Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F.
The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.
Sirangelo
Ivana
I
Dipartimento di Biochimica e Biofisica, Seconda Università degli Studi di Napoli, via L. De Crecchio 7, 80138 Naples, Italy.
Malmo
Clorinda
C
Iannuzzi
Clara
C
Mezzogiorno
Antonio
A
Bianco
Maria Rosaria
MR
Papa
Michele
M
Irace
Gaetano
G
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics*; D052160:Benzothiazoles; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002942:Circular Dichroism; D004396:Coloring Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics*; D041681:NIH 3T3 Cells; D010590:Phalloidine / Q000494:pharmacology; D011419:Propidium / Q000494:pharmacology; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D052160:Benzothiazoles; D004396:Coloring Agents; D005456:Fluorescent Dyes; D009211:Myoglobin; D013778:Tetrazolium Salts; D013844:Thiazoles; C010857:apomyoglobin; D010590:Phalloidine; C009462:thioflavin T; D011419:Propidium; C027132:1-anilino-8-naphthalenesulfonate; C022616:thiazolyl blue