Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells

Abstract Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation i...

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Autores principales:	Rebecca San Gil, Tracey Berg, Heath Ecroyd
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c5b805513f5c4a0f924c87f420a182b2
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spelling	oai:doaj.org-article:c5b805513f5c4a0f924c87f420a182b22021-12-02T12:31:46ZUsing bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells10.1038/s41598-017-02459-92045-2322https://doaj.org/article/c5b805513f5c4a0f924c87f420a182b22017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02459-9https://doaj.org/toc/2045-2322Abstract Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25–46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.Rebecca San GilTracey BergHeath EcroydNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rebecca San Gil Tracey Berg Heath Ecroyd Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
description	Abstract Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25–46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.
format	article
author	Rebecca San Gil Tracey Berg Heath Ecroyd
author_facet	Rebecca San Gil Tracey Berg Heath Ecroyd
author_sort	Rebecca San Gil
title	Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
title_short	Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
title_full	Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
title_fullStr	Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
title_full_unstemmed	Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
title_sort	using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/c5b805513f5c4a0f924c87f420a182b2
work_keys_str_mv	AT rebeccasangil usingbicistronicconstructstoevaluatethechaperoneactivitiesofheatshockproteinsincells AT traceyberg usingbicistronicconstructstoevaluatethechaperoneactivitiesofheatshockproteinsincells AT heathecroyd usingbicistronicconstructstoevaluatethechaperoneactivitiesofheatshockproteinsincells
_version_	1718394304594968576

Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells

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