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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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Limited protease digestion analysis for folding status of PrP mutants.Total detergent lysates of the indicated PrP constructs were digested with various concentrations of trypsin on ice before separation into soluble and insoluble fractions that were analyzed by immunoblots. Note that these digestion conditions are significantly milder than that used for analysis of surface exposure (e.g., in Fig. 4A and 5A), where trypsin fully digests the PrP mutants.
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ppat-1000479-g006: Limited protease digestion analysis for folding status of PrP mutants.Total detergent lysates of the indicated PrP constructs were digested with various concentrations of trypsin on ice before separation into soluble and insoluble fractions that were analyzed by immunoblots. Note that these digestion conditions are significantly milder than that used for analysis of surface exposure (e.g., in Fig. 4A and 5A), where trypsin fully digests the PrP mutants.

Mentions: Altered localization, detergent-insolubility, and incomplete glycan modification are all indirect indicators of protein misfolding. To directly assess whether the specific subpopulation of mutant PrPs that display these characteristics are indeed folded differently, we employed limited trypsin digestion (Fig. 6). In this experiment, total detergent lysates from PrP-expressing cells were treated on ice with increasing concentrations of trypsin, followed by separation into soluble and insoluble fractions. As expected, essentially all mature wild type PrP was completely in the supernatant fraction, and this population was highly sensitive to trypsin. By contrast, unglycosylated PrP (presumably a cytosolic form that is folded differently) was quantitatively in the insoluble fraction and showed notably more trypsin resistance. A small amount of immature glycosylated PrP was also seen in the insoluble fraction and displayed modest trypsin resistance. The differences in trypsin sensitivity among the different PrP forms illustrates the utility of this assay in discriminating among them.


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

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

Limited protease digestion analysis for folding status of PrP mutants.Total detergent lysates of the indicated PrP constructs were digested with various concentrations of trypsin on ice before separation into soluble and insoluble fractions that were analyzed by immunoblots. Note that these digestion conditions are significantly milder than that used for analysis of surface exposure (e.g., in Fig. 4A and 5A), where trypsin fully digests the PrP mutants.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000479-g006: Limited protease digestion analysis for folding status of PrP mutants.Total detergent lysates of the indicated PrP constructs were digested with various concentrations of trypsin on ice before separation into soluble and insoluble fractions that were analyzed by immunoblots. Note that these digestion conditions are significantly milder than that used for analysis of surface exposure (e.g., in Fig. 4A and 5A), where trypsin fully digests the PrP mutants.
Mentions: Altered localization, detergent-insolubility, and incomplete glycan modification are all indirect indicators of protein misfolding. To directly assess whether the specific subpopulation of mutant PrPs that display these characteristics are indeed folded differently, we employed limited trypsin digestion (Fig. 6). In this experiment, total detergent lysates from PrP-expressing cells were treated on ice with increasing concentrations of trypsin, followed by separation into soluble and insoluble fractions. As expected, essentially all mature wild type PrP was completely in the supernatant fraction, and this population was highly sensitive to trypsin. By contrast, unglycosylated PrP (presumably a cytosolic form that is folded differently) was quantitatively in the insoluble fraction and showed notably more trypsin resistance. A small amount of immature glycosylated PrP was also seen in the insoluble fraction and displayed modest trypsin resistance. The differences in trypsin sensitivity among the different PrP forms illustrates the utility of this assay in discriminating among them.

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