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Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates
Published on: December 6, 2017
Bcl-2 family interactome analysis using bacterial surface display
1Department of Chemical and Biological Engineering, A207 Engineering Quadrangle, Princeton University, Princeton, NJ 08544, USA.
Abstract:
Members of the Bcl-2 family of proteins have opposing roles in programmed cell death; family members can play either pro-apoptotic or anti-apoptotic roles. Heterodimeric interactions between pro-apoptotic and anti-apoptotic members of the Bcl-2 family are critical for the regulation of apoptosis and are important targets for cancer therapeutics. Bcl-2 family interactions are mediated by the highly-conserved BH3 domain, corresponding to a single amphipathic α-helix, which binds in a hydrophobic cleft of its Bcl-2 family interaction partner. Here, using a high-throughput peptide-protein interaction assay based on bacterial cell surface display and flow cytometry, we present quantitative data for a near-complete set of 17 BH3 domains from the human genome binding to each of the 5 anti-apoptotic Bcl-2 family members. Biophysical insights into the affinity and specificity of these interactions are provided by analysis of the interactome data. In addition we carried out a truncation study of the Bim BH3 domain to define the core residues responsible for anti-apoptotic protein binding. The interactome data from this study has implications both in basic research on apoptosis and in the design of peptidic cancer therapeutics.
Insights
This study quantifies interactions between BH3 domains and anti-apoptotic Bcl-2 proteins, revealing insights into programmed cell death regulation. This data aids in developing new cancer therapeutics targeting apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Bcl-2 protein family regulates programmed cell death (apoptosis) through pro-apoptotic and anti-apoptotic members.
- Interactions between these family members are crucial for apoptosis control and represent therapeutic targets in cancer.
- Bcl-2 family interactions are mediated by the conserved BH3 domain, an alpha-helix that binds to a hydrophobic cleft.
Purpose of the Study:
- To quantitatively map the binding affinities and specificities of 17 human BH3 domains to 5 anti-apoptotic Bcl-2 proteins.
- To provide biophysical insights into these critical protein-protein interactions.
- To identify the core residues of the Bim BH3 domain essential for binding to anti-apoptotic partners.
Main Methods:
- A high-throughput peptide-protein interaction assay utilizing bacterial cell surface display and flow cytometry.
- Quantitative analysis of binding data to generate an interactome map.
- Truncation studies of the Bim BH3 domain to pinpoint key binding residues.
Main Results:
- Generated quantitative binding data for a near-complete set of 17 human BH3 domains against 5 anti-apoptotic Bcl-2 family members.
- Provided biophysical insights into the affinity and specificity landscape of these crucial interactions.
- Defined the minimal sequence of the Bim BH3 domain required for interaction with anti-apoptotic proteins.
Conclusions:
- The comprehensive interactome data offers a valuable resource for understanding apoptosis regulation.
- Findings have direct implications for the rational design of novel peptidic therapeutics targeting the Bcl-2 family in cancer.
- This work advances both fundamental knowledge of apoptosis and the development of targeted cancer therapies.
