Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases.
The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.
Holinger
E P
EP
Apoptosis Technology, Inc., Cambridge, Massachusetts 02139, USA.
Chittenden
T
T
Lutz
R J
RJ
D051943:Antennapedia Homeodomain Protein; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000494:pharmacology*; D008928:Mitochondria / Q000378:metabolism; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D014157:Transcription Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein
D051943:Antennapedia Homeodomain Protein; C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; D020169:Caspases