Demonstration of thein vivointeraction of key cell death regulators by structure-based design of second-site suppressors

Abstract

Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4͞CED-9 complexes. We show here that an unusual gain-offunction mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4͞CED-9 complexes. Based on a modeled EGL-1͞CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4͞CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes. P rogrammed cell death is a tightly regulated cellular process crucial for metazoan development and homeostasis (1, 2). Improper regulation of programmed cell death can lead to a variety of diseases, including cancer and degenerative disorders (3). Genetic analysis of programmed cell death in Caenorhabditis elegans has identified four genes whose activities are essential for proper activation and execution of programmed cell death (4). Three of them promote cell death (ced-3, ced-4, and egl-1), and the fourth, ced-9, protects against cell death (5-7). Importantly, these genes encode proteins that share significant sequence and functional homology with mammalian cell death regulators. EGL-1 is similar to BH3-only pro-apoptotic proteins (7, 8), CED-3 is a member of the aspartate-specific cysteine protease family (caspases) (9, 10), CED-4 is similar to one of the apoptotic protease-activating factors (Apaf-1) (11, 12), and CED-9 is a member of a family of anti-apoptotic proteins first defined by the mammalian Bcl-2 protein (8,13,14). Some of the C. elegans proteins and their mammalian homologues have been shown to be functionally interchangeable (4), indicating that the cell death pathway is evolutionarily conserved.Bcl-2 was first identified as an inhibitor of apoptosis by virtue of its ability to protect against lymphocyte cell death (14-16). Subsequently, a family of Bcl-2-related proteins, ...

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

“…29 While we cannot eliminate the possibility that the CED-4/CED-9 complex known to be associated with the mitochondrial membrane in the worm exists in a different oligomeric form to that described here for the E. coli-derived complex, our biochemical data for different complexes, including mutants, are consistent with the features of the protein complex characterised previously. 5,6,[8][9][10]12,15 For example, we have shown that the formation of the complex is not dependent on the presence of the CARD of CED-4, consistent with the requirement that it be free to recruit CED-3 to a trimolecular complex of CED-3/CED-4/CED-9. 7 This result agrees with other studies using yeast two-hybrid assays showing a strong interaction between CED-9 and CED-4 with its CARD deleted.…”

“…Importantly, the release of the CED-4 from the complex appeared to be dependent upon a specific interaction of the EGL-1 BH3 peptide with wild-type CED-9. A single point mutation in CED-9, substituting the highly conserved glycine at position 169 with glutamate, which has been reported to effect EGL-1 binding, 8,15 completely prevented the EGL-1 BH3 domain peptide from displacing CED-4 (Figure 3d,e). However, the ability of this mutant CED-9 to bind CED-4 was unaffected (Figure 3d), which may explain why this mutant is a more potent inhibitor of cell death 4 since it can no longer be restrained by EGL-1.…”

“…In contrast, attempts to purify similar levels of CED-4 alone were hindered by the protein's insolubility and tendency to aggregate into inclusion bodies. Previously, the expression of soluble GST-fusions of CED-4 has been reported, 8,14,24 although the amounts of protein produced in these studies is unclear, while other attempts to coexpress CED-4 and CED-3 resulted only in the production of insoluble proteins. 29 While we cannot eliminate the possibility that the CED-4/CED-9 complex known to be associated with the mitochondrial membrane in the worm exists in a different oligomeric form to that described here for the E. coli-derived complex, our biochemical data for different complexes, including mutants, are consistent with the features of the protein complex characterised previously.…”

“…22 This mutation substitutes the highly conserved glycine residue at position 169 with glutamic acid and prevents EGL-1-induced release of CED-4, but preserves CED-4 binding to CED-9. 5,8,15 The CED-9/EGL-1 complex structure shows that the replacement of glycine with glutamic acid would introduce a steric clash with EGL-1, thereby negatively affecting its binding. 14 The three-dimensional structure of the mammalian homologue of CED-4, apoptotic protease-activating factor 1 (APAF-1), with WD40 domains deleted, has also recently been solved.…”

Noncanonical cell death programs in the nematode Caenorhabditis elegans

“…In animals, homozygous for a temperature-sensitive ced-9(lf) allele, the number of cell deaths observed in the germ line is nearly three times than that observed in wild-type animals. Surprisingly, however, neither egl-1(lf) mutations nor a ced-9(gf) mutation, reported to disrupt binding to EGL-1, 23,35 affects germ cell death. Thus, in the germ line, egl-1 cannot serve to integrate signals that control cell death onset.…”