The AGAAAAGA Palindrome in PrP Is Required to Generate a Productive PrPSc-PrPC Complex That Leads to Prion Propagation

Eric M Norstrom,James A Mastrianni

doi:10.1074/jbc.m413441200

Eric M Norstrom, James A Mastrianni

Open Access

https://doi.org/10.1074/jbc.m413441200

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Abstract

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.

Highlights

The prion diseases, the best known of which include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cows, result from the generation and propagation of a conformational variant (PrPSc) of the normal prion protein (PrPC), a predominantly brain-derived glycoprotein of unknown function [1]
ScN2a cells were transiently transfected with wt PrP or that lacking the hydrophobic core sequence (PrP⌬112–119) and, after 48 h of expression, the cells were assayed for newly synthesized proteinase K (PK)-resistant PrPSc by Western blot
Defining the regional domains or residues within PrP that are essential for the de novo generation of PrPSc and/or the binding of PrPSc to PrPC is crucial, to better understand the process of prion biogenesis and to clarify other features of prion biology, such as the species barrier and host susceptibility, in addition to assisting in the development of therapeutic strategies

Summary

Introduction

The prion diseases, the best known of which include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cows, result from the generation and propagation of a conformational variant (PrPSc) of the normal prion protein (PrPC), a predominantly brain-derived glycoprotein of unknown function [1]. While the above studies support the palindrome as critical to prion generation, direct mechanistic evidence for this is currently lacking To provide such evidence and further define the role of the AGAAAAGA palindrome in prion processing, we applied a well documented neuroblastoma cell line chronically infected with prions (ScN2a) to model PrPSc replication and a heterologous yeast expression system [15] to model de novo PrPSc generation. The AGAAAAGA Palindrome in Prion Generation for PrP aggregation; it is critical, for the de novo generation of the specific PrPSc conformation and for the proper association of PrPSc and PrPC molecules necessary for the conformational transfer These results confirm the palindrome segment as an essential component to prion genesis and highlight the utility of this approach in resolving the specifics of prion conversion and propagation

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