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Direct visualization of Bcl-2 family protein interactions using live cell fluorescent protein redistribution assays
C Wong1, D J Anderson, E F Lee
1Department of Research Oncology, Genentech, Inc., South San Francisco, CA 90480, USA.
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.
Insights
Researchers developed new live-cell assays to study interactions between Bcl-2 family proteins, crucial for cancer therapy. These tools help assess drug candidates targeting cancer-driving protein interactions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Bcl-2 family proteins regulate cell death and are implicated in cancer development and treatment resistance.
- Interactions between pro-survival and pro-death Bcl-2 proteins are key therapeutic targets.
- Existing methods for studying these interactions in cells are limited.
Purpose of the Study:
- To develop novel quantitative, high-throughput microscopy assays for assessing Bcl-2 and BH3-only protein interactions in live cells.
- To provide tools for characterizing the cellular activity of therapeutic molecules targeting these interactions.
- To advance the understanding of regulatory mechanisms governing these critical protein-protein interactions.
Main Methods:
- Utilized fluorescent proteins for labeling Bcl-2 and BH3-only proteins.
- Developed live-cell microscopy assays for visualizing and quantifying mitochondrial interactions.
- Employed tool compounds to validate assay performance in drug discovery contexts.
Main Results:
- Successfully established quantitative, high-throughput live-cell microscopy assays.
- Demonstrated the assays' capability to visualize and quantify mitochondria-localized Bcl-2 and BH3-only protein interactions.
- Validated the assays' utility in assessing the cellular effects of compounds targeting these protein-protein interactions.
Conclusions:
- The developed assays address a critical need for tools to study Bcl-2 family protein interactions in live cells.
- These assays are suitable for drug discovery efforts aimed at targeting cancer-related protein-protein interactions.
- This work provides a foundation for a deeper understanding of the cellular regulation of Bcl-2 family protein interactions.
