Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Summary

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-β peptide.

Abstract

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-β peptide variants with their respective intrinsic aggregation propensities.

Introduction

Proteostasis is a fundamental determinant of cell fitness and aging processes. In cells, protein homeostasis is maintained by sophisticated protein quality control networks aimed to ensure the correct refolding of misfolded protein conformers by chaperones and/or their targeted proteolysis with several well-conserved mechanisms^1,2,3,4,5. A large number of studies provide support to the link between the onset and progression of a broad range of human diseases and the failure of proteostasis, leading to prote....

Protocol

1. S. cerevisiae Cultures and Protein Expression

Note: Aβ variants exhibit different relative aggregation propensities due to a mutation in a single residue at position 19 (Phe19) of the Aβ42 peptide (Figure 1A). These peptide variants are tagged with green fluorescent protein (GFP), which acts as an aggregation reporter (Figure 1)²⁹.

1. Transform plasmids encoding for 20 Aβ42-GFP variants into yeast cells with a BY4741 parental background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0)³⁰. Ea....

Results

This protocol describes how to employ a collection of 20 variants of the Aβ42 peptide where Phe19 has been mutated to all natural proteinogenic amino acids²⁸. The theoretical aggregation propensities of these proteins can be analyzed using two different bioinformatic algorithms (AGGRESCAN and TANGO^31,32). In both cases, this analysis renders a progressive gradation of aggregation tendencies, ascribing, as a.......

Discussion

A wide range of diseases is linked to the accumulation of misfolded proteins into cellular deposits^6,7,8,33. Many efforts have been made to unravel the molecular mechanisms that trigger the onset of these diseases using computational approaches, which do not take into account protein concentrations, or in vitro approaches, in which the protein concentration remains constant during the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yeast cells BY4741	ATCC	201388	Genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
pESC(-Ura) plasmid	Agilent Genomics	217454	Yeast expression plasmid with a Gal promotor. Selectable marker URA3
Yeast Synthetic Drop-out Medium Supplements	Sigma	Y1501	Powder
Yeast Nitrogen Base Without Amino Acids	Sigma	Y0626	Powder
Raffinose	Sigma	R7630	Powder
Glucose	Sigma	G7021	Powder
Galactose	Sigma	G0750	Powder
Phosphate Buffered Saline (PBS)	Fisher Scientific	BP3991	Solution 10X
CellROX Deep Red Reagent	Life Technologies	C10422	Free radical cell-permeant fluorescent sensor, non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS), with absorption/emission maxima at 644/665 nm.
Y-PER protein extraction reagent	Thermo Scientific	78990	Liquid cell lysis buffer
Acrylamide/Bis-acrylamide	Sigma	A6050	Solution
Bradford dye reagent	Bio-Rad	5000205	Dye reagent for one-step determination of protein concentration
β-amyloid antibody 6E10	BioLegend	803001	Mouse IgG1. The epitope lies within amino acids 3-8 of beta amyloid (EFRHDS).
Goat anti-mouse IgG-HRP conjugate	Bio-Rad	1721011
Membrane Immobilon-P, PVDF	Millipore	IPVH00010
Luminata forte	Merk	WBLUF0100	Premixed, ready to use chemiluminescent HRP detection reagent
Phenylmethanesulfonyl fluoride solution (PMSF)	Sigma	93482	Protease inhibitor. Dissolved at 0.1 M in ethanol
FACSCanto flow cytometer	BD Biosciences	657338	Equipped with a 488 nm blue laser for the detection of GFP, and 635 nm red laser / 530/30 nm BP filter and 660/20 BP filter
Mini Trans-Blot Electrophoresis Transfer cell	Bio-Rad	1703930	Protein transference system
Mini-PROTEAN Tetra Handcast Systems	Bio-Rad	1658000FC	Electrophoresis system