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Recent studies indicate that the pathogenesis of Alzheimer disease may be related to the interaction between prion protein (PrP) and certain oligomeric species of Aβ peptide. However, the mechanism of this interaction remains unclear and controversial. Here we provide direct experimental evidence that, in addition to previously demonstrated binding to Aβ oligomers, PrP also interacts with mature Aβ fibrils. However, contrary to the recent claim that PrP causes fragmentation of Aβ fibrils into oligomeric species, no evidence for such a disassembly could be detected in the present study. In contrast, our data indicate that the addition of PrP to preformed Aβ fibrils results in a lateral association of individual fibrils into larger bundles. These findings have potentially important implications for understanding the mechanism by which PrP might impact Aβ toxicity as well as for the emerging efforts to use PrP-derived compounds as inhibitors of Aβ-induced neurodegeneration.

Nucleic acid induces conformational changes in the prion protein (23-231 amino acids) to a structure resembling its pathological isoform. The prion protein, in turn, facilitates DNA strand transfer and acts as a DNA chaperone which is modulated by the N-terminal unstructured basic segment of the protein. Here we have studied the prion protein induced conformational changes in DNA using oligonucleotides covalently labeled with the energy donor fluorescein and the acceptor rhodamine moieties by fluorescence resonance energy transfer (FRET) and by thermal stability of the unlabeled oligonucleotides. The protein induces a strong FRET effect in the oligonucleotides evidenced from the simultaneous quenching of fluorescence intensity of the donor and increase in the fluorescence intensity of the acceptor, which indicate bending of the oligonucleotides by the prion protein. The energy transfer efficiency induced by the protein is greater for the larger oligonucleotide. The prion protein also induces significant structural destabilization of the oligonucleotides observed from the lowering of their melting temperatures in the presence of the protein. The truncated globular prion protein 121-231 fragment neither induces FRET effect on the oligonucleotides nor destabilizes their structures, indicating that the N-terminal segment of the prion protein is essential for the DNA bending process. Equilibrium binding and kinetics of FRET show that the protein binding to the oligonucleotides and their bending occur simultaneously. The DNA structural changes observed in the presence of the prion protein are similar to those caused by proteins involved in initiation and regulation for protein synthesis.

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with an N-myristoylamidomaleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core but controls the mode of lateral assembly of filaments into higher order fibrils.

In this paper, the density functional theory (DFT) method B3LYP/LANL2DZ was used to calculate binding energies and electron affinities for various Cu(II) binding modes of mammalian normal prion protein (PrP(c)). The calculation results not only provide solid evidence to support one of the experimentally determined Cu(II) binding modes of PrP(c) but also shed new light on the normal function of the elusive protein; that is, PrP(c) is rather a Cu(II) transporter than an antioxidant. In addition, the employed theoretical methodology is also useful to investigate the metal chelating properties of other proteins and to rationally design Cu,Zn-superoxide dismutase mimics.

Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.