Copper(II) Inhibits in Vitro Conversion of Prion Protein into Amyloid Fibrils

• Published in: Biochemistry (Easton) 2005-05, Vol.44 (18), p.6776-6787
• Main Authors: Bocharova, Olga V, Breydo, Leonid, Salnikov, Vadim V, Baskakov, Ilia V
• Format: Article
• Language: English
• Subjects: Amyloid - antagonists & inhibitors; Amyloid - chemistry; Amyloid - metabolism; Animals; Binding Sites; Cations, Divalent; Copper - chemistry; Drug Resistance; Endopeptidase K - chemistry; Hydrogen-Ion Concentration; Hydrolysis; Macromolecular Substances - antagonists & inhibitors; Macromolecular Substances - chemistry; Macromolecular Substances - metabolism; Manganese - chemistry; Mice; Peptide Fragments - antagonists & inhibitors; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Prions - antagonists & inhibitors; Prions - chemistry; Prions - metabolism; Protein Binding; Protein Structure, Secondary; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Repetitive Sequences, Amino Acid; Zinc - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi050251q

In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89−230 (rPrP 89−230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673−676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu2+ on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu2+ inhibited conversion of full-length recombinant PrP (rPrP 23−230) into amyloid fibrils. This effect was most pronounced for Cu2+, and less so for Zn2+, while Mn2+ had no effect on the conversion. Cu2+-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23−230 displays lower Cu2+-binding capacity. Using rPrP 89−230, we found that Cu2+-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu2+ inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of α-rPrP. Remarkably, Cu2+ also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu2+ induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu2+ induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu2+ in the pathogenesis of prion diseases is complex. Because Cu2+ may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.