High-throughput methods to detect dimerization of Bcl-2 family proteins.
Members of the Bcl-2 family are critical regulators of apoptosis. Antiapoptotic family proteins such as Bcl-2 and Bcl-x(L) function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby preventing release of apoptotic proteins, including cytochrome c, from the mitochondria. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerizations by small-molecule inhibitors could be used to modulate cell death in both cancer (to increase apoptosis) and degenerative disorders (to decrease apoptosis), and assays are necessary to screen compound libraries. Fluorescence polarization and enzyme-linked immunosorbent assay-based methods to detect Bcl-2 protein interactions have been described. Here, two further methods that are rapid, "mix and read," homogeneous reactions, insensitive to compound autofluorescence, and amenable to high-throughput screening, are described: a scintillation proximity assay and a time-resolved fluorescence resonance energy transfer assay (HTRF). The assays are designed using tags such that different Bcl-2 family members or BH3 domain peptides can be readily applied to either format, as exemplified by the use here of histidine-tagged Bcl-x(L) and biotinylated BH3 peptides.
Eisai London Research Laboratories Ltd., Bernard Katz Building, University College London, Gower Street, WC1E 6BT, London, UK.
Jonathan
J
Whitfield
Kokichi
K
Harada
Catherine
C
Bardelle
James M
JM
Staddon
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D019921:Biotinylation; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor; D012588:Scintillation Counting / Q000379:methods*; D015534:Trans-Activators / Q000378:metabolism; D051020:bcl-X Protein
C474007:Bax protein (53-86); D002614:Chelating Agents; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D015534:Trans-Activators; D051020:bcl-X Protein; D006639:Histidine; D003300:Copper