Assays for the Degradation of Misfolded Proteins in Cells.
Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.
Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine; Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine; guolili@mail.med.upenn.edu.
Lili
L
Guo
Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine.
Wil
W
Prall
Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine; xyang@mail.med.upenn.edu.
Xiaolu
X
Yang
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