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EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.
- Source :
-
Cell death and differentiation [Cell Death Differ] 2008 Oct; Vol. 15 (10), pp. 1609-18. Date of Electronic Publication: 2008 Jun 20. - Publication Year :
- 2008
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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.
- Subjects :
- Animals
Apoptosis Regulatory Proteins genetics
Apoptosis Regulatory Proteins metabolism
Bcl-2-Like Protein 11
Caenorhabditis elegans genetics
Caenorhabditis elegans Proteins genetics
Cell Survival
Cells, Cultured
Fibroblasts cytology
Fibroblasts physiology
Humans
Membrane Proteins genetics
Membrane Proteins metabolism
Mice
Mice, Knockout
Myeloid Cell Leukemia Sequence 1 Protein
Proto-Oncogene Proteins genetics
Proto-Oncogene Proteins metabolism
Proto-Oncogene Proteins c-bcl-2 genetics
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Repressor Proteins genetics
bcl-X Protein genetics
Caenorhabditis elegans metabolism
Caenorhabditis elegans Proteins metabolism
Cell Death physiology
Proto-Oncogene Proteins c-bcl-2 metabolism
Repressor Proteins metabolism
Signal Transduction physiology
bcl-X Protein metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1350-9047
- Volume :
- 15
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- Cell death and differentiation
- Publication Type :
- Academic Journal
- Accession number :
- 18566606
- Full Text :
- https://doi.org/10.1038/cdd.2008.86