The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2.
Since the over-expression of Bcl-2 is a common cause of multi-drug resistance, cytotoxic peptides that overcome the effects of Bcl-2 may be clinically useful. We harnessed the death-promoting alpha helical properties of the BH3 domain of BAD by fusing it to the Antennapedia (ANT) domain, which allows for cell entry (ANTBH3BAD). Treatment of 32D cells with the ANTBH3BAD peptide results in a 99% inhibition of colony formation. No significant toxicity is observed after treatment with ANT or BH3BAD alone. A mutant fusion peptide unable to bind Bcl-2 induces cell death as effectively as the wild-type ANTBH3BAD. Furthermore, 32D cells over-expressing Bcl-2 show no resistance to the ANTBH3BAD peptide. Therefore, the toxicity of the peptide was independent of the Bcl-2 pathway. We demonstrate that the toxicity of the peptide is due to its alpha helicity that disrupts mitochondrial function. Since this peptide overcomes major forms of drug resistance, it may be therapeutically useful if appropriately targeted to malignant cells.
Schimmer
A D
AD
University Health Network, Princess Margaret Hospital, 610 University Ave., Toronto, Ontario M5G 2M9, Canada.
Hedley
D W
DW
Chow
S
S
Pham
N A
NA
Chakrabartty
A
A
Bouchard
D
D
Mak
T W
TW
Trus
M R
MR
Minden
M D
MD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D017209:Apoptosis; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D005260:Female; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014157:Transcription Factors; D051025:bcl-Associated Death Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D051943:Antennapedia Homeodomain Protein; C494819:BAD protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases