An N-terminal Polybasic Domain and Cell Surface Localization Are Required for Mutant Prion Protein Toxicity

Isaac H Solomon,Natasha Khatri,Emiliano Biasini,Tania Massignan,James E Huettner,David A Harris

doi:10.1074/jbc.m110.214973

Isaac H Solomon, Natasha Khatri + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.214973

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Abstract

There is evidence that alterations in the normal physiological activity of PrP(C) contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP(C) has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of Δ105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP(Sc) formation, suggesting that common structural features may govern both the functional activity of PrP(C) and its conversion to PrP(Sc).

Highlights

Progress in investigating this mechanism has been hampered by a lack of understanding of the physiological role of PrPC and the absence of assays to measure the functional activity of the protein
Several of the sequence domains identified in our study as being essential for PrP toxicity have been previously reported to be critical for PrPSc formation, suggesting that common structural features may govern both the functional activity of PrPC and its conversion to PrPSc
It is known that structural changes occur during the transition from PrPC to PrPSc, notably in the central region of the protein [40, 41], but how these changes are related to prion toxicity remains to be determined

Summary

Introduction

Progress in investigating this mechanism has been hampered by a lack of understanding of the physiological role of PrPC and the absence of assays to measure the functional activity of the protein. These two assays, which measure functional activities of PrPC, provide powerful tools for identifying structural domains and cellular compartments that are essential for PrP toxicity, information that could otherwise be obtained only by the creation and characterization of multiple transgenic mouse lines. In both the DBCA and patch clamp assay, the largest effects are observed for ⌬105–125 PrP (referred to as ⌬CR PrP), a deletion mutant that is missing 21 highly conserved residues in the central region of the protein. Requirements for Prion Protein Toxicity molecules capable of blocking mutant PrP toxicity in our cellular assays may be valuable as anti-prion therapeutics

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