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Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays

Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein–protein interactions attractive therapeutic targets. Although these interaction...

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Detalles Bibliográficos
Autores principales: Wong, C, Anderson, D J, Lee, E F, Fairlie, W D, Ludlam, M J C
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424096/
https://www.ncbi.nlm.nih.gov/pubmed/22460384
http://dx.doi.org/10.1038/cddis.2012.28
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author Wong, C
Anderson, D J
Lee, E F
Fairlie, W D
Ludlam, M J C
author_facet Wong, C
Anderson, D J
Lee, E F
Fairlie, W D
Ludlam, M J C
author_sort Wong, C
collection PubMed
description Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein–protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein–protein interactions within the cell.
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spelling pubmed-54240962017-05-19 Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays Wong, C Anderson, D J Lee, E F Fairlie, W D Ludlam, M J C Cell Death Dis Original Article Bcl-2 family proteins have important roles in tumor initiation, progression and resistance to therapy. Pro-survival Bcl-2 proteins are regulated by their interactions with pro-death BH3-only proteins making these protein–protein interactions attractive therapeutic targets. Although these interactions have been extensively characterized biochemically, there is a paucity of tools to assess these interactions in cells. Here, we address this limitation by developing quantitative, high throughput microscopy assays to characterize Bcl-2 and BH3-only protein interactions in live cells. We use fluorescent proteins to label the interacting proteins of interest, enabling visualization and quantification of their mitochondria-localized interactions. Using tool compounds, we demonstrate the suitability of our assays to characterize the cellular activity of putative therapeutic molecules that target the interaction between pro-survival Bcl-2 and pro-death BH3-only proteins. In addition to the relevance of our assays for drug discovery, we anticipate that our work will contribute to an improved understanding of the mechanisms that regulate these important protein–protein interactions within the cell. Nature Publishing Group 2012-03 2012-03-29 /pmc/articles/PMC5424096/ /pubmed/22460384 http://dx.doi.org/10.1038/cddis.2012.28 Text en Copyright © 2012 Macmillan Publishers Limited http://creativecommons.org/licenses/by-nc-nd/3.0/ This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Original Article
Wong, C
Anderson, D J
Lee, E F
Fairlie, W D
Ludlam, M J C
Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title_full Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title_fullStr Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title_full_unstemmed Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title_short Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
title_sort direct visualization of bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424096/
https://www.ncbi.nlm.nih.gov/pubmed/22460384
http://dx.doi.org/10.1038/cddis.2012.28
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