Imunidade Adaptativa , Complexo CD3/metabolismo , Citometria de Fluxo , Proteína Adaptadora GRB2/metabolismo , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ativadoras de Esfingolipídeos/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo
8.
Proc Jpn Acad Ser B Phys Biol Sci ; 88(10): 554-82, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23229750

RESUMO

Analysis of lipid storage in postmortem brains of patients with amaurotic idiocy led to the recognition of five lysosomal ganglioside storage diseases and identification of their inherited metabolic blocks. Purification of lysosomal acid sphingomyelinase and ceramidase and analysis of their gene structures were the prerequisites for the clarification of Niemann-Pick and Farber disease. For lipid catabolism, intraendosomal vesicles are formed during the endocytotic pathway. They are subjected to lipid sorting processes and were identified as luminal platforms for cellular lipid and membrane degradation. Lipid binding glycoproteins solubilize lipids from these cholesterol poor membranes and present them to water-soluble hydrolases for digestion. Biosynthesis and intracellular trafficking of lysosomal hydrolases (hexosaminidases, acid sphingomyelinase and ceramidase) and lipid binding and transfer proteins (GM2 activator, saposins) were analyzed to identify the molecular and metabolic basis of several sphingolipidoses. Studies on the biosynthesis of glycosphingolipids yielded the scheme of Combinatorial Ganglioside Biosynthesis involving promiscuous glycosyltransferases. Their defects in mutagenized mice impair brain development and function.


Assuntos
Esfingolipidoses/metabolismo , Esfingolipídeos/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Endocitose , Humanos , Lisossomos/enzimologia , Lisossomos/metabolismo , Proteínas Ativadoras de Esfingolipídeos/química , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Esfingolipidoses/enzimologia , Esfingolipidoses/genética , Esfingolipidoses/patologia , Esfingolipídeos/biossíntese
9.
Exp Neurol ; 235(2): 513-6, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22507308

RESUMO

Serum antibodies to different gangliosides have been identified in some Guillain-Barré (GBS) subtypes and variants. In the January issue of Experimental Neurology Susuki and colleagues (2012) showed that in experimental neuropathies associated with antibodies to GM1, GD1a and GD1b the common mechanism is a complement mediated dysfunction and disruption of the nodes of Ranvier which causes a pathophysiological continuum from early reversible conduction failure to axonal degeneration. These observations, correlated and integrated with electrophysiological and pathological findings in humans indicate that the GBS subtypes acute motor conduction block neuropathy, acute motor axonal neuropathy, acute motor and sensory neuropathy and acute sensory neuropathy and possibly also a chronic disorder as multifocal motor neuropathy represent a spectrum of the same immunopathologic process. Being the nodal axolemma and the paranode the focus of the nerve injury, these immune mediated neuropathies could be more properly classified as nodo-paranodopathies.


Assuntos
Autoanticorpos/fisiologia , Gangliosídeos/imunologia , Polineuropatias/classificação , Polineuropatias/imunologia , Animais , Autoanticorpos/classificação , Síndrome de Guillain-Barré/classificação , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/metabolismo , Humanos , Polineuropatias/metabolismo , Proteínas Ativadoras de Esfingolipídeos/imunologia
10.
J Child Neurol ; 24(12): 1513-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19955343

RESUMO

Mutated PSAP gene resulting in sphingolipid activator protein B deficiency is known to cause metachromatic leukodystrophy variant in which arylsulfatase A is normal. Of 16 patients with metachromatic leukodystrophy that were evaluated in our center, 7 patients were diagnosed with arylsulfatase A-deficient metachromatic leukodystrophy, whereas 9 children from 4 unrelated Saudi families were found to have sphingolipid activator protein B deficiency. PSAP analysis found that the 4 families segregate the same homozygous mutation that was a g.722G>C transversion resulting in C241S change, which was previously reported in an Arab patient. Our work, which reports the largest series of patients with sphingolipid activator protein B deficiency, suggests that this variant is likely to be more common than arylsulfatase A-deficient metachromatic leukodystrophy in Arabs, a notion that has potential diagnostic and preventive implications.


