The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function.
The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold - Stefin A quadruple mutant - to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-apoptotic effector protein observed in this study.
1] Section of Experimental Therapeutics, Leeds LS9 7TF, UK [2] Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
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Stadler
Section of Experimental Oncology, Leeds Institute of Molecular Medicine, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK.
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DC
Tomlinson
Section of Experimental Therapeutics, Leeds LS9 7TF, UK.
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Lee
Section of Experimental Oncology, Leeds Institute of Molecular Medicine, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK.
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MA
Knowles
Section of Experimental Therapeutics, Leeds LS9 7TF, UK.
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Ko Ferrigno
D017209:Apoptosis; D052158:Aptamers, Peptide / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052156:SELEX Aptamer Technique
D052158:Aptamers, Peptide; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2