Characterization of the PrPSc-PrPSc binding interface using PrP peptide-grafted aptamers.

摘要

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are always fatal neurodegenerative disorders. According to the widely accepted "Protein only" hypothesis, prion diseases are caused by a misfolded isoform (PrPSc) of the host-encoded cellular prion protein (PrPc), and are characterized by spongiform degeneration in pathology. The central events are the direct physical contact and specific binding interaction between PrPc and PrPSc. As a result, PrPc is converted into a protease-resistant state, then febrile or amyloid forms. The detailed molecular mechanisms of conversion, such as binding sites of PrPc and PrPSc or dynamics of the conformational change, are not well known yet. The characterizations of transmission barriers and prion strains indicate that a few amino acid substitutions in specific regions may affect the conversion leading to the transmission barriers. Knowledge of the domains that are responsible for the interaction with PrPSc would help in understanding of the different conformational states among strains and would be valuable targets for diagnosis and therapy.;In this study, we developed a new set of PrP peptide-grafted aptamers binding to PrPSc which were used to screen the specific binding sites responsible for the conversion. To achieve this, PrP primary structure was divided into 14 overlapping peptides (20mers) and inserted into thioredoxin A (TrxA) which was used as the constant backbone. These PrP peptides cover PrP 23-231 which is the mature form without a signal peptide and the GPI anchor. Using these PrP peptide-grafted aptamers, we have a systematic tool to map and characterize the PrPc and PrPSc binding interface to find out the important binding sites in PrPc.;The results demonstrate that PrP peptide-grafted aptamers have differential effects on PrPSc propagation in mouse neuroblastoma cells persistently infected with prion strain 22L- and RML-stain. These aptamers intervene with PrPSc propagation by binding to PrPSc , but not PrPc, in a dose-dependent manner. Potential specific binding sites and interaction domains which could differentially interfere with prion conversion between 22L and RML strains were identified. These specific domains may be targets for development of new diagnostic tools for prion strains and new therapeutic strategies.