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 is partly PK resistant. (a) Cells treated with PBS or β–hPrP90-231 (1 μM) for 24 and 48 h were collected in non-denaturing lysis buffer, without protease inhibitors. Proteins for each sample (100 μg) were digested with increasing concentrations of PK (1, 2, and 10 μg/ml) at 37°C for 30 min. PrPC and β–hPrP90-231 digestion fragments were resolved by 15% SDS-PAGE using the anti-PrP antibody 3F4. β–hPrP90-231 (100 ng) was loaded to compare the electrophoretic run of PrP fragment before and after intracellular uptake (lane C). Control cells evidence a faint 3F4-immunoreactive signal between 35 and 45 kDa, corresponding to PrPC, that is almost fully digested by PK. Cell treatment with β–hPrP90-231 for 24 and 48 h induced the appearance of a 16 kDa band corresponding to internalized PrP fragment that showed a full digestion only at 10 μg/ml PK, indicating that internalized β–hPrP90-231 is significantly more protease-resistant than PrPC. (b) Densitometric analysis of 3F4 signal in untreated (white columns) and PK-digested samples (light gray, gray, and black columns). Time 0, in the absence of β–hPrP90-231, indicates PK effects on PrPC (MW 35–45 kDa). Plot shows that intracellular β–hPrP90-231 resistance is higher than that of PrPc and increases with treatment time. Values (n=3) represent OD intensity/mm2 and expressed as percentage of respective PK-undigested samples. *P<0.05 and **P<0.01 versus respective undigested controls; OP<0.05, OOP<0.01 versus corresponding PK digestion of vehicle-treated cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Internalized β–hPrP90-231 is partly PK resistant. (a) Cells treated with PBS or β–hPrP90-231 (1 μM) for 24 and 48 h were collected in non-denaturing lysis buffer, without protease inhibitors. Proteins for each sample (100 μg) were digested with increasing concentrations of PK (1, 2, and 10 μg/ml) at 37°C for 30 min. PrPC and β–hPrP90-231 digestion fragments were resolved by 15% SDS-PAGE using the anti-PrP antibody 3F4. β–hPrP90-231 (100 ng) was loaded to compare the electrophoretic run of PrP fragment before and after intracellular uptake (lane C). Control cells evidence a faint 3F4-immunoreactive signal between 35 and 45 kDa, corresponding to PrPC, that is almost fully digested by PK. Cell treatment with β–hPrP90-231 for 24 and 48 h induced the appearance of a 16 kDa band corresponding to internalized PrP fragment that showed a full digestion only at 10 μg/ml PK, indicating that internalized β–hPrP90-231 is significantly more protease-resistant than PrPC. (b) Densitometric analysis of 3F4 signal in untreated (white columns) and PK-digested samples (light gray, gray, and black columns). Time 0, in the absence of β–hPrP90-231, indicates PK effects on PrPC (MW 35–45 kDa). Plot shows that intracellular β–hPrP90-231 resistance is higher than that of PrPc and increases with treatment time. Values (n=3) represent OD intensity/mm2 and expressed as percentage of respective PK-undigested samples. *P<0.05 and **P<0.01 versus respective undigested controls; OP<0.05, OOP<0.01 versus corresponding PK digestion of vehicle-treated cells

Mentions: We measured PK sensitivity of internalized hPrP90-231 to obtain an index of resistance to lysosomal degradation. Cells treated with vehicle or hPrP90-231 were lysed under non-denaturing conditions, in the absence of protease inhibitors, and proteins incubated with increasing amounts of PK (Figure 5a). In the absence of PK treatment, endogenous PrPC was detected without alterations induced by hPrP90-231 treatment. Conversely, a large 3F4-immunoreactive band was detectable in cell lysates after treatment with hPrP90-231, showing an apparent molecular weight matching with that of hPrP90-231 (16 kDa). Although PrPC was completely degraded by PK (1 μg), the complete digestion of the bona fide intracellular hPrP90-231 required a 10-fold higher PK concentration. The marked difference in proteolysis resistance between PrPC and internalized hPrP90-231 was further demonstrated using the cell blotting technique. Treated cells were firmly pressed on lysis buffer-soaked nitrocellulose membrane to transfer their protein content as a sort of fingerprint; membranes were then treated with PK before being probed with 3F4 and 8B4 antibodies (Figure 6). Immunoblots revealed that cells exposed to hPrP90-231 retain a 3F4-immunoreactive signal, resistant to PK digestion (up to 0.5 μg/ml, upper panel), whereas vehicle-treated cells showed an almost complete loss of 3F4 signal. Using the 8B4 antibody (lower panel), PK treatment markedly reduced the immunoreactive signal in both vehicle and hPrP90-231-treated samples, indicating a high PrPC sensitivity to proteolysis that is not modified by cell exposure to hPrP90-231.


