title: Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays.
abstract: 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.
authors.affiliation: Department of Research Oncology, Genentech, Inc., South San Francisco, CA 90480, USA.
authors.forename: C
authors.initials: C
authors.lastname: Wong
authors.forename: D J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: E F
authors.initials: EF
authors.lastname: Lee
authors.forename: W D
authors.initials: WD
authors.lastname: Fairlie
authors.forename: M J C
authors.initials: MJ
authors.lastname: Ludlam
mesh_terms: D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D025941:Protein Interaction Mapping; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins