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1.
Protein Pept Lett ; 27(8): 782-792, 2020.
Article in English | MEDLINE | ID: mdl-32096738

ABSTRACT

BACKGROUND: It has been previously found that PrP23-98, which contains four highly conserved octarepeats (residues 60-91) and one partial repeat (residues 92-96), polymerizes into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions. OBJECTIVE: We aimed to determine the requirements for the formation of these aggregates. METHODS: In this study, we performed an aggregation experiment using N-acetylated and Camidated PrP fragments of the N-terminal domain, Octa1, Octa2, Octa3, Octa4, PrP84-114, and PrP76-114, in the presence of NADPH with copper ions, and focused on the effect of the number of copper-binding sites on aggregation. RESULTS: Among these PrP fragments, Octa4, containing four copper-binding sites, was particularly effective in forming aggregates. We also tested the effect of other pyridine nucleotides and adenine nucleotides on the aggregation of Octa4. ATP was equally effective, but NADH, NADP, ADP, and AMP had no effect. CONCLUSION: The phosphate group on the adenine-linked ribose moiety of adenine nucleotides and pyridine nucleotides is presumed to be essential for the observed effect on aggregation. Efficient aggregation requires the presence of the four octarepeats. These insights may be helpful in the eventual development of therapeutic agents against prion-related disorders.


Subject(s)
Copper/chemistry , Endopeptidase K/chemistry , Peptide Fragments/chemistry , Prion Proteins/chemistry , Purine Nucleotides/chemistry , Humans
2.
Protein Pept Lett ; 23(11): 988-993, 2016.
Article in English | MEDLINE | ID: mdl-27633855

ABSTRACT

Recent studies have indicated that PrP23-98, an N-terminal portion of PrP, polymerizes into amyloid-like and proteinase K (PK)-resistant aggregates in the presence of NADPH with copper ions [19], and then that CRT suppressed aggregation of PrP23-98 and also promoted solubilization of the aggregates [18]. As it is interesting to find out whether other chaperones can inhibit aggregation of PrP23-98 in vitro similar to CRT, this study was conducted to determine whether BiP, Grp94, PDI Grp58 and heat shock cognate protein70 (Hsc70) can inhibit aggregation of PrP23-98 in vitro. The present results indicated that Grp94 suppressed aggregation of PrP23-98, but that Grp94 could not mediate solubilization occurred in the aggregates in contrast to CRT. Other chaperons induced aggregation of PrP23-98 in the absence of NADPH.


Subject(s)
HSC70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Prion Proteins/metabolism , Protein Aggregates/physiology , Protein Disulfide-Isomerases/metabolism , Endoplasmic Reticulum Chaperone BiP , NADP/metabolism , Peptide Fragments/metabolism , Protein Binding
3.
Biosci Biotechnol Biochem ; 75(8): 1625-7, 2011.
Article in English | MEDLINE | ID: mdl-21821925

ABSTRACT

Because prion protein PrP-(23-98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23-98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23-98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.


Subject(s)
Calreticulin/therapeutic use , Copper/antagonists & inhibitors , NADP/antagonists & inhibitors , Peptide Fragments/metabolism , Plaque, Amyloid/prevention & control , Prions/metabolism , Protein Binding/drug effects , Recombinant Proteins/metabolism , Amyloid/metabolism , Calreticulin/pharmacology , Copper/adverse effects , Copper/pharmacology , Dose-Response Relationship, Drug , Endopeptidase K/metabolism , Humans , NADP/adverse effects , NADP/pharmacology , Plaque, Amyloid/metabolism , Prion Diseases/drug therapy , Prion Diseases/pathology , Protein Structure, Quaternary , Recombinant Proteins/genetics , Solutions , Spectrophotometry
4.
Protein Pept Lett ; 16(1): 91-6, 2009.
Article in English | MEDLINE | ID: mdl-19149680

ABSTRACT

Here, we show for the first time that non-fibrillar and spherical aggregates produced from PrP-(23-98) in the presence of NADPH plus copper ions are toxic to cultured cells and induce apoptotic signals. It is also confirmed that endogenous cellular PrP isoform is not required for toxicity to occur.


Subject(s)
Apoptosis/drug effects , Endopeptidase K/metabolism , Peptide Fragments/toxicity , Prions/toxicity , Recombinant Proteins/toxicity , Animals , Cell Line , Hippocampus/drug effects , Mice , Protein Structure, Quaternary
5.
J Biol Chem ; 281(46): 34880-7, 2006 Nov 17.
Article in English | MEDLINE | ID: mdl-16990274

ABSTRACT

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.


Subject(s)
Copper/metabolism , Endopeptidase K/metabolism , NADP/pharmacology , Prions/chemistry , Prions/metabolism , Animals , Copper/pharmacology , Humans , Mice , NADP/metabolism , Prions/genetics , Prions/ultrastructure , Protein Structure, Quaternary/drug effects , Recombinant Proteins/metabolism
6.
J Nutr Sci Vitaminol (Tokyo) ; 51(6): 398-405, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16521698

ABSTRACT

It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H2O2. This H2O2-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with L-ascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.


Subject(s)
Ascorbic Acid/pharmacology , Hydrogen Peroxide/pharmacology , Oxidative Stress/drug effects , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Ascorbic Acid/analysis , Bacteria/enzymology , CHO Cells , Cricetinae , Cricetulus , Diamide/pharmacology , Gene Expression , Glutathione/metabolism , Oxidation-Reduction , Sulfhydryl Compounds/metabolism , Transfection
7.
Biochem J ; 387(Pt 1): 247-55, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15554874

ABSTRACT

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.


Subject(s)
Copper/metabolism , Peptide Fragments/metabolism , Prions/metabolism , Amino Acid Motifs/drug effects , Animals , Catalase/pharmacology , Catalysis , Catecholamines/pharmacology , Cations, Divalent/metabolism , Dimerization , Dopamine/metabolism , Hydrogen Peroxide/metabolism , Mice , Oxidation-Reduction , Peptides/metabolism , Prions/chemistry , Protein Structure, Tertiary/drug effects , Superoxide Dismutase/pharmacology , Superoxides/metabolism
8.
FEBS Lett ; 511(1-3): 118-22, 2002 Jan 30.
Article in English | MEDLINE | ID: mdl-11821060

ABSTRACT

The amino-terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion. This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline. In this study, we have investigated copper-catalyzed oxidation of an N-terminal part of human PrP, PrP23-98, that was prepared by the recombinant DNA technique. Carbonyl formations on copper-bound PrP23-98 induced by dopamine and L-ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23-98-bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.


Subject(s)
Copper/metabolism , Dopamine/metabolism , Oxidoreductases/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Prions/chemistry , Prions/metabolism , Amino Acids/analysis , Ascorbic Acid/metabolism , Blotting, Western , Chelating Agents/metabolism , Chelating Agents/pharmacology , Escherichia coli , Humans , Oxidation-Reduction , Oxidoreductases/chemistry
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