Abstract
Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis*
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BH3 Interacting Domain Death Agonist Protein
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COS Cells
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism
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Caspase 8
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Caspase 9
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Caspases / metabolism
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Cytochrome c Group / metabolism
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DNA, Complementary / metabolism
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HeLa Cells
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Humans
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Immunoblotting
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Jurkat Cells
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Membrane Proteins / metabolism*
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Mice
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Microscopy, Confocal
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Microscopy, Fluorescence
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Minor Histocompatibility Antigens
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Mitochondria / metabolism
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Mitochondria, Liver / metabolism
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Plasmids / metabolism
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Precipitin Tests
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Protein Binding
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Protein Structure, Tertiary
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Proto-Oncogene Proteins c-bcl-2 / physiology*
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Receptors, TNF-Related Apoptosis-Inducing Ligand
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Receptors, Tumor Necrosis Factor / metabolism
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Recombinant Proteins / metabolism
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Time Factors
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Two-Hybrid System Techniques
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
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fas Receptor / biosynthesis
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fas Receptor / metabolism
Substances
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BAK1 protein, human
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BAX protein, human
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BCL2-related protein A1
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BH3 Interacting Domain Death Agonist Protein
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BID protein, human
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Bak1 protein, mouse
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Bax protein, mouse
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Bid protein, mouse
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Carrier Proteins
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Cytochrome c Group
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DNA, Complementary
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Membrane Proteins
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Minor Histocompatibility Antigens
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Receptors, TNF-Related Apoptosis-Inducing Ligand
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Receptors, Tumor Necrosis Factor
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Recombinant Proteins
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TNFRSF10A protein, human
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
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fas Receptor
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CASP8 protein, human
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CASP9 protein, human
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Casp8 protein, mouse
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Casp9 protein, mouse
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Caspase 8
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Caspase 9
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Caspases