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Selective processing and metabolism of disease-causing mutant prion proteins.

Ashok A, Hegde RS - PLoS Pathog. (2009)

Bottom Line: The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER.Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments.These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C)). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc) form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C) to PrP(Sc), our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc) in familial prion disease.

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Steady state localization of wtPrP and various disease-causing mutants.(A) Indirect immunofluorescent localization of PrP using the 3F4 antibody in N2a cells transiently transfected with wtPrP or any of 7 different PrP mutants. Identical detector settings were used to image representative fields of cells. (B) Enlarged images of single cells chosen from the corresponding fields from panel A illustrate the overall subtle differences in localization of C-terminal mutant PrPs compared to wtPrP and PrP(A117V). (C) Cells co-expressing fluorescently tagged wtPrP and PrP(H187R) were imaged. Pseudocolored depiction of the mutant∶wt fluorescence ratio in different cellular locales is shown in the last panel (scale is below the image).
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ppat-1000479-g001: Steady state localization of wtPrP and various disease-causing mutants.(A) Indirect immunofluorescent localization of PrP using the 3F4 antibody in N2a cells transiently transfected with wtPrP or any of 7 different PrP mutants. Identical detector settings were used to image representative fields of cells. (B) Enlarged images of single cells chosen from the corresponding fields from panel A illustrate the overall subtle differences in localization of C-terminal mutant PrPs compared to wtPrP and PrP(A117V). (C) Cells co-expressing fluorescently tagged wtPrP and PrP(H187R) were imaged. Pseudocolored depiction of the mutant∶wt fluorescence ratio in different cellular locales is shown in the last panel (scale is below the image).

Mentions: Wild type human PrP (wtPrP) and several human disease-causing PrP mutants were expressed in mouse N2a cells and selectively visualized by indirect immunofluorescence with the human-specific 3F4 PrP antibody (Fig. 1A–B). As expected, wtPrP was found predominantly on the cell surface, with varying amounts of an intracellular pool in the ER, Golgi, and endosomal system (Fig. S1A). This pattern is consistent with PrP trafficking through the secretory pathway en route to the cell surface, its constitutive recycling through the endosomal system, and its eventual degradation in lysosomes. This same general pattern was seen in all cells regardless of expression level, although there was some heterogeneity in the relative amounts found in each of the different cellular compartments (Fig. S1A). All PrP mutants also showed localization in the same compartments, including the ER, Golgi, endosomes and the cell surface (Fig. S1B). A panel of representative cells, all at comparable expression levels, illustrates the generally similar patterns of localization for wtPrP and each of the mutants (Fig. 1B).


Selective processing and metabolism of disease-causing mutant prion proteins.

Ashok A, Hegde RS - PLoS Pathog. (2009)

Steady state localization of wtPrP and various disease-causing mutants.(A) Indirect immunofluorescent localization of PrP using the 3F4 antibody in N2a cells transiently transfected with wtPrP or any of 7 different PrP mutants. Identical detector settings were used to image representative fields of cells. (B) Enlarged images of single cells chosen from the corresponding fields from panel A illustrate the overall subtle differences in localization of C-terminal mutant PrPs compared to wtPrP and PrP(A117V). (C) Cells co-expressing fluorescently tagged wtPrP and PrP(H187R) were imaged. Pseudocolored depiction of the mutant∶wt fluorescence ratio in different cellular locales is shown in the last panel (scale is below the image).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691595&req=5

ppat-1000479-g001: Steady state localization of wtPrP and various disease-causing mutants.(A) Indirect immunofluorescent localization of PrP using the 3F4 antibody in N2a cells transiently transfected with wtPrP or any of 7 different PrP mutants. Identical detector settings were used to image representative fields of cells. (B) Enlarged images of single cells chosen from the corresponding fields from panel A illustrate the overall subtle differences in localization of C-terminal mutant PrPs compared to wtPrP and PrP(A117V). (C) Cells co-expressing fluorescently tagged wtPrP and PrP(H187R) were imaged. Pseudocolored depiction of the mutant∶wt fluorescence ratio in different cellular locales is shown in the last panel (scale is below the image).
Mentions: Wild type human PrP (wtPrP) and several human disease-causing PrP mutants were expressed in mouse N2a cells and selectively visualized by indirect immunofluorescence with the human-specific 3F4 PrP antibody (Fig. 1A–B). As expected, wtPrP was found predominantly on the cell surface, with varying amounts of an intracellular pool in the ER, Golgi, and endosomal system (Fig. S1A). This pattern is consistent with PrP trafficking through the secretory pathway en route to the cell surface, its constitutive recycling through the endosomal system, and its eventual degradation in lysosomes. This same general pattern was seen in all cells regardless of expression level, although there was some heterogeneity in the relative amounts found in each of the different cellular compartments (Fig. S1A). All PrP mutants also showed localization in the same compartments, including the ER, Golgi, endosomes and the cell surface (Fig. S1B). A panel of representative cells, all at comparable expression levels, illustrates the generally similar patterns of localization for wtPrP and each of the mutants (Fig. 1B).

Bottom Line: The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER.Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments.These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C)). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc) form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C) to PrP(Sc), our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc) in familial prion disease.

Show MeSH
Related in: MedlinePlus