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 is partly PK resistant. (a) Cells treated with PBS or β–hPrP90-231 (1 μM) for 24 and 48 h were collected in non-denaturing lysis buffer, without protease inhibitors. Proteins for each sample (100 μg) were digested with increasing concentrations of PK (1, 2, and 10 μg/ml) at 37°C for 30 min. PrPC and β–hPrP90-231 digestion fragments were resolved by 15% SDS-PAGE using the anti-PrP antibody 3F4. β–hPrP90-231 (100 ng) was loaded to compare the electrophoretic run of PrP fragment before and after intracellular uptake (lane C). Control cells evidence a faint 3F4-immunoreactive signal between 35 and 45 kDa, corresponding to PrPC, that is almost fully digested by PK. Cell treatment with β–hPrP90-231 for 24 and 48 h induced the appearance of a 16 kDa band corresponding to internalized PrP fragment that showed a full digestion only at 10 μg/ml PK, indicating that internalized β–hPrP90-231 is significantly more protease-resistant than PrPC. (b) Densitometric analysis of 3F4 signal in untreated (white columns) and PK-digested samples (light gray, gray, and black columns). Time 0, in the absence of β–hPrP90-231, indicates PK effects on PrPC (MW 35–45 kDa). Plot shows that intracellular β–hPrP90-231 resistance is higher than that of PrPc and increases with treatment time. Values (n=3) represent OD intensity/mm2 and expressed as percentage of respective PK-undigested samples. *P<0.05 and **P<0.01 versus respective undigested controls; OP<0.05, OOP<0.01 versus corresponding PK digestion of vehicle-treated cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Internalized β–hPrP90-231 is partly PK resistant. (a) Cells treated with PBS or β–hPrP90-231 (1 μM) for 24 and 48 h were collected in non-denaturing lysis buffer, without protease inhibitors. Proteins for each sample (100 μg) were digested with increasing concentrations of PK (1, 2, and 10 μg/ml) at 37°C for 30 min. PrPC and β–hPrP90-231 digestion fragments were resolved by 15% SDS-PAGE using the anti-PrP antibody 3F4. β–hPrP90-231 (100 ng) was loaded to compare the electrophoretic run of PrP fragment before and after intracellular uptake (lane C). Control cells evidence a faint 3F4-immunoreactive signal between 35 and 45 kDa, corresponding to PrPC, that is almost fully digested by PK. Cell treatment with β–hPrP90-231 for 24 and 48 h induced the appearance of a 16 kDa band corresponding to internalized PrP fragment that showed a full digestion only at 10 μg/ml PK, indicating that internalized β–hPrP90-231 is significantly more protease-resistant than PrPC. (b) Densitometric analysis of 3F4 signal in untreated (white columns) and PK-digested samples (light gray, gray, and black columns). Time 0, in the absence of β–hPrP90-231, indicates PK effects on PrPC (MW 35–45 kDa). Plot shows that intracellular β–hPrP90-231 resistance is higher than that of PrPc and increases with treatment time. Values (n=3) represent OD intensity/mm2 and expressed as percentage of respective PK-undigested samples. *P<0.05 and **P<0.01 versus respective undigested controls; OP<0.05, OOP<0.01 versus corresponding PK digestion of vehicle-treated cells
Mentions: We measured PK sensitivity of internalized hPrP90-231 to obtain an index of resistance to lysosomal degradation. Cells treated with vehicle or hPrP90-231 were lysed under non-denaturing conditions, in the absence of protease inhibitors, and proteins incubated with increasing amounts of PK (Figure 5a). In the absence of PK treatment, endogenous PrPC was detected without alterations induced by hPrP90-231 treatment. Conversely, a large 3F4-immunoreactive band was detectable in cell lysates after treatment with hPrP90-231, showing an apparent molecular weight matching with that of hPrP90-231 (16 kDa). Although PrPC was completely degraded by PK (1 μg), the complete digestion of the bona fide intracellular hPrP90-231 required a 10-fold higher PK concentration. The marked difference in proteolysis resistance between PrPC and internalized hPrP90-231 was further demonstrated using the cell blotting technique. Treated cells were firmly pressed on lysis buffer-soaked nitrocellulose membrane to transfer their protein content as a sort of fingerprint; membranes were then treated with PK before being probed with 3F4 and 8B4 antibodies (Figure 6). Immunoblots revealed that cells exposed to hPrP90-231 retain a 3F4-immunoreactive signal, resistant to PK digestion (up to 0.5 μg/ml, upper panel), whereas vehicle-treated cells showed an almost complete loss of 3F4 signal. Using the 8B4 antibody (lower panel), PK treatment markedly reduced the immunoreactive signal in both vehicle and hPrP90-231-treated samples, indicating a high PrPC sensitivity to proteolysis that is not modified by cell exposure to hPrP90-231.

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