Assuntos
Predisposição Genética para Doença/genética , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/metabolismo , Mutação/genética , Proteínas Ativadoras de Esfingolipídeos/deficiência , Proteínas Ativadoras de Esfingolipídeos/genética , Substituição de Aminoácidos/genética , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Humanos , Lactente , Leucodistrofia Metacromática/fisiopatologia , Masculino , Arábia Saudita
11.
Cent Nerv Syst Agents Med Chem ; 9(2): 119-31, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20021345

RESUMO

Prosaposin, a 66 kDa glycoprotein, was identified initially as the precursor of the sphingolipid activator proteins, saposins A-D, which are required for the enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. While mature saposins are distributed to lysosomes, prosaposin exists in secretory body fluids and plasma membranes. In addition to its role as the precursor, prosaposin shows a variety of neurotrophic and myelinotrophic activities through a receptor-mediated mechanism. In studies in vivo, prosaposin was demonstrated to exert a variety of neuro-efficacies capable of preventing neuro-degeneration following neuro-injury and promoting the amelioration of allodynia and hyperalgesia in pain models. Collective findings indicate that prosaposin is not a simple house-keeping precursor protein; instead, it is a protein essentially required for the development and maintenance of the central and peripheral nervous systems. Accumulating evidence over the last decade has attracted interests in exploring and developing new therapeutic approaches using prosaposin for human disorders associated with neuro-degeneration. In this review we detail the structure characteristics, cell biological feature, in vivo efficacy, and neuro-therapeutic potential of prosaposin, thereby providing future prospective in clinical application of this multifunctional protein.


Assuntos
Bioquímica , Transporte Biológico/fisiologia , Glicoproteínas/uso terapêutico , Neurobiologia , Sistema Nervoso Periférico/fisiologia , Precursores de Proteínas/metabolismo , Saposinas/fisiologia , Proteínas Ativadoras de Esfingolipídeos/uso terapêutico , Processamento Alternativo/genética , Sequência de Aminoácidos , Sequência Conservada/genética , Sequência Conservada/fisiologia , Feminino , Humanos , Lactação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Dados de Sequência Molecular , Fatores de Crescimento Neural/fisiologia , Precursores de Proteínas/genética , Saposinas/genética , Saposinas/metabolismo , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Distribuição Tecidual/fisiologia , Transfecção
12.
Histol Histopathol ; 24(4): 481-92, 2009 04.
Artigo em Inglês | MEDLINE | ID: mdl-19224451

RESUMO

The delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptor (MPR). The MPR has been demonstrated to attain the early endosomes via a process that requires the interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins. Additionally, the MPR can be recycled back to the trans-Golgi network (TGN) through its interaction with the retromer complex. Interestingly, in I-cell disease (ICD), in which the MPR pathway is non-functional, many soluble lysosomal proteins continue to traffic to the lysosomes. This observation led to the discovery that sortilin is responsible for the MPR-independent targeting of the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More recently, our laboratory has tested the hypothesis that sortilin is also capable of sorting a variety of cathepsins that exhibit varying degrees of MPR-independent transport. We have demonstrated that the transport of cathepsin D is partially dependent upon sortilin, that cathepsin H requires sortilin, and that cathepsins K and L attain the lysosomes in a sortilin-independent fashion. As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It has been observed that like the MPR, the anterograde trafficking of sortilin and its cargo require both GGAs and AP-1. Similarly, the retrograde recycling pathway of sortilin also involves an interaction with retromer through a YXXphi site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of sortilin and MPR possess a high degree of functional homology and both receptors share a conserved trafficking mechanism.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Lisossomos/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Catepsina D/metabolismo , Catepsina H , Catepsina K , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Humanos , Hidrolases/metabolismo , Mucolipidoses/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Receptor IGF Tipo 2/fisiologia , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo
13.
Acta Neuropathol ; 117(2): 201-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18762956

RESUMO

The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessively inherited disorders collectively considered to be one among the most common pediatric neurodegenerative lysosomal storage diseases. Four main clinical subtypes have been described based on the age at presentation: infantile, late infantile, juvenile and adult types. In addition, rare congenital cases of NCL have been reported in the literature. Previously, a homozygous mutation in the cathepsin D gene has been shown to cause congenital NCL in a patient of Pakistani origin. We report a case of a 39-week estimated gestational age female infant with severe microcephaly and hypertonia, whereas MRI showed generalized hypoplasia of the cerebral and cerebellar hemispheres. The infant died on day two after birth. Postmortem examination revealed a small, firm brain with extensive neuronal loss and gliosis. Remaining neurons, astrocytes and macrophages contained PAS-positive storage material with granular ultrastructure and immunoreactivity against sphingolipid activator protein D. A diagnosis of congenital NCL was rendered with a novel mutation, c.299C > T (p.Ser100Phe) in exon 3 of the cathepsin D gene. In the patient fibroblasts, cathepsin D activity was marginal, but the protein appeared stable and normally processed. This was confirmed in overexpression studies. Importantly, by identification of the mutation in the family, we were able to confirm the first prenatal diagnosis excluding cathepsin D deficiency in the younger sibling of the patient.


