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|Title:||A fluorescence based sensor assay that monitors general protein aggregation in human cells|
Domingues, Ana S.
Varanda, Ana S.
Santos, Manuel A. S.
Soares, Ana R.
|Keywords:||Fluorescence sensor assay|
GFP Green Fluorescent Proteins
HSP27 Heat-Shock Proteins
|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.|
|Appears in Collections:||IBIMED - Artigos|
DCM - Artigos
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