EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency |
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Authors: | Lee E F Chen L Yang H Colman P M Huang D C S Fairlie W D |
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Institution: | 1The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. |
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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.Cell Death and Differentiation (2008) 15, 1609-1618; doi:10.1038/cdd.2008.86; published online 20 June 2008. |
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