Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP(Sc) of a normal cellular glycoprotein, PrPc, which is characterized by a high content of beta-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109-122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrPc molecule, should play a role in promoting the conversion into PrP(Sc). By using persistently scrapie-infected mouse neuroblastoma (Sc+-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrPc that are capable of blocking the conversion of endogenous, wild-type PrPc into PrP(Sc). We constructed a deletion mutant (PrPc delta114-121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109-122). Transient transfections of mammalian expression vectors encoding either wild-type PrPc or PrPc delta114-121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrPc versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc+-MNB cells revealed that PrPc delta114-121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP(Sc) derived from endogenous PrPc. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrPc plays an important role in the conversion process of PrPc into PrP(Sc) and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP(Sc) accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.
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PMID:Overexpression of nonconvertible PrPc delta114-121 in scrapie-infected mouse neuroblastoma cells leads to trans-dominant inhibition of wild-type PrP(Sc) accumulation. 944 12

A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of beta-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first beta-strand, the second beta-strand, or the first alpha-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc(+)-MNB cells) and a cell-free conversion assay. Removal of the second beta-strand or the first alpha-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first beta-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc(+)-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the beta-strands and the first alpha-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.
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PMID:Deletion of beta-strand and alpha-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform. 1158 71