RESUMEN
Transmissible spongiform encephalopathies (TSEs) are a group of zoonotic and fatal neurodegenerative disorders that affect humans and animals. The pathogenesis of TSEs involves a conformational conversion of the cellular prion protein (PrP) into abnormal isoforms. Currently, cellular and pathological forms of PrP are differentiated using specific antibody-based analyses that are resource intensive and not applicable to all species and strains. Thus, there is an urgent need for sensitive and efficient assays that can detect pathological forms of PrP. Using systematic evolution of ligands by exponential enrichment, we developed DNA aptamers that can differentiate normal and abnormal PrP isoforms. These aptamers represent the first reagents that can identify pathological isoforms of PrP across multiple host species. Second, they are able to distinguish different strains of prions. Third, they can be used to detect prions in peripheral blood cells, which are otherwise undetectable using conventional antibody-based detection methods. Thus, DNA aptamers offer promise for the development of presymptomatic screens of tissue, blood and other body fluids for prion contamination.
Asunto(s)
Aptámeros de Nucleótidos , Proteínas PrPSc/genética , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Ligandos , Proteínas PrPSc/metabolismoRESUMEN
The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.
Asunto(s)
Enfermedades por Prión/orina , Priones/orina , Procesamiento Proteico-Postraduccional , Humanos , Priones/patogenicidadRESUMEN
Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of a misfolded form (PrP(Sc)) of the cellular prion protein (PrP(C)) in the brains of affected individuals. The conversion of PrP(C) to PrP(Sc) is thought to involve a change in protein conformation from a normal, primarily alpha-helical structure into a beta-sheet conformer. Few proteins have been identified that differentially interact with the two forms of PrP. It has been reported that plasminogen binds to PrP(Sc) from a variety of prion phenotypes. We have examined potential motifs within the kringle region that may be responsible for binding to PrP. We synthesized 12-15-mer peptides that contain small, repetitive stretches of amino acid residues found within the kringle domains of plasminogen. These synthetic peptides were found to capture PrP(Sc) from the brain homogenates of bovine spongiform encephalopathy affected cattle, chronic wasting disease affected elk, experimental scrapie of hamsters and that of subjects affected by Creutzfeldt-Jakob disease, without binding to PrP(C) in unaffected controls. Therefore, we have identified critical peptide motifs that may be important for protein-protein interactions in prion disease pathogenesis. The ability of these synthetic peptides to bind preferentially to PrP(Sc) suggests a potential application in the diagnosis of prion diseases.