Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrP Sc , which is enriched in the -sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrP Sc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.Prion diseases, also called transmissible spongiform encephalopathies, are neurodegenerative diseases affecting a variety of mammalian species from mink to cow, with human being no exception. In these diseases, cellular prion protein (PrP C ) 5 converts into the aggregated form PrP Sc , which is the main component of the infectious agents, the prions (1). PrP C is a glycosylphosphatidylinositol-anchored protein found on the membranes of neurons and many other cells (2). The N-terminal half of the protein, which is devoid of a defined tertiary structure (3), is followed by a C-terminal globular domain composed of three ␣-helices (H1, H2, H3) and a short antiparallel -sheet (composed of two strands, B1 and B2) (Fig. 1A) (4). High resolution structures of the C-terminal domain of PrP from different species revealed the conservation of the protein fold (5). In contrast to the availability of structural information on PrP C , the characteristics of PrP Sc make it inaccessible to high-resolution structural techniques (x-ray crystallography and high resolution NMR). PrP Sc is characterized by the increased content of the -secondary structure in comparison to PrP C (6 -8). Epitope accessibility (9 -12), deuterium exchange (13-15), limited proteolysis and mas...
J. Neurochem. (2008) 104, 1553–1564.
Abstract
Conversion of the native, predominantly α‐helical conformation of prion protein (PrP) into the β‐stranded conformation is characteristic for the transmissible spongiform encephalopathies such as Creutzfeld–Jakob disease. Curcumin, an extended planar molecule and a dietary polyphenol, inhibits in vitro conversion of PrP and formation of protease resistant PrP in neuroblastoma cell lines. Curcumin recognizes the converted β‐form of the PrP both as oligomers and fibrils but not the native form. Curcumin binds to the prion fibrils in the left‐handed chiral arrangement as determined by circular dichroism. We show that curcumin labels the plaques of the brain sections of variant Creutzfeld–Jakob disease cases and stains the same structures as antibodies against the PrP. In contrast to thioflavin T, curcumin also binds to the α‐helical intermediate of PrP present at acidic pH at stoichiometry of 1 : 1. Congo red competes with curcumin for binding to the α‐intermediate as well as to the β‐form of PrP but is toxic and binds also to the native form of PrP. We therefore show that the partially unfolded structural intermediate of the PrP can be targeted by non‐toxic compound of natural origin.
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