Assuntos
Catepsina D/deficiência , Catepsina D/genética , Amostra da Vilosidade Coriônica , Lipofuscinoses Ceroides Neuronais/genética , Adulto , Astrócitos/química , Astrócitos/ultraestrutura , Encéfalo/patologia , Química Encefálica , Morte Celular , Feminino , Fibroblastos/enzimologia , Gliose/genética , Gliose/patologia , Humanos , Recém-Nascido , Macrófagos/química , Macrófagos/ultraestrutura , Microcefalia/genética , Hipertonia Muscular/genética , Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/ultraestrutura , Mutação Puntual , Gravidez , Proteínas Ativadoras de Esfingolipídeos/análise
14.
Immunol Lett ; 116(2): 149-55, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18207250

RESUMO

Cellular membranes contain microdomains known as 'lipid rafts' or detergent-insoluble microdomains (DRM), enriched in cholesterol and sphingolipids. DRM can play an important role in many cellular processes, including signal transduction, cytoskeletal organization, and pathogen entry. Many receptors like T cell receptors, B cell receptors and IgE receptors have been shown to reside in DRM. The majority of these receptors depend on multivalent ligand interaction to associate with these microdomains. We, here, study association between the high affinity IgG receptor, FcgammaRI (CD64), and membrane microdomains. FcgammaRI is a 72kDa type I glycoprotein that can mediate phagocytosis of opsonized pathogens, but can also effectively capture small immune complexes, and facilitates antigen presentation. We found FcgammaRI to predominantly reside within detergent-insoluble buoyant membranes, together with FcRgamma-chain, but independent of cross-linking ligand. With the use of confocal imaging, FcgammaRI was found to co-patch with GM1, a microdomain-enriched glycolipid. Depletion of cellular cholesterol, furthermore, modulated FcgammaRI-ligand interactions. These data indicated FcgammaRI to reside within lipid rafts without prior triggering of the receptor.


Assuntos
Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Receptores de IgG/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Colesterol/deficiência , Colesterol/metabolismo , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Camundongos , Receptores de IgG/química , Receptores de IgG/genética , Proteínas Ativadoras de Esfingolipídeos/imunologia
15.
Biol Chem ; 388(6): 617-26, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17552909

RESUMO

Niemann-Pick type C disease is an inherited fatal disorder characterized by the accumulation of unesterified cholesterol and other lipids in the endosomal/lysosomal compartment. Two independent genes responsible for this neurodegenerative disorder have been identified, but the precise functions of the encoded Niemann-Pick C1 (NPC1) and C2 (NPC2) proteins are not yet known. We developed a cell-free assay for measuring intermembrane lipid transport and examined the ability of bovine NPC2 (bNPC2) for intermembrane cholesterol transfer. NPC2 specifically extracts cholesterol from phospholipid bilayers and catalyzes intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity is dependent on temperature, pH, ionic strength, lipid composition of the model membranes, and the ratio of donor to acceptor vesicles. In model membranes, the presence of the lysosomal anionic phospholipids bis(monooleoylglycero)phosphate and phosphatidyl inositol significantly stimulated cholesterol transfer by NPC2, whereas bis(monomyristoylglycero)phosphate, phosphatidyl serine, and phosphatidic acid had no effect. Moreover, ceramide stimulated cholesterol transfer slightly, whereas sphingomyelin reduced cholesterol transfer rates. With our assay system we identified for the first time the ability of other lysosomal proteins, most notably the GM2-activator protein, to mediate intermembrane cholesterol transfer. This assay system promises to be a valuable tool for further quantitative and mechanistic studies of protein-mediated lipid transfer.


Assuntos
Bioensaio , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Glicoproteínas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Ceramidas/farmacologia , Humanos , Lipossomos/metabolismo , Lisofosfolipídeos/farmacologia , Monoglicerídeos/farmacologia , Palmitatos/farmacologia , Ácidos Fosfatídicos/farmacologia , Fosfatidilserinas/farmacologia , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Esfingomielinas/farmacologia , Vesículas Transportadoras/metabolismo
16.
Immunology ; 120(4): 536-43, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17250589

RESUMO

Monocytes constitute 5-10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14(+) GM1(low) population and a CD14(+) GM1(high) population comprising about 97.5% and 2.5% of total CD14(+) cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14(+) GM1(low) cells proved to be less endocytic and more responsive to LPS, whereas CD14(+) GM1(high) cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14(+) GM1(high) cells increases from about 2.5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1(low) and GM1(high) monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1(high) favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process.


