Conformation dependent pro-apoptotic activity of the recombinant human prion protein fragment 90-231.
The transition of prion protein from a mainly alpha-structured isoform (PrPC) to a beta sheet-containing protein (PrPSc) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in alpha and beta conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53 degrees C, 1h) we converted hPrP90-231 in a structural isoform displaying PrPSc-related characteristics: high beta sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38- dependent apoptosis. Conversely, in the native alpha-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a beta-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.
Corsaro
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A
Section of Pharmacology, Dept. Oncology Biology and Genetics, University of Genoa, Viale Benedetto XV 2, 16132 Genoa, Italy.
Paludi
D
D
Villa
V
V
D'Arrigo
C
C
Chiovitti
K
K
Thellung
S
S
Russo
C
C
Di Cola
D
D
Ballerini
P
P
Patrone
E
E
Schettini
G
G
Aceto
A
A
Florio
T
T
D000682:Amyloid / Q000096:biosynthesis; D017209:Apoptosis / Q000187:drug effects*; D052160:Benzothiazoles; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000737:chemistry; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D009336:Necrosis; D018621:PrPC Proteins / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013778:Tetrazolium Salts; D013844:Thiazoles / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D018621:PrPC Proteins; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C009462:thioflavin T; D007770:L-Lactate Dehydrogenase; D048051:p38 Mitogen-Activated Protein Kinases; D019286:Endopeptidase K; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; C022616:thiazolyl blue