Impairment of the Ubiquitin-Proteasome System by Protein Aggregation
Tóm tắt
Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.
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Tài liệu tham khảo
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Cells were labeled with 35 S-Met and 35 S-Cys for 10 min chased and lysed by sonication in phosphate-buffered saline containing 1% Triton X-100. Lysates were immunoprecipitated with antibody to GFP (anti-GFP) and resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). For immunoblotting cells were lysed in 10 mM tris (pH 7.4) 5 mM EDTA with 1% Triton X-100.
Clasto-lactacystin β-lactone the active form of lactacystin was used in this study.
For GFP u -1 characterization 10 000 to 15 000 cells were analyzed on a flow cytometer.
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For analysis of ubiquitin conjugates ∼200 000 cells collected by fluorescence-activated cell sorting (FACS) from the top 10% and lowest 10% of cells expressing Q25-GFP and Q103-GFP were lysed in SDS sample buffer and resolved by SDS-PAGE.
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For cell cycle analyses HEK 293 cells were fixed in 4% paraformaldehyde 72 hours after transfection and treated with 5 μg/ml ribonuclease A for 2 hours at room temperature stained with 10 μg/ml propidium iodide and analyzed for DNA and GFP content by flow cytometry.
Overexpression of GFP u itself a short-lived protein does not cause cell cycle arrest indicating that the observed increase is not due simply to the presence of an abundant UPS substrate.
We thank R. Frizzell for FLAG-CFTR A. Tobin for huntingtin plasmids M. Bucci for the GFP cell line M. Rexach for anti-GFP and C. Carswell-Crumpton for assistance with flow cytometry. We also thank R. Rajan and other members of the Kopito laboratory for stimulating discussions. Supported in part by a research grant from the National Institutes of Health a Howard Hughes Summer Fellowship from the Department of Biological Sciences at Stanford University and an NIH predoctoral training grant.