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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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The N-terminus of PrP modulates mutant PrP metabolism.(A) The full length (FL) or N-terminally deleted (ΔN, lacking residues 23–48) constructs for wild type and mutant PrPs were analyzed by the detergent solubility assay. In each case, deletion of the N-terminus resulted in decreased insoluble forms. A corresponding increase in the fully mature soluble form is apparent in most cases. (B) Pulse chase analyses (as in Fig. 7A) of wtPrP, PrP(H187R), wtPrPΔN and PrP(H187R)ΔN were quantified by phosphorimaging. The left panel plots the appearance of fully mature species (as a proportion of total PrP) over time. The middle panel shows the time course of disappearance for immature glycosylated species (plotted as a percent of the amount present at pulse). The inclusion of 5 µM MG132, a proteasome inhibitor, had no effect on the immature species for PrP(H187R)ΔN. The right panel shows the fate of unglycosylated PrP, without or with 5 µM MG132. (C) Lysates harvested after pulse labeling with S35-methionine from cells expressing wtPrP, wtPrPΔN, PrP(H187R) and PrP(H187R)ΔN were separated into detergent soluble (S) and insoluble (P) fractions, immunoprecipitated, and analyzed by autoradiography. The percent of labeled PrP that is insoluble is indicated below the respective panels.
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ppat-1000479-g010: The N-terminus of PrP modulates mutant PrP metabolism.(A) The full length (FL) or N-terminally deleted (ΔN, lacking residues 23–48) constructs for wild type and mutant PrPs were analyzed by the detergent solubility assay. In each case, deletion of the N-terminus resulted in decreased insoluble forms. A corresponding increase in the fully mature soluble form is apparent in most cases. (B) Pulse chase analyses (as in Fig. 7A) of wtPrP, PrP(H187R), wtPrPΔN and PrP(H187R)ΔN were quantified by phosphorimaging. The left panel plots the appearance of fully mature species (as a proportion of total PrP) over time. The middle panel shows the time course of disappearance for immature glycosylated species (plotted as a percent of the amount present at pulse). The inclusion of 5 µM MG132, a proteasome inhibitor, had no effect on the immature species for PrP(H187R)ΔN. The right panel shows the fate of unglycosylated PrP, without or with 5 µM MG132. (C) Lysates harvested after pulse labeling with S35-methionine from cells expressing wtPrP, wtPrPΔN, PrP(H187R) and PrP(H187R)ΔN were separated into detergent soluble (S) and insoluble (P) fractions, immunoprecipitated, and analyzed by autoradiography. The percent of labeled PrP that is insoluble is indicated below the respective panels.

Mentions: To gain insight into the misfolding-selective sorting operating on mutant PrP, we sought to identify potential sorting determinant(s) by analyzing PrP deletion mutants. We found that deletion of residues 23–48 (the ΔN constructs) markedly reduced the amount of detergent-insoluble, immaturely glycosylated species of several PrP mutants relative to their full length (FL) counterparts (Fig. 10A). In most cases, this decrease was accompanied by a relative increase in the fully mature, detergent-soluble species (Fig. 10A). In all but the most severe mutant [e.g., PrP(D202N)], deleting the N-terminal domain normalized the behavior in the solubility assay to near wild type levels. Even for PrP(D202N), a significant degree of normalization was observed. A predominantly cell surface localization of the fully mature species for the ΔN constructs was confirmed by both trypsin accessibility assays and indirect immunofluorescence (data not shown).


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

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

The N-terminus of PrP modulates mutant PrP metabolism.(A) The full length (FL) or N-terminally deleted (ΔN, lacking residues 23–48) constructs for wild type and mutant PrPs were analyzed by the detergent solubility assay. In each case, deletion of the N-terminus resulted in decreased insoluble forms. A corresponding increase in the fully mature soluble form is apparent in most cases. (B) Pulse chase analyses (as in Fig. 7A) of wtPrP, PrP(H187R), wtPrPΔN and PrP(H187R)ΔN were quantified by phosphorimaging. The left panel plots the appearance of fully mature species (as a proportion of total PrP) over time. The middle panel shows the time course of disappearance for immature glycosylated species (plotted as a percent of the amount present at pulse). The inclusion of 5 µM MG132, a proteasome inhibitor, had no effect on the immature species for PrP(H187R)ΔN. The right panel shows the fate of unglycosylated PrP, without or with 5 µM MG132. (C) Lysates harvested after pulse labeling with S35-methionine from cells expressing wtPrP, wtPrPΔN, PrP(H187R) and PrP(H187R)ΔN were separated into detergent soluble (S) and insoluble (P) fractions, immunoprecipitated, and analyzed by autoradiography. The percent of labeled PrP that is insoluble is indicated below the respective panels.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691595&req=5

ppat-1000479-g010: The N-terminus of PrP modulates mutant PrP metabolism.(A) The full length (FL) or N-terminally deleted (ΔN, lacking residues 23–48) constructs for wild type and mutant PrPs were analyzed by the detergent solubility assay. In each case, deletion of the N-terminus resulted in decreased insoluble forms. A corresponding increase in the fully mature soluble form is apparent in most cases. (B) Pulse chase analyses (as in Fig. 7A) of wtPrP, PrP(H187R), wtPrPΔN and PrP(H187R)ΔN were quantified by phosphorimaging. The left panel plots the appearance of fully mature species (as a proportion of total PrP) over time. The middle panel shows the time course of disappearance for immature glycosylated species (plotted as a percent of the amount present at pulse). The inclusion of 5 µM MG132, a proteasome inhibitor, had no effect on the immature species for PrP(H187R)ΔN. The right panel shows the fate of unglycosylated PrP, without or with 5 µM MG132. (C) Lysates harvested after pulse labeling with S35-methionine from cells expressing wtPrP, wtPrPΔN, PrP(H187R) and PrP(H187R)ΔN were separated into detergent soluble (S) and insoluble (P) fractions, immunoprecipitated, and analyzed by autoradiography. The percent of labeled PrP that is insoluble is indicated below the respective panels.
Mentions: To gain insight into the misfolding-selective sorting operating on mutant PrP, we sought to identify potential sorting determinant(s) by analyzing PrP deletion mutants. We found that deletion of residues 23–48 (the ΔN constructs) markedly reduced the amount of detergent-insoluble, immaturely glycosylated species of several PrP mutants relative to their full length (FL) counterparts (Fig. 10A). In most cases, this decrease was accompanied by a relative increase in the fully mature, detergent-soluble species (Fig. 10A). In all but the most severe mutant [e.g., PrP(D202N)], deleting the N-terminal domain normalized the behavior in the solubility assay to near wild type levels. Even for PrP(D202N), a significant degree of normalization was observed. A predominantly cell surface localization of the fully mature species for the ΔN constructs was confirmed by both trypsin accessibility assays and indirect immunofluorescence (data not shown).

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