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The 37/67 kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67 kDa LR cell surface localization and interaction with the cellular prion protein.

Sarnataro D, Pepe A, Altamura G, De Simone I, Pesapane A, Nitsch L, Montuori N, Lavecchia A, Zurzolo C - Sci Rep (2016)

Bottom Line: A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet.We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C).These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", 80131, Naples, Italy.

ABSTRACT
The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrP(C)) and to be required for pathological PrP (PrP(Sc)) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrP(C) interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C). Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrP(C) interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrP(C) on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.

No MeSH data available.


Related in: MedlinePlus

NSC47924 affects the intracellular fate of 37/67 kDa LR and PrPC.GT1 cells were grown on coverslips, cooled at 4 °C on ice, and stained with SAF32 mAb (1:100) and 5004 pAb (1:50) to label cell surface PrPC and 37/67 kDa LR, respectively. To allow membrane trafficking and internalization, GT1 cells were warmed at 37 °C in the culture medium with or without NSC47924 for indicated times. After that, the cells were washed, fixed in PFA 4% and permeabilized in 0.1% TX-100 for 10 min. To detect internalized proteins the cells were labelled with secondary antibodies (PrPC, green; 37/67 kDa LR, red) and their colocalization was measured. RT: Room Temperature; Scale bars, 10 μm.
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f5: NSC47924 affects the intracellular fate of 37/67 kDa LR and PrPC.GT1 cells were grown on coverslips, cooled at 4 °C on ice, and stained with SAF32 mAb (1:100) and 5004 pAb (1:50) to label cell surface PrPC and 37/67 kDa LR, respectively. To allow membrane trafficking and internalization, GT1 cells were warmed at 37 °C in the culture medium with or without NSC47924 for indicated times. After that, the cells were washed, fixed in PFA 4% and permeabilized in 0.1% TX-100 for 10 min. To detect internalized proteins the cells were labelled with secondary antibodies (PrPC, green; 37/67 kDa LR, red) and their colocalization was measured. RT: Room Temperature; Scale bars, 10 μm.

Mentions: In order to specifically investigate the effect of NSC47924 on the intracellular fate of the two proteins, we performed double indirect immunofluorescence assays under permeabilized conditions, following specifically the cell surface laminin receptor and PrPC pools (Fig. 5). GT1 cells, were first incubated on ice (t = 0′ at 4 °C, which blocks membrane trafficking)51 with the primary antibodies against both laminin receptor and PrPC and then treated at 37 °C (which allows membrane trafficking) with or without the inhibitor, from 30 min up to 180 min, fixed and permeabilized. Accordingly to Co-IP assays, the two proteins colocalized on the cell surface and already after 30 min at room temperature (RT), both 37/67 kDa LR and PrPC entered the cells, as shown by the punctate staining. The presence of the inhibitor, (t = 30′ RT, room temperature, + NSC47924) induced internalization of 37/67 kDa LR and a decrement of PrPC colocalization, which was totally abolished with increasing times of inhibitor treatment (t = 90′ and 180′). The disappearance of punctate laminin receptor staining which resulted somewhat sparse into the cells and sharply less intense compared to control, supports the results from biotinylation kinetics (Fig. 4), confirming that the inhibitor stimulates 37/67 kDa LR internalization and possibly its degradation, while PrPC was stabilized on the cell surface due to its prevented internalization. This effect could be explained by assuming that PrPC becomes not able to be internalized because of the absence of its receptor.


The 37/67 kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67 kDa LR cell surface localization and interaction with the cellular prion protein.

Sarnataro D, Pepe A, Altamura G, De Simone I, Pesapane A, Nitsch L, Montuori N, Lavecchia A, Zurzolo C - Sci Rep (2016)

NSC47924 affects the intracellular fate of 37/67 kDa LR and PrPC.GT1 cells were grown on coverslips, cooled at 4 °C on ice, and stained with SAF32 mAb (1:100) and 5004 pAb (1:50) to label cell surface PrPC and 37/67 kDa LR, respectively. To allow membrane trafficking and internalization, GT1 cells were warmed at 37 °C in the culture medium with or without NSC47924 for indicated times. After that, the cells were washed, fixed in PFA 4% and permeabilized in 0.1% TX-100 for 10 min. To detect internalized proteins the cells were labelled with secondary antibodies (PrPC, green; 37/67 kDa LR, red) and their colocalization was measured. RT: Room Temperature; Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829897&req=5

f5: NSC47924 affects the intracellular fate of 37/67 kDa LR and PrPC.GT1 cells were grown on coverslips, cooled at 4 °C on ice, and stained with SAF32 mAb (1:100) and 5004 pAb (1:50) to label cell surface PrPC and 37/67 kDa LR, respectively. To allow membrane trafficking and internalization, GT1 cells were warmed at 37 °C in the culture medium with or without NSC47924 for indicated times. After that, the cells were washed, fixed in PFA 4% and permeabilized in 0.1% TX-100 for 10 min. To detect internalized proteins the cells were labelled with secondary antibodies (PrPC, green; 37/67 kDa LR, red) and their colocalization was measured. RT: Room Temperature; Scale bars, 10 μm.
Mentions: In order to specifically investigate the effect of NSC47924 on the intracellular fate of the two proteins, we performed double indirect immunofluorescence assays under permeabilized conditions, following specifically the cell surface laminin receptor and PrPC pools (Fig. 5). GT1 cells, were first incubated on ice (t = 0′ at 4 °C, which blocks membrane trafficking)51 with the primary antibodies against both laminin receptor and PrPC and then treated at 37 °C (which allows membrane trafficking) with or without the inhibitor, from 30 min up to 180 min, fixed and permeabilized. Accordingly to Co-IP assays, the two proteins colocalized on the cell surface and already after 30 min at room temperature (RT), both 37/67 kDa LR and PrPC entered the cells, as shown by the punctate staining. The presence of the inhibitor, (t = 30′ RT, room temperature, + NSC47924) induced internalization of 37/67 kDa LR and a decrement of PrPC colocalization, which was totally abolished with increasing times of inhibitor treatment (t = 90′ and 180′). The disappearance of punctate laminin receptor staining which resulted somewhat sparse into the cells and sharply less intense compared to control, supports the results from biotinylation kinetics (Fig. 4), confirming that the inhibitor stimulates 37/67 kDa LR internalization and possibly its degradation, while PrPC was stabilized on the cell surface due to its prevented internalization. This effect could be explained by assuming that PrPC becomes not able to be internalized because of the absence of its receptor.

Bottom Line: A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet.We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C).These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", 80131, Naples, Italy.

ABSTRACT
The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrP(C)) and to be required for pathological PrP (PrP(Sc)) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrP(C) interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C). Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrP(C) interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrP(C) on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.

No MeSH data available.


Related in: MedlinePlus