EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.
Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
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Lee
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Chen
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Yang
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Colman
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Huang
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Fairlie
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D029742:Caenorhabditis elegans Proteins; C112486:EGL-1 protein, C elegans; C579094:Mcl1 protein, mouse; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; C408009:Pmaip1 protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D051020:bcl-X Protein