RESUMO
BACKGROUND: Osteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving blindness and deafness, and bone marrow failure. In â¼70% of the patients there is a molecularly defined failure to maintain an acid pH at the osteoclast-bone interface (the ruffled border) which is necessary for the bone resorptive activity. METHODS AND RESULTS: In eight patients with infantile osteopetrosis which could be cured by bone marrow transplantation, the study identified by homozygosity mapping in distantly related consanguineous pedigrees a missense mutation in a highly conserved residue in the SNX10 gene. The mutation segregated with the disease in the families and was carried by one of 211 anonymous individuals of the same ethnicity. In the patients' osteoclasts, the mutant SNX10 protein was abnormally abundant and its distribution altered. The patients' osteoclasts were fewer and smaller than control cells, their resorptive capacity was markedly deranged, and the endosomal pathway was perturbed as evidenced by the distribution of internalised dextran. CONCLUSIONS: SNX10 was recently shown to interact with vacuolar type H(+)-ATPase (V-ATPase) which pumps protons at the osteoclast-bone interface. Mutations in TCIRG1, the gene encoding a subunit of the V-ATPase complex, account for the majority of cases of osteopetrosis. It is speculated that SNX10 is responsible for the vesicular sorting of V-ATPase from Golgi or for its targeting to the ruffled border. A mutation in SNX10 may therefore result in 'secondary V-ATPase deficiency' with a failure to acidify the resorption lacuna. Determination of the sequence of the SNX10 gene is warranted in molecularly undefined patients with recessive 'pure' osteopetrosis of infancy.
Assuntos
Mutação , Osteopetrose/genética , Nexinas de Classificação/genética , Sequência de Bases , Consanguinidade , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteopetrose/patologia , Linhagem , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(ß-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.
Assuntos
Citomegalovirus/enzimologia , Citomegalovirus/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Montagem de Vírus , Células Cultivadas , Citomegalovirus/ultraestrutura , Fibroblastos/virologia , Deleção de Genes , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Prions are infectious agents resulting from the conversion of a normal cellular protein, PrP(C), to a misfolded species, PrP(Sc). Iatrogenic transmission of prions is known from surgical procedures involving stainless steel materials. Here, it was shown that stainless steel containing nickel and molybdenum binds PrP(Sc) more efficiently and transmits infection to cells in culture to a higher degree than if these elements are not present. Furthermore, both nickel and molybdenum alone adsorbed PrP(Sc), and nickel powder could be used to extract PrP(Sc) from dilute solutions, thus providing a simple approach to concentration of PrP(Sc). The fact that nickel and molybdenum in steel alloys increased the binding affinity, and bound infectivity, of PrP(Sc) is an important issue to consider in the manufacture of surgical instruments and abattoir tools.
Assuntos
Cirurgia Geral/instrumentação , Molibdênio/metabolismo , Níquel/metabolismo , Doenças Priônicas/transmissão , Príons/metabolismo , Aço Inoxidável , Adsorção , Animais , Humanos , Ligação ProteicaRESUMO
Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority ( www.prionpriority.eu ) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper ( www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies.
Assuntos
Cadeia Alimentar , Doenças Priônicas/epidemiologia , Doenças Priônicas/prevenção & controle , Príons/análise , Ração Animal/efeitos adversos , Animais , Bovinos , Diagnóstico Precoce , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/epidemiologia , Encefalopatia Espongiforme Bovina/prevenção & controle , Encefalopatia Espongiforme Bovina/transmissão , Europa (Continente)/epidemiologia , Humanos , Doenças Priônicas/diagnóstico , Doenças Priônicas/transmissão , Príons/isolamento & purificação , Príons/metabolismo , Príons/patogenicidade , Scrapie/diagnóstico , Scrapie/epidemiologia , Scrapie/prevenção & controle , Scrapie/transmissãoRESUMO
Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination and tested for antiprion activity. Polycations of dextran, pullulan and arabinogalactan grafted with oligoamines of 2 to 4 amino groups were investigated for their ability to eliminate PrP(Sc), the protease-resistant isoform of the prion protein, from chronically infected neuroblastoma cells, ScN2a-M. The proteinase K (PK)-resistant PrP elimination depends on both the concentration of the reagent and the duration of exposure. The most potent compound was found to be dextran-spermine that caused depletion of PrP(Sc) to undetectable levels at concentration of 31 ng/mL after 4 days of exposure. Activity analysis revealed that grafted oligoamine indentity of the polycation plays a significant role in elimination of PK-resistant PrP from chronically infected N2a-M cells, regardless of the polysaccharide used. Dextran-spermine conjugates were modified with oleic acid and with methoxypoly(ethylene glycol) (MPEG) at various degrees of substitution for further studies and their antiprion activity was examined. Substitution of dextran-spermine with MPEG or oleic acid slightly decreases its activity as a function of MPEG/oleic acid content. These findings confirm previous reports that polycations are effective in eliminating PrP(Sc) in vitro.
Assuntos
Poliaminas/síntese química , Polissacarídeos/síntese química , Proteínas PrPSc/antagonistas & inibidores , Animais , Cátions , Linhagem Celular Tumoral , Dextranos/química , Endopeptidase K/metabolismo , Galactanos/química , Glucanos/química , Camundongos , Poliéster Sulfúrico de Pentosana/química , Poliéster Sulfúrico de Pentosana/farmacologia , Poliaminas/química , Poliaminas/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into ß-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.
Assuntos
Amiloide/metabolismo , Imageamento Tridimensional , Microdomínios da Membrana/metabolismo , Proteínas PrPSc/metabolismo , Actinas/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Animais , Anticorpos/metabolismo , Benzotiazóis , Sobrevivência Celular , Endocitose , Hipocampo/metabolismo , Camundongos , Modelos Biológicos , Fosfoinositídeo Fosfolipase C/metabolismo , Polissacarídeos/metabolismo , Proteínas PrPSc/química , Ligação Proteica , Desnaturação Proteica , Coloração e Rotulagem , Tiazóis/metabolismoRESUMO
Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for â¼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of parkinsonism.
Assuntos
Auxilinas/genética , Proteínas de Choque Térmico HSP40/genética , Mutação , Transtornos Parkinsonianos/genética , Adolescente , Auxilinas/metabolismo , Sequência de Bases , Clatrina/metabolismo , Análise Mutacional de DNA/métodos , Saúde da Família , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , LinhagemAssuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Metilfenidato/farmacologia , Doenças Priônicas/tratamento farmacológico , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Masculino , Metilfenidato/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (DeltaUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the DeltaUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or DeltaUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.
Assuntos
Citomegalovirus/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Montagem de Vírus , Membrana Celular/química , Núcleo Celular/química , Núcleo Celular/virologia , Células Cultivadas , Citomegalovirus/genética , Citoplasma/química , Citoplasma/virologia , Fibroblastos/virologia , Deleção de Genes , Complexo de Golgi/virologia , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Frações Subcelulares/química , Vacúolos/química , Vacúolos/virologia , Montagem de Vírus/genéticaRESUMO
Prions replicate in the host cell by the self-propagating refolding of the normal cell surface protein, PrP(C), into a beta-sheet-rich conformer, PrP(Sc). Exposure of cells to prion-infected material and subsequent endocytosis can sometimes result in the establishment of an infected culture. However, the relevant cell surface receptors have remained unknown. We have previously shown that cellular heparan sulfates (HS) are involved in the ongoing formation of scrapie prion protein (PrP(Sc)) in chronically infected cells. Here we studied the initial steps in the internalization of prions and in the infection of cells. Purified prion "rods" are arguably the purest prion preparation available. The only proteinaceous component of rods is PrP(Sc). Mouse neuroblastoma N2a, hypothalamus GT1-1, and Chinese hamster ovary cells efficiently bound both hamster and mouse prion rods (at 4 degrees C) and internalized them (at 37 degrees C). Treating cells with bacterial heparinase III or chlorate (a general inhibitor of sulfation) strongly reduced both binding and uptake of rods, whereas chondroitinase ABC was inactive. These results suggested that the cell surface receptor of prion rods involves sulfated HS chains. Sulfated glycans inhibited both binding and uptake of rods, probably by competing with the binding of rods to cellular HS. Treatments that prevented endocytosis of rods also prevented the de novo infection of GT1-1 cells when applied during their initial exposure to prions. These results indicate that HS are an essential part of the cellular receptor used both for prion uptake and for cell infection. Cellular HS thus play a dual role in prion propagation, both as a cofactor for PrP(Sc) synthesis and as a receptor for productive prion uptake.
Assuntos
Heparitina Sulfato/química , Heparitina Sulfato/fisiologia , Príons/química , Animais , Ânions , Encéfalo/metabolismo , Células CHO , Linhagem Celular Tumoral , Cloratos/química , Cricetinae , Relação Dose-Resposta a Droga , Endocitose , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Biológicos , Polissacarídeo-Liases/química , Polissacarídeos/química , Príons/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , TemperaturaRESUMO
Prion diseases are infectious, sporadic and inherited fatal neurodegenerations that are propagated by an abnormal refolding of the cellular prion protein PrP(C). Which chaperones assist the normal folding of PrP(C) is unknown. The linkage of familial Gerstmann- Sträussler-Scheinker (GSS) syndrome with proline substitutions in PrP raised the prospect that peptidylprolyl cis-trans isomerases (PPIases) may play a role in normal PrP metabolism. Here we used cyclo sporin A (CsA), an immunosuppressant, to inhibit the cyclophilin family of PPIases in cultured cells. CsA-treated cells accumulated proteasome-resistant, 'prion-like' PrP species, which deposited in long-lived aggresomes. PrP aggresomes also formed with disease-linked proline mutants when proteasomes were inhibited. These results suggest mechanisms whereby abnormally folded cytosolic PrP may in some cases participate in the development of spontaneous and inherited prion diseases.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/metabolismo , Corpos de Inclusão/metabolismo , Proteínas PrPC/metabolismo , Animais , Células CHO/metabolismo , Células CHO/ultraestrutura , Cricetinae , Ciclosporina/metabolismo , Cisteína Endopeptidases/metabolismo , Corpos de Inclusão/química , Modelos Biológicos , Complexos Multienzimáticos/metabolismo , Mutação , Proteínas PrPC/genética , Proteínas PrPSc/metabolismo , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Tubulina (Proteína)/metabolismo , Ubiquitina/metabolismo , Vimentina/metabolismoRESUMO
During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Endocitose/efeitos dos fármacos , Heparitina Sulfato/química , Heparitina Sulfato/farmacologia , Neuroblastoma/metabolismo , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/química , Animais , Materiais Biomiméticos/síntese química , Biomimética/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Camundongos , Neuroblastoma/patologia , Biblioteca de Peptídeos , Proteínas PrPSc/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.
Assuntos
Amiloide/química , Ácidos Cólicos/química , Detergentes/farmacologia , Microdomínios da Membrana/metabolismo , Octoxinol/farmacologia , Animais , Western Blotting , Calnexina/química , Caveolina 1 , Caveolinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Centrifugação , Colesterol/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosilação , Iohexol/farmacologia , Metabolismo dos Lipídeos , Lipídeos/química , Microdomínios da Membrana/química , Camundongos , Príons/química , Células Tumorais CultivadasRESUMO
We recently reported that immunization of mice with certain self-prion protein peptides induced specific T-cell and B-cell immune responses; importantly, this immunization was associated with a decrease in the number of protease-resistant PrP(Sc) particles recoverable in a transplanted, scrapie-infected syngeneic tumor. The present study was carried out to determine whether immunization with the immunogenic PrP peptides might influence the natural history of experimental scrapie in mice. We immunized C57BL/6 mice with self-prion peptides in complete Freund's adjuvant (CFA) or with CFA alone as a control and then infected the mice with mouse-adapted scrapie by injection either intraperitoneally or intracerebrally. We report here that immunization with CFA, irrespective of whether prion peptides were present in the inoculum, resulted in marked prolongation of survival of the mice, whether the challenge was intracerebral or intraperitoneal. Mice in the immunized and control groups that died contained equivalent amounts of PrP(Sc). Thus, CFA immunization has a therapeutic effect in experimental scrapie in mice, possibly by reducing the rate of PrP(Sc) accumulation in the brain.
