Limits...
Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

Thellung S, Corsaro A, Villa V, Simi A, Vella S, Pagano A, Florio T - Cell Death Dis (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Department of Oncology, Biology and Genetics University of Genova, Genova, Italy.

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.

Show MeSH

Related in: MedlinePlus

Internalized β–hPrP90-231 forms SDS-stable aggregates. Cells were treated with vehicle (lanes 2 a–d) or β–hPrP90-231 (1 μM) for 1 (lanes 3 a–d) and 4 days (lanes 4 a–d). Total cell lysates (100 μg) were resolved by semi-denaturing detergent-agarose gel electrophoresis (a and b) and SDS-PAGE (c and d). Blots were probed with anti-PrP antibodies 3F4 (a and c) and 8B4 (b and d). Purified β–hPrP90-231 (100 ng, lanes 1 a–c) was subjected to both electrophoretic conditions, to evidence its apparent molecular weight (c, lane 1), and native aggregation state (a, lane 1). No signal is detected in lane 1b, as 8B4 antibody does not recognize β–hPrP90-231. Blot a shows 3F4-immunoreactive aggregates after 1 and 4 days of treatment with hPrP90-231. Blot b shows that no significant 8B4-reactive aggregates are induced by β–hPrP90-231 treatment. SDS-PAGE shows that, although β–hPrP90-231 monomer is recovered within SH-SY5Y cytoplasm after 1 and 4 days of treatment (blot c), the expression of endogenous PrPC is unmodified (blot d)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3101817&req=5

fig3: Internalized β–hPrP90-231 forms SDS-stable aggregates. Cells were treated with vehicle (lanes 2 a–d) or β–hPrP90-231 (1 μM) for 1 (lanes 3 a–d) and 4 days (lanes 4 a–d). Total cell lysates (100 μg) were resolved by semi-denaturing detergent-agarose gel electrophoresis (a and b) and SDS-PAGE (c and d). Blots were probed with anti-PrP antibodies 3F4 (a and c) and 8B4 (b and d). Purified β–hPrP90-231 (100 ng, lanes 1 a–c) was subjected to both electrophoretic conditions, to evidence its apparent molecular weight (c, lane 1), and native aggregation state (a, lane 1). No signal is detected in lane 1b, as 8B4 antibody does not recognize β–hPrP90-231. Blot a shows 3F4-immunoreactive aggregates after 1 and 4 days of treatment with hPrP90-231. Blot b shows that no significant 8B4-reactive aggregates are induced by β–hPrP90-231 treatment. SDS-PAGE shows that, although β–hPrP90-231 monomer is recovered within SH-SY5Y cytoplasm after 1 and 4 days of treatment (blot c), the expression of endogenous PrPC is unmodified (blot d)

Mentions: We used semi-denaturing detergent-agarose gel electrophoresis to assess the formation of hPrP90-231 intracellular aggregates.20, 21, 22 SH-SY5Y cells, treated with vehicle or hPrP90-231 (1 μM) were lysed and protein electrophoresis performed in non-denaturing conditions to preserve hPrP90-231 intermolecular bonds and separate aggregates according to the their dimensions (Figures 3a and b). A separate set of samples was denatured and resolved by a standard SDS-PAGE to identify the molecular weight of endogenous PrPC and internalized hPrP90-231 (Figures 3c and d). We also used two antibodies able to discriminate hPrP90-231 from PrPC; 3F4 antibody recognizes both proteins, 8B4 antibody labels the first 60–80 a.a. of PrP and does not detect hPrP90-231. In control cells, 3F4 and 8B4 evidenced two discrete bands, likely corresponding to endogenous PrPC (Figures 3a and b, lanes 2). In treated cells, large hPrP90-231 aggregates were detected as 3F4-reactive proteins of high molecular weight (Figure 3a, lanes 3, 4), but distributed throughout the electrophoretic lanes, indicating that internalized hPrP90-231 is recovered in macroaggregated complexes of different size. hPrP90-231 intracellular content and electrophoretic mobility were similar after 1 or 4 days of treatment (Figure 3a, lanes 3, 4), demonstrating that hPrP90-231 aggregation state is not significantly affected by cell catabolic machinery. 8B4-immunoreactive signal in hPrP90-231-treated cells was not changed with respect to controls (Figure 3b, lanes 3, 4) signifying that PrPC does not aggregate after hPrP90-231 internalization. SDS-PAGE showed that intracellular hPrP90-231 aggregates are composed by 16 kDa monomers (Figure 3c) and do not alter PrPC expression (Figure 3d).


Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

Thellung S, Corsaro A, Villa V, Simi A, Vella S, Pagano A, Florio T - Cell Death Dis (2011)

Internalized β–hPrP90-231 forms SDS-stable aggregates. Cells were treated with vehicle (lanes 2 a–d) or β–hPrP90-231 (1 μM) for 1 (lanes 3 a–d) and 4 days (lanes 4 a–d). Total cell lysates (100 μg) were resolved by semi-denaturing detergent-agarose gel electrophoresis (a and b) and SDS-PAGE (c and d). Blots were probed with anti-PrP antibodies 3F4 (a and c) and 8B4 (b and d). Purified β–hPrP90-231 (100 ng, lanes 1 a–c) was subjected to both electrophoretic conditions, to evidence its apparent molecular weight (c, lane 1), and native aggregation state (a, lane 1). No signal is detected in lane 1b, as 8B4 antibody does not recognize β–hPrP90-231. Blot a shows 3F4-immunoreactive aggregates after 1 and 4 days of treatment with hPrP90-231. Blot b shows that no significant 8B4-reactive aggregates are induced by β–hPrP90-231 treatment. SDS-PAGE shows that, although β–hPrP90-231 monomer is recovered within SH-SY5Y cytoplasm after 1 and 4 days of treatment (blot c), the expression of endogenous PrPC is unmodified (blot d)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101817&req=5

fig3: Internalized β–hPrP90-231 forms SDS-stable aggregates. Cells were treated with vehicle (lanes 2 a–d) or β–hPrP90-231 (1 μM) for 1 (lanes 3 a–d) and 4 days (lanes 4 a–d). Total cell lysates (100 μg) were resolved by semi-denaturing detergent-agarose gel electrophoresis (a and b) and SDS-PAGE (c and d). Blots were probed with anti-PrP antibodies 3F4 (a and c) and 8B4 (b and d). Purified β–hPrP90-231 (100 ng, lanes 1 a–c) was subjected to both electrophoretic conditions, to evidence its apparent molecular weight (c, lane 1), and native aggregation state (a, lane 1). No signal is detected in lane 1b, as 8B4 antibody does not recognize β–hPrP90-231. Blot a shows 3F4-immunoreactive aggregates after 1 and 4 days of treatment with hPrP90-231. Blot b shows that no significant 8B4-reactive aggregates are induced by β–hPrP90-231 treatment. SDS-PAGE shows that, although β–hPrP90-231 monomer is recovered within SH-SY5Y cytoplasm after 1 and 4 days of treatment (blot c), the expression of endogenous PrPC is unmodified (blot d)
Mentions: We used semi-denaturing detergent-agarose gel electrophoresis to assess the formation of hPrP90-231 intracellular aggregates.20, 21, 22 SH-SY5Y cells, treated with vehicle or hPrP90-231 (1 μM) were lysed and protein electrophoresis performed in non-denaturing conditions to preserve hPrP90-231 intermolecular bonds and separate aggregates according to the their dimensions (Figures 3a and b). A separate set of samples was denatured and resolved by a standard SDS-PAGE to identify the molecular weight of endogenous PrPC and internalized hPrP90-231 (Figures 3c and d). We also used two antibodies able to discriminate hPrP90-231 from PrPC; 3F4 antibody recognizes both proteins, 8B4 antibody labels the first 60–80 a.a. of PrP and does not detect hPrP90-231. In control cells, 3F4 and 8B4 evidenced two discrete bands, likely corresponding to endogenous PrPC (Figures 3a and b, lanes 2). In treated cells, large hPrP90-231 aggregates were detected as 3F4-reactive proteins of high molecular weight (Figure 3a, lanes 3, 4), but distributed throughout the electrophoretic lanes, indicating that internalized hPrP90-231 is recovered in macroaggregated complexes of different size. hPrP90-231 intracellular content and electrophoretic mobility were similar after 1 or 4 days of treatment (Figure 3a, lanes 3, 4), demonstrating that hPrP90-231 aggregation state is not significantly affected by cell catabolic machinery. 8B4-immunoreactive signal in hPrP90-231-treated cells was not changed with respect to controls (Figure 3b, lanes 3, 4) signifying that PrPC does not aggregate after hPrP90-231 internalization. SDS-PAGE showed that intracellular hPrP90-231 aggregates are composed by 16 kDa monomers (Figure 3c) and do not alter PrPC expression (Figure 3d).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Department of Oncology, Biology and Genetics University of Genova, Genova, Italy.

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.

Show MeSH
Related in: MedlinePlus