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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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Related in: MedlinePlus

All globular domain PrP mutants display altered localization.Cells expressing wtPrP and the indicated mutants were stained, imaged and analyzed as in Fig. 2. The surface∶intracellular ratio of PrP is plotted for each of the mutants for comparison with wtPrP. Data points represent individual cells from a single representative experiment. All mutants were analyzed together on the same day. Horizontal black bars indicate the mean values for each data set. Each mutant dataset was compared to wtPrP by the Student's t-test and found to be statistically significant in all cases (p<10−8), except PrP(A117V).
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ppat-1000479-g003: All globular domain PrP mutants display altered localization.Cells expressing wtPrP and the indicated mutants were stained, imaged and analyzed as in Fig. 2. The surface∶intracellular ratio of PrP is plotted for each of the mutants for comparison with wtPrP. Data points represent individual cells from a single representative experiment. All mutants were analyzed together on the same day. Horizontal black bars indicate the mean values for each data set. Each mutant dataset was compared to wtPrP by the Student's t-test and found to be statistically significant in all cases (p<10−8), except PrP(A117V).

Mentions: It is noteworthy that the absolute proportion of intracellular PrP changed among different experiments and appeared to be influenced by culture conditions (e.g., batches of serum, age of cells, relative confluence, and time after transfection). Despite this variability, the relative differences among the mutants remained reproducibly consistent when sufficient numbers of cells were analyzed. Shown in Fig. 3 is the tabulated data from a single experiment comparing wtPrP with seven mutants, showing consistently decreased surface-to-intracellular ratio among each of the globular domain mutants, but not PrP(A117V). The wide heterogeneity among cells, the effect of different culture conditions, the expression level-dependence of localization patterns, and the substantial overlap in distribution between wtPrP and the mutants are likely to explain the previously diverse (and sometimes conflicting) results regarding the effects of PrP mutants. Indeed, many of the previous localization patterns (ER, aggregates, Golgi, etc.) were readily observed in our experiments in subpopulations of cells, emphasizing the importance of more quantitative population studies in identifying overall systematic differences.


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

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

All globular domain PrP mutants display altered localization.Cells expressing wtPrP and the indicated mutants were stained, imaged and analyzed as in Fig. 2. The surface∶intracellular ratio of PrP is plotted for each of the mutants for comparison with wtPrP. Data points represent individual cells from a single representative experiment. All mutants were analyzed together on the same day. Horizontal black bars indicate the mean values for each data set. Each mutant dataset was compared to wtPrP by the Student's t-test and found to be statistically significant in all cases (p<10−8), except PrP(A117V).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000479-g003: All globular domain PrP mutants display altered localization.Cells expressing wtPrP and the indicated mutants were stained, imaged and analyzed as in Fig. 2. The surface∶intracellular ratio of PrP is plotted for each of the mutants for comparison with wtPrP. Data points represent individual cells from a single representative experiment. All mutants were analyzed together on the same day. Horizontal black bars indicate the mean values for each data set. Each mutant dataset was compared to wtPrP by the Student's t-test and found to be statistically significant in all cases (p<10−8), except PrP(A117V).
Mentions: It is noteworthy that the absolute proportion of intracellular PrP changed among different experiments and appeared to be influenced by culture conditions (e.g., batches of serum, age of cells, relative confluence, and time after transfection). Despite this variability, the relative differences among the mutants remained reproducibly consistent when sufficient numbers of cells were analyzed. Shown in Fig. 3 is the tabulated data from a single experiment comparing wtPrP with seven mutants, showing consistently decreased surface-to-intracellular ratio among each of the globular domain mutants, but not PrP(A117V). The wide heterogeneity among cells, the effect of different culture conditions, the expression level-dependence of localization patterns, and the substantial overlap in distribution between wtPrP and the mutants are likely to explain the previously diverse (and sometimes conflicting) results regarding the effects of PrP mutants. Indeed, many of the previous localization patterns (ER, aggregates, Golgi, etc.) were readily observed in our experiments in subpopulations of cells, emphasizing the importance of more quantitative population studies in identifying overall systematic differences.

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