RESUMO
Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.
Assuntos
Proteínas PrPSc/genética , Proteínas Priônicas/genética , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/transmissão , Animais , Animais Geneticamente Modificados , Cervos , Camundongos , América do Norte , NoruegaRESUMO
Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease.
Assuntos
Cervos , Doenças das Cabras , Doenças Priônicas , Príons , Rena , Scrapie , Doenças dos Ovinos , Doença de Emaciação Crônica , Animais , Ovinos , Príons/genética , Rena/metabolismo , Doenças Priônicas/veterinária , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Doença de Emaciação Crônica/genética , Noruega/epidemiologia , Cabras/metabolismoRESUMO
BACKGROUND: Endothelial cells have important functions in e.g. regulating blood pressure, coagulation and host defense reactions. Serglycin is highly expressed by endothelial cells, but there is limited data on the roles of this proteoglycan in immune reactions. METHODS: Cultured primary human endothelial cells were exposed to proinflammatory agents lipopolysaccharide (LPS) and interleukin 1ß (IL-1ß). The response in serglycin synthesis, secretion and intracellular localization and effect on the proteoglycan binding chemokines CXCL-1 and CXCL-8 were determined by qRT-PCR, Western blotting, immunocytochemistry, ELISA and serglycin knockdown experiments. RESULTS: Both LPS and IL-1ß increased the synthesis and secretion of serglycin, while only IL-1ß increased serglycin mRNA expression. Stimulation increased the number of serglycin containing vesicles, with a greater portion of large vesicles after LPS treatment. Also, increased intracellular and secreted levels of CXCL-1 and CXCL-8 were observed. The increase in CXCL-8 secretion was unchanged in serglycin knockdown cells. However, the increase in CXCL-1 secretion from IL-1ß stimulation was reduced 27% in serglycin knockdown cells; while the LPS-induced secretion was not affected. In serglycin expressing cells CXCL-1 positive vesicles were evenly distributed throughout the cytoplasm, while confided to the Golgi region in serglycin knockdown cells. This was the case only for IL-1ß stimulated cells. LPS-induced CXCL-1 distribution was unaffected by serglycin expression. CONCLUSIONS: These results suggest that different signaling pathways are involved in regulating secretion of serglycin and partner molecules in activated endothelial cells. GENERAL SIGNIFICANCE: This knowledge increases our understanding of the roles of serglycin in immune reactions. This article is part of a Special Issue entitled: Matrix-mediated cell behaviour and properties.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Inflamação/metabolismo , Inflamação/patologia , Proteoglicanas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células Cultivadas , Quimiocina CXCL1/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Interleucina-8/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Transporte Proteico/efeitos dos fármacosRESUMO
The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub(4)) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub(4) chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub(4) was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub(4) was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub(4) was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis.
Assuntos
Endocitose/fisiologia , Lisossomos/metabolismo , Proteólise , Receptor ErbB-2/metabolismo , Ubiquitinação/fisiologia , Animais , Benzoquinonas/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Endocitose/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Lisossomos/efeitos dos fármacos , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteólise/efeitos dos fármacos , Receptor ErbB-2/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Suínos , Ubiquitina/metabolismoRESUMO
Epsin consists of an epsin NH(2)-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Motivos de Aminoácidos , Linhagem Celular , Invaginações Revestidas da Membrana Celular/ultraestrutura , Endocitose , Humanos , Microscopia Eletrônica , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Receptores da Transferrina/metabolismoRESUMO