Assuntos
Endocitose , Microdomínios da Membrana/metabolismo , Monócitos/metabolismo , Proteínas Ativadoras de Esfingolipídeos/sangue , Diferenciação Celular/imunologia , Células Cultivadas , Suscetibilidade a Doenças , Humanos , Receptores de Lipopolissacarídeos , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Monócitos/imunologia , Monócitos/fisiologia , Mycobacterium bovis , Proteínas Ativadoras de Esfingolipídeos/imunologia , Receptor 4 Toll-Like/sangue , Tuberculose/imunologia
17.
Neurobiol Aging ; 28(1): 8-17, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16332401

RESUMO

Synapse loss in Alzheimer's disease (AD) is poorly understood but evidence suggests it is a key pathological event. In order to precisely detect stable synaptic changes, we have developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved AD samples, and have previously shown that amyloid-beta (Abeta) accumulates in surviving presynaptic terminals in AD cortex. In the present experiments we have examined amyloid-containing terminals in more detail, first dual labeling synaptosomes from AD cortex for Abeta and a series of markers, and then using quadrant analysis to compare amyloid-positive and amyloid-negative terminals. Amyloid-positive synaptosomes were larger in size than amyloid-negatives (p<0.007), and significant increases were observed in mean fluorescence for the lipid raft markers cholesterol (27%; p<0.0005) and GM1 ganglioside (24%; p<0.005). SNAP-25 immunofluorescence was increased by 31% (p<0.0001) in amyloid-bearing terminals, consistent with a sprouting response to amyloid accumulation. These results suggest that Abeta accumulation in synaptic terminals may underly dysfunction prior to or independent of extracellular amyloid deposition.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Colesterol/metabolismo , Neocórtex/metabolismo , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptossomos/metabolismo , Adaptação Fisiológica , Idoso , Feminino , Humanos , Técnicas In Vitro , Masculino , Microdomínios da Membrana/metabolismo
18.
J Exp Med ; 203(8): 1951-61, 2006 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-16847070

RESUMO

Oxidative stress generated by ischemia/reperfusion is known to prime inflammatory cells for increased responsiveness to subsequent stimuli, such as lipopolysaccharide (LPS). The mechanism(s) underlying this effect remains poorly elucidated. These studies show that alveolar macrophages recovered from rodents subjected to hemorrhagic shock/resuscitation expressed increased surface levels of Toll-like receptor 4 (TLR4), an effect inhibited by adding the antioxidant N-acetylcysteine to the resuscitation fluid. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of RAW 264.7 macrophages similarly caused an increase in surface TLR4. The H2O2-induced increase in surface TLR4 was prevented by depleting intracellular calcium or disrupting the cytoskeleton, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy transfer between TLR4 and the raft marker GM1 as well as biochemical analysis of the raft components demonstrated that oxidative stress redistributes TLR4 to lipid rafts in the plasma membrane. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-beta-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system.


Assuntos
Macrófagos Alveolares/citologia , Microdomínios da Membrana/metabolismo , Estresse Oxidativo , Choque Hemorrágico/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Cultivadas , Colesterol/deficiência , Apresentação Cruzada/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Peróxido de Hidrogênio/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Fator 88 de Diferenciação Mieloide , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteínas Ativadoras de Esfingolipídeos/metabolismo
19.
Virol J ; 3: 21, 2006 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-16579862

RESUMO

BACKGROUND: We have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. But due to the size and omega-like shape of caveolae it is possible that A-MLV initially binds cells outside of caveolae. Rafts have been suggested to be pre-caveolae and we here investigate whether A-MLV initially binds to its receptor Pit2, a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains. RESULTS: Here, we show that a high amount of cell-bound A-MLV was attached to large rafts of NIH3T3 at the time of investigation. These large rafts were not enriched in caveolin-1, a major structural component of caveolae. In addition, they are rather of natural occurrence in NIH3T3 cells than a result of patching of smaller rafts by A-MLV. Thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped MLV particles showed the same pattern of large rafts as cells incubated with A-MLV, but VSV-G pseudotyped MLV particles did not show any preference to attach to these large microdomains. CONCLUSION: The high concentration of A-MLV particles bound to large rafts of NIH3T3 cells suggests a role of these microdomains in early A-MLV binding events.


Assuntos
Colesterol/metabolismo , Fibroblastos/virologia , Vírus da Leucemia Murina/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Animais , Cavéolas/metabolismo , Cavéolas/virologia , Membrana Celular/química , Membrana Celular/virologia , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo/métodos , Microdomínios da Membrana/química , Camundongos , Células NIH 3T3 , Proteínas Ativadoras de Esfingolipídeos/metabolismo
20.
Annu Rev Cell Dev Biol ; 21: 81-103, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16212488

RESUMO

Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family.


Assuntos
Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Ativadoras de Esfingolipídeos/metabolismo , Esfingolipídeos/metabolismo , Animais , Ânions/metabolismo , Humanos , Lisossomos/química , Lipídeos de Membrana/química , Modelos Biológicos , Proteínas Ativadoras de Esfingolipídeos/química , Esfingolipídeos/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA