RESUMO
Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrP(Sc) (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrP(Sc). Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.
Assuntos
Glucuronidase/genética , Glucuronidase/metabolismo , Doenças Priônicas/prevenção & controle , Animais , Linhagem Celular , Cricetinae , Progressão da Doença , Suscetibilidade a Doenças , Feminino , Heparitina Sulfato/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidade , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Scrapie/etiologia , Scrapie/metabolismo , Scrapie/prevenção & controle , Fatores de Tempo , Regulação para CimaRESUMO
Prion diseases propagate by converting a normal glycoprotein of the host, PrP(C), into a pathogenic "prion" conformation. Several misfolding mutants of PrP(C) are degraded through the ER-associated degradation (ERAD)-proteasome pathway. In their infectious form, prion diseases such as bovine spongiform encephalopathy involve PrP(C) of wild-type sequence. In contrast to mutant PrP, wild-type PrP(C) was hitherto thought to be stable in the ER and thus immune to ERAD. Using proteasome inhibitors, we now show that approximately 10% of nascent PrP(C) molecules are diverted into the ERAD pathway. Cells incubated with N-acetyl-leucinal-leucinal-norleucinal (ALLN), lactacystin or MG132 accumulated both detergent-soluble and insoluble PrP species. The insoluble fraction included an unglycosylated 26 kDa PrP species with a protease-resistant core, and a M(r) "ladder" that contained ubiquitylated PrP. Our results show for the first time that wild-type PrP(C) molecules are subjected to ERAD, in the course of which they are dislocated into the cytosol and ubiquitylated. The presence of wild-type PrP molecules in the cytosol may have potential pathogenic implications.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas PrPC/metabolismo , Ubiquitina/metabolismo , Animais , Brefeldina A/farmacologia , Células CHO , Cricetinae , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Leupeptinas/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Complexos Multienzimáticos/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional , Solubilidade , Células Tumorais CultivadasRESUMO
The presence of at least three distinct polygalacturonases (PGase) in callus of Orobanche was demonstrated. The PGase activity is labile and at pH 4.5 does not require activation by cations. It can be partially purified on Biogel P 100 columns and can be resolved by PAGE into several bands. Broomrape callus tissue also contains inhibitors of PGase activity. One of these is a low M(r) compound, stable to boiling and removable by dialysis. An additional inhibitor precipitable by ammonium sulphate is also present.