Assuntos
Adjuvante de Freund/uso terapêutico , Doenças Priônicas/terapia , Sequência de Aminoácidos , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/imunologia , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Proteínas PrPSc/metabolismo , Doenças Priônicas/imunologia , Doenças Priônicas/mortalidade , Príons/efeitos dos fármacos , Príons/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Scrapie/metabolismo , Taxa de Sobrevida , Fatores de TempoRESUMO
Dendritic cells (DC) of the CD11c(+) myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c(+) DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP(Sc)) in vitro, indicating a possible role of these cells in the clearance of PrP(Sc). To determine the mechanisms of PrP(Sc) degradation, CD11c(+) DC that had been exposed to PrP(Sc) derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP(Sc) was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP(Sc) in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP(C)) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP(Sc) is proteolytically cleaved preferentially by cysteine proteases in both CD11c(+) DC and ScGT1-1 cells and that the degradation of PrP(Sc) by proteases is different from that of PrP(C). Interference by protease inhibitors with DC-induced processing of PrP(Sc) has the potential to modify prion spread, clearance, and immunization in a host.
Assuntos
Antígeno CD11c/metabolismo , Cisteína Endopeptidases/metabolismo , Células Dendríticas/metabolismo , Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Gonadotropinas/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Scrapie/metabolismoRESUMO
During prion diseases, the host protein PrPC is refolded into an abnormal conformer "prion" PrPSc. Histological and pharmacological data have suggested that glycosaminoglycans may be involved in the development of prion diseases. Here we present the first direct evidence that cellular glycosaminoglycans play a role in the biogenesis of PrPSc in prion-infected ScN2a cells. When ScN2a cells were incubated with estradiol beta-d-xyloside to inhibit the glycosylation of proteoglycans, PrPSc was vastly reduced. Treating ScN2a-M cells with heparinase III, but not with heparinase I or chondroitinase ABC, caused a profound reduction of PrPSc. In contrast, neither the amount of PrPC nor its subcellular distribution were affected as assayed by immunofluorescence microscopy and flotation procedures. In vitro treatment of ScN2a membranes with heparinase III at either neutral or acidic pH did not reduce the level of protease-resistant PrPSc. The inhibitor of sulfation, sodium chlorate, vastly reduces PrPSc in ScN2a cells (Gabizon, R., Meiner, Z., Halimi, M., and Ben-Sasson, S. A. (1993) J. Cell. Physiol. 157, 319-325). Both soluble heparan sulfate and chondroitin sulfate partially restored the level of PrPSc in chlorate-treated cells. We conclude that heparinase III-sensitive, presumably undersulfated, cellular heparan sulfate plays a significant role in the biogenesis of PrPSc in ScN2a cells.
Assuntos
Heparitina Sulfato/metabolismo , Proteínas PrPSc/metabolismo , Animais , Glicosaminoglicanos/metabolismo , Glicosídeos/farmacologia , Glicosilação , Heparina Liase/farmacologia , Heparitina Sulfato/química , Técnicas In Vitro , Camundongos , Modelos Biológicos , Polissacarídeo-Liases/farmacologia , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Doenças Priônicas/metabolismoRESUMO
The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c(+) myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrP(Sc). In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrP(Sc), which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c(+) DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrP(Sc). Thus, the migrating and highly proteolytic CD11c(+) myeloid DC may affect the balance between propagation and clearance of PrP(Sc) in the organism.
Assuntos
Células Dendríticas/metabolismo , Proteínas PrPSc/metabolismo , Animais , Antígeno CD11c/metabolismo , Diferenciação Celular , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Scrapie/imunologia , Scrapie/metabolismoRESUMO
The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.