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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 medium for assays using microplate fluorescence reader. Mix 25 mM glucose, 0.4 mM glycine, 0.4 mM arginine, 0.2 mM cysteine, 4.0 mM glutamine, 0.2 mM....

Representative Results

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,.......

Discussion

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 .......

Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
Dulbecco's Modified Eagle MediumLife Technologies11995-092
Fetal Bovine SerumLife Technologies10082147
Lipofectamine 2000Life Technologies11668019
NP-40Sigma-AldrichNP40S-500ML
SDSSigma-AldrichL3771
MG132Sigma-AldrichM8699
CycloheximideSigma-AldrichC7698
Dithiothreitol (DTT)Fisher Scientific45000232
Complete Protease Inhibitor Cocktail TabletsRoche Boehringer Mannheim4693159001
Bio-Dot Apparatus Bio-Rad1706545
Living Colors GFP Monoclonal AntibodyClonetech632375
Anti-Actin mAb Rabbit, IgGSigma-AldrichA45060-200UL
Amino acidsSigma-AldrichAmino acids are used for making low fluorecence culturing medium
Olympus IX-81  Inverted Fluorescence MicroscopeOlympusIX71/IX81
96 Well Black TC Plate w/ Transluscent Clear BottomSigma-Greiner89135-048
Fluorescence Bottom Plate Reader Infinite 200® PROTECANInfinite 200® PRO
Cellulose acetate membrane 0.2 µmSterlitechCA023001
Prism 5GraphPadStatistical analysis software

References

  1. Dobson, C. M. Protein folding and misfolding. Nature. 426, 884-890 (2003).
  2. Goldberg, A. L. Protein degradation and protection against misfolded or damaged proteins. Nature. 426, 895-899 (2003).
  3. ....

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Misfolded ProteinsProtein DegradationProtein Quality ControlNeurodegenerative DiseasesAtxn1 82QNuclear LuciferaseCell FractionationCycloheximideProteasome InhibitorMG132Cell LysisCentrifugation

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