Assays for the Degradation of Misfolded Proteins in Cells

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 cells^1,2. 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....

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 .......

Materials

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