Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis Regulatory Proteins / genetics
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Apoptosis Regulatory Proteins / metabolism
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Bcl-2-Like Protein 11
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Caenorhabditis elegans / genetics
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Caenorhabditis elegans / metabolism*
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Caenorhabditis elegans Proteins / genetics
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Caenorhabditis elegans Proteins / metabolism*
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Cell Death / physiology*
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Cell Survival
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Cells, Cultured
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Fibroblasts / cytology
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Fibroblasts / physiology
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Humans
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mice
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Mice, Knockout
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Myeloid Cell Leukemia Sequence 1 Protein
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / metabolism*
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Signal Transduction / physiology*
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bcl-X Protein / genetics
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bcl-X Protein / metabolism*
Substances
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Apoptosis Regulatory Proteins
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BCL2L11 protein, human
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Bcl-2-Like Protein 11
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Bcl2l11 protein, mouse
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Caenorhabditis elegans Proteins
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EGL-1 protein, C elegans
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Mcl1 protein, mouse
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Membrane Proteins
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Myeloid Cell Leukemia Sequence 1 Protein
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Pmaip1 protein, mouse
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Recombinant Fusion Proteins
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Repressor Proteins
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bcl-X Protein