RESUMO
This review assesses problems that confound attempts to isolate 'raft' domains from cell membranes, focusing in particular upon the isolation of detergent resistant membrane (DRM). Despite its widespread use, this technique is rightly viewed with skepticism by many membrane biochemists and biophysics for reasons that include the inability to isolate DRMs at 37°C, the temperature at which their lipids are supposed to be ordered and so exclude detergents. If solubilization is done in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids, DRMs can readily be isolated at 37°C, and these have many properties expected of lipid rafts. However, to date these DRMs have remained somewhat larger than current concepts of rafts. We describe an adaptation of this method that purifies nano-meso scale DRMs, and could be a significant step towards purifying the membrane of individual 'rafts'.
Assuntos
Detergentes/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Animais , Biofísica , Humanos , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/ultraestrutura , Nanocompostos/ultraestruturaRESUMO
For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics them to separate fates within sensory neurons. The binding of both to ligand cluster 4 should enable genetic modification of PrP binding without disrupting other roles of LRP1 essential to neuronal viability and function, thereby enabling in vivo analysis of the role of this interaction in controlling both prion and LRP1 biology.
Assuntos
Endocitose , Príons/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Endossomos/metabolismo , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Príons/ultraestrutura , Ligação Proteica , Estrutura Secundária de Proteína , Células Receptoras Sensoriais/ultraestruturaRESUMO
The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 degrees C undermines the claim that DRMs consist of lipid nanodomains that exist in an L(o) (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg(2+), K(+)) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 degrees C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 degrees C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 degrees C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.
Assuntos
Detergentes/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Temperatura , Animais , Soluções Tampão , Linhagem Celular , Glicoproteínas/metabolismo , Microdomínios da Membrana/ultraestrutura , Microscopia Eletrônica , Príons/metabolismo , Ratos , Ratos Wistar , Solubilidade , UltracentrifugaçãoRESUMO
The trafficking of normal cellular prion protein (PrPC) is believed to control its conversion to the altered conformation (designated PrPSc) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrPC on neurons is rapidly and constitutively endocytosed by means of coated pits, a property dependent upon basic amino acids at its N-terminus. Here, we show that low-density lipoprotein receptor-related protein 1 (LRP1), which binds to multiple ligands through basic motifs, associates with PrPC during its endocytosis and is functionally required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrPC in biosynthetic compartments, with a concomitant lowering of surface PrPC, suggesting that LRP1 expedites the trafficking of PrPC to the neuronal surface. PrPC and LRP1 can be co-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrPC binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 muM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface, and biosynthetic, trafficking of PrPC in neurons.
Assuntos
Endocitose , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Animais , Membrana Celular/química , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Camundongos , Chaperonas Moleculares/farmacologia , Neurônios Aferentes/química , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Proteínas PrPC/análise , Proteínas PrPC/biossíntese , Interferência de RNA , RatosRESUMO
The key mechanism in prion disease is the conversion of cellular prion protein into an altered, pathogenic conformation, in which cellular mechanisms play a poorly understood role. Both forms of prion protein are lipid-anchored and reside in rafts that appear to protect the native conformation against conversion. Neurons rapidly traffic their cellular prion protein out of its lipid rafts to be endocytosed via coated pits before recycling back to the cell surface. It is argued in this review that understanding the mechanism of this trafficking holds the key to understanding the cellular role in the conformational conversion of prion protein.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/transmissão , Príons/metabolismo , Animais , Endocitose , Humanos , Doenças Priônicas/patologia , Dobramento de Proteína , Transporte ProteicoRESUMO
Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (=3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained approximately 20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.
Assuntos
Encéfalo/ultraestrutura , Membrana Celular/química , Glicosilfosfatidilinositóis/química , Lipídeos de Membrana/análise , Príons/metabolismo , Antígenos Thy-1/metabolismo , Animais , Sítios de Ligação , Detergentes , Feminino , Glucosilceramidas/análise , Glicosilação , Glicosilfosfatidilinositóis/metabolismo , Lipídeos de Membrana/química , Neurônios/ultraestrutura , Príons/análise , Ratos , Ratos Sprague-Dawley , Solubilidade , Antígenos Thy-1/análiseRESUMO
The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid 'raft' domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23-107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.
Assuntos
Endocitose , Glicosilfosfatidilinositóis/metabolismo , Neurônios Aferentes/metabolismo , Proteínas PrPC/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Dissulfetos/química , Cinética , Microdomínios da Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Neurônios Aferentes/citologia , Neurônios Aferentes/ultraestrutura , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Receptores da Transferrina/metabolismo , Receptores da Transferrina/ultraestrutura , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Antígenos Thy-1/metabolismo , Antígenos Thy-1/ultraestruturaRESUMO
We have studied the neurotoxicity of amyloid-beta (Abeta) after a single unilateral intravitreal injection. Within the retina apoptotic cells were seen throughout the photoreceptor layer and the inner nuclear layer but not in the ganglion cell layer at 48 h after injection of Abeta(1-42) compared to vehicle control and control peptide. At 5 months, there was a significant reduction in total cell numbers in the ganglion cell layer in Nissl stained retinas. There was glial cell dysfunction with upregulation of glial fibrillary acidic protein and a reduction in the expression of Müller cell associated proteins in the injected retinas. These results suggest an indirect cytotoxic effect of Abeta on retinal neurons and an important role for dysfunction of Müller glia in mediating Abeta neurotoxicity.