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Biology

Assays for the Degradation of Misfolded Proteins in Cells

Published: August 28th, 2016

DOI:

10.3791/54266

1Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, 2Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

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.

Proteins are the most abundant macromolecules in cells, and they play an essential role in virtually all biological processes. The biological activity of most proteins requires their folding into, and maintaining, the native three-dimensional structures. Proteins with aberrant conformations not only lose their normal functions, but also frequently form soluble oligomeric species or aggregates that impair the functions of other proteins and are toxic to cells1,2. To counteract protein misfolding, cells employ both molecular chaperones, which assist unfolded or partially folded polypeptides to reach their native conformation, and degradation pathways, which e....

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1. Preparation of Reagent

  1. Prepare cell lysis buffer (50 mM Tris, pH 8.8, 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40). Supplement 2 mM DTT, 1x complete protease cocktail, and 250 IU/ml benzonase before use.
  2. Prepare pellet buffer (20 mM Tris, pH 8.0, 15 mM MgCl2). Supplement 2 mM DTT, 1x complete protease cocktail and 250 IU/ml benzonase before use.
  3. Prepare 3x boiling buffer (6% SDS, 20 mM Tris, pH 8.0). Supplement 150 mM DTT before use.
  4. Prepare low-fluorescence DMEM.......

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In a steady state analysis, microscopically visible Atxn1 82Q-GFP nuclear aggregates can be observed in 30 - 50% of HeLa cells 20 hr after transfection (Figure 1A). Western blot analysis of NS and SS fractions using anti-GFP antibody shows a distinct band of Atxn1 82Q-GFP between 100 kDa and 150 kDa markers, corresponding to the protein's molecular weight (Figure 1B). Atxn1 82Q-GFP in the SR fraction can be detected either by filter retardation assay,.......

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Mechanisms that regulate the degradation of misfolded proteins are essential for maintaining the homeostasis of cellular proteins, and they likely represent valuable drug targets for treating neurodegenerative disorders and other protein-misfolding diseases. Here, assays that examine the degradation of misfolded proteins are described, using a pathogenic Atxn1 protein (Atxn1 82Q) and a nuclear localized luciferase mutant (NLS-LucDM) as examples.

To examine the degradation Atxn1 82Q, which has .......

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We thank S. Raychaudhuri for providing the destabilized firefly luciferase mutant plasmid, and A. Glavis-bloom and N. Charan for technical assistance. This work was supported, in part, by grants from NIH (CA088868, GM060911, and CA182675).

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Name Company Catalog Number Comments
Dulbecco's Modified Eagle Medium Life Technologies 11995-092
Fetal Bovine Serum Life Technologies 10082147
Lipofectamine 2000 Life Technologies 11668019
NP-40 Sigma-Aldrich NP40S-500ML
SDS Sigma-Aldrich L3771
MG132 Sigma-Aldrich M8699
Cycloheximide Sigma-Aldrich C7698
Dithiothreitol (DTT) Fisher Scientific 45000232
Complete Protease Inhibitor Cocktail Tablets Roche Boehringer Mannheim 4693159001
Bio-Dot Apparatus  Bio-Rad 1706545
Living Colors GFP Monoclonal Antibody Clonetech 632375
Anti-Actin mAb Rabbit, IgG Sigma-Aldrich A45060-200UL
Amino acids Sigma-Aldrich Amino acids are used for making low fluorecence culturing medium
Olympus IX-81  Inverted Fluorescence Microscope Olympus IX71/IX81
96 Well Black TC Plate w/ Transluscent Clear Bottom Sigma-Greiner 89135-048
Fluorescence Bottom Plate Reader Infinite 200® PRO TECAN Infinite 200® PRO
Cellulose acetate membrane 0.2 µm Sterlitech CA023001
Prism 5 GraphPad Statistical analysis software

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