The endothelial glycocalyx (EG) is essential for proper function of the endothelium and for vascular integrity, but its role in premature atherogenesis in rheumatoid arthritis (RA) has not been studied yet. EG impairment can play a role in pathogenesis of vascular disease, and one of its characteristics is shedding of syndecan-1 from endothelial cells. Syndecan-1 shedding is mediated by matrix metalloproteinase-9 (MMP-9) and counteracted by tissue inhibitor of metalloproteinases (TIMP)-1. Cardiovascular disease risk in RA is reversible by disease modifying antirheumatic drugs (DMARDs), but the exact modes of action are still unclear. Therefore, we examined effects of DMARDs on syndecan-1, MMP-9 and TIMP-1 in RA patients, and searched for associations between these parameters and inflammatory activity. From the observational PSARA study, we examined 39 patients starting with methotrexate (MTX) monotherapy (in MTX naïve patients, n = 19) or tumor necrosis factor inhibitors (TNFi) in combination with MTX (in MTX non-responders, n = 20) due to active RA. Serum syndecan-1, MMP-9 and TIMP-1 were measured at baseline and after six weeks of treatment. Serum syndecan-1 (p = 0.008) and TIMP-1 (p<0.001) levels decreased after six weeks of anti-rheumatic treatment. Levels of MMP-9 also decreased, but the difference was not statistically significant. The improvement in syndecan-1 levels were independent of changes in inflammatory activity. There was no significant difference in changes in syndecan-1 levels from baseline to 6 weeks between the MTX and TNFi groups, however the change was significant within the MTX group. Six weeks of antirheumatic treatment was associated with reduction in serum levels of syndecan-1, which might reflect reduced syndecan-1 shedding from EG. Thus, it is possible that EG-preserving properties of DMARDs might contribute to their cardioprotective effects. These effects may be at least partly independent of their anti-inflammatory actions. Our findings do not support the notion that syndecan-1 shedding in RA is mediated mainly by increased MMP-9 or decreased TIMP-9 serum concentration.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Sindecana-1/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Estudos Prospectivos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.
Assuntos
Encéfalo/metabolismo , Cervos , Proteínas Priônicas/análise , Príons , Rena , Doença de Emaciação Crônica/diagnóstico , Animais , Arvicolinae , Bioensaio , Fezes , Feminino , Fluorescência , Tecido Linfoide , Masculino , Mesocricetus , Noruega , Risco , Saliva , Doença de Emaciação Crônica/sangue , Doença de Emaciação Crônica/urinaRESUMO
Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1ß (IL-1ß). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Sindecana-4/metabolismo , Cicatrização , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , RNA Interferente Pequeno/genética , Sindecana-2/metabolismo , Sindecana-3/metabolismo , Sindecana-4/genéticaRESUMO
Monocytes play multiple roles in the immune system, and are active in both acute and chronic diseases. Patients exposed to bacterial infections depend on monocytes in defense reactions, but excessive immune reactions may also cause morbidity through systemic inflammatory responses. Few studies have addressed the importance of proteoglycans, and in particular, the hematopoietic serglycin, in such monocyte immune reactions. Adherent primary monocytes were cultured in absence and presence of LPS. Media were analyzed by ELISA for detection of serglycin. Lysed cell fractions were used to determine the mRNA level of serglycin. Monocytes were also cultured on chamber slides to investigate if serglycin could be detected intracellularly by immunocytochemistry. Monocytes secreted serglycin, and LPS-stimulation increased the secretion. Secretion of inflammatory cytokines increased to a larger extent than serglycin. mRNA levels of serglycin were also increased, suggesting both increased expression and secretion. Immunocytochemistry revealed the presence of serglycin in intracellular vesicles, many destined for secretion. Serglycin containing vesicles increased in number and size when the cells were exposed to LPS. Intracellular vesicle localization and secretion of the proteoglycan serglycin is shown for the first time in primary human monocytes. Monocyte activation by LPS increased the expression and secretion of serglycin, suggesting roles for serglycin in inflammatory processes.
RESUMO
Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.
Assuntos
Sulfatos de Condroitina/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Proteoglicanas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Cloratos/farmacologia , Sulfatos de Condroitina/biossíntese , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Rim/citologia , Modificação Traducional de Proteínas/efeitos dos fármacos , Modificação Traducional de Proteínas/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteoglicanas/biossíntese , Proteoglicanas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genéticaRESUMO
Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin-Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.
Assuntos
Brefeldina A/farmacologia , Glicosaminoglicanos/biossíntese , Heparitina Sulfato/biossíntese , Animais , Linhagem Celular , Dissacarídeos/química , Cães , Glucosamina/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/isolamento & purificação , Rim , Sulfatos/metabolismo , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.