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J. Biol. Chem., Vol. 281, Issue 46, 34880-34887, November 17, 2006

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Combination of NADPH and Copper Ions Generates Proteinase K-resistant Aggregates from Recombinant Prion Protein^*

Noriyuki Shiraishi¹, Hirotoshi Utsunomiya, and Morimitsu Nishikimi

From the Department of Biochemistry and Central Research Laboratory, Wakayama Medical University, Wakayama 641-8509, Japan

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23–98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23–98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23–231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.

Received for publication, July 11, 2006 , and in revised form, September 20, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan. Tel.: 81-73-441-0628; Fax: 81-73-441-0628; E-mail: nshirais{at}wakayama-med.ac.jp.

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