Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry.
Proteomics
; 6(1): 26-34, 2006 Jan.
Article
em En
| MEDLINE
| ID: mdl-16294306
ABSTRACT
The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrP(C) interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.
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Base de dados:
MEDLINE
Assunto principal:
Príons
/
Eletroforese em Gel Bidimensional
/
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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Far-Western Blotting
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Proteínas de Membrana
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article