Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET.
Increased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initiation, progression and resistance to traditional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to chemotherapy. Recently, we applied fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pattern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the corresponding BimEL mutant showed no decrease in binding to these anti-apoptotic proteins. Therefore, in MCF-7 cells, Bim has unique binding properties compared with other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics.
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
Qian
Q
Liu
Brian
B
Leber
David W
DW
Andrews
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D001713:Biphenyl Compounds / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009596:Nitrophenols / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D051025:bcl-Associated Death Protein / Q000737:chemistry / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism; D000097573:Red Fluorescent Protein
D000814:Aniline Compounds; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D000072224:Bcl-2-Like Protein 11; D001713:Biphenyl Compounds; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009596:Nitrophenols; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D000097573:Red Fluorescent Protein; C501332:ABT-737; C000606775:BCL2L11 protein, human; C528561:navitoclax