A Fluorescence-Based Sensor Assay that Monitors General Protein Aggregation in Human Cells.

Marisa Pereira,Ana R Soares,Ana S Domingues,Diogo Tomé,Cristiana Paulo,Manuel A S Santos,Ana S Varanda

doi:10.1002/biot.201700676

Abstract

Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation.

Highlights

In order to identify misfolding conditions in human cells, we have developed a human specific heat shock protein 27 (HSP27):green fluorescence protein (GFP) chimeric sensor (Figure 1A)
To construct the human HSP27:GFP chimeric protein, the HSP27 coding region without its stop codon and 5’ flanking region containing the promotor were amplified as a %1900 bp product from HeLa cells genomic DNA (gDNA) preparations
Our results show that our sensor detects general protein aggregation in an easy manner and has advantages relatively to the available protein-aggregation reporters

Summary

Introduction

The accumulation of protein aggregates is a common feature of protein conformational disorders and is generally correlated with the onset of diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD) disease, and other age-related diseases (ARD).[1,2,3,4,5] Aggregation of proteins like amyloid-beta peptide (Aβ), α-synuclein (α-syn), or huntingtin (Htt) is commonly found in AD, PD, and HD, respectively.[6,7,8] Aggregates of non-amyloid proteins are Thioflavin (ThT) is a molecular probe that is commonly used to detect amyloid fibrils, in particular β-sheet-rich structures of amyloid.[15,16,17].

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Conclusion