title: Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.
abstract: To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.
authors: Laboratory of Pharmacology, Department of Oncology, Biology and Genetics University of Genova, Genova, Italy.
authors: S
authors: S
authors: Thellung
authors: A
authors: A
authors: Corsaro
authors: V
authors: V
authors: Villa
authors: A
authors: A
authors: Simi
authors: S
authors: S
authors: Vella
authors: A
authors: A
authors: Pagano
authors: T
authors: T
authors: Florio
mesh_terms: D017209:Apoptosis; D016923:Cell Death; D045744:Cell Line, Tumor; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism*; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017096:Prion Diseases / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D012995:Solubility
chemical_list: D018621:PrPC Proteins; D019286:Endopeptidase K