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'Edgetic' perturbation of a C. elegans BCL2 ortholog.

Dreze M, Charloteaux B, Milstein S, Vidalain PO, Yildirim MA, Zhong Q, Svrzikapa N, Romero V, Laloux G, Brasseur R, Vandenhaute J, Boxem M, Cusick ME, Hill DE, Vidal M - Nat. Methods (2009)

Bottom Line: Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively.Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein.This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

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Positioning edgetic residues in CED-9 structures. (a) Positions of edgetic residues in the CED-9 sequence. The portion of CED-9 present in the crystal (PDB ID code 1OHU)19 and the α-helices observed in the corresponding structure are indicated above the sequence; BCL2 Homology (BH) domains19 are indicated under the sequence. Edgetic and non-edgetic residues are in bold font, edgetic residues are colored as indicated. (b) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left); residues mutated in edgetic alleles defective for CED-4 and/or SPD-5 interaction are shown as sticks. Helix α4 (the region undergoing EGL-1-induced conformational changes) is indicated18. Van der Waals surface of the same structure in identical orientation (right). The CED-4 binding site and the hypothetical SPD-5 binding site are shown. (c) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left) at opposite orientation with respect to (b). Residues mutated in edgetic alleles defective for SPD-5 and/or F25F8.1 interactions are shown as sticks. Van der Waals surface of the same structure in identical orientation (right). The EGL-1 binding site is shown.
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Figure 5: Positioning edgetic residues in CED-9 structures. (a) Positions of edgetic residues in the CED-9 sequence. The portion of CED-9 present in the crystal (PDB ID code 1OHU)19 and the α-helices observed in the corresponding structure are indicated above the sequence; BCL2 Homology (BH) domains19 are indicated under the sequence. Edgetic and non-edgetic residues are in bold font, edgetic residues are colored as indicated. (b) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left); residues mutated in edgetic alleles defective for CED-4 and/or SPD-5 interaction are shown as sticks. Helix α4 (the region undergoing EGL-1-induced conformational changes) is indicated18. Van der Waals surface of the same structure in identical orientation (right). The CED-4 binding site and the hypothetical SPD-5 binding site are shown. (c) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left) at opposite orientation with respect to (b). Residues mutated in edgetic alleles defective for SPD-5 and/or F25F8.1 interactions are shown as sticks. Van der Waals surface of the same structure in identical orientation (right). The EGL-1 binding site is shown.

Mentions: Since there is no obvious clustering of edgetic residues for any specific partner on the CED-9 primary sequence (Fig. 5a), suggesting that the binding sites for SPD-5, F25F8.1 and CED-4 are conformational, we used sets of edgetic residues to map the putative binding sites for SPD-5 and F25F8.1 (Fig. 5b,c, Supplementary Figs. 9 and 10; see Supplementary Data 4 for detail). Our edgetic strategy enabled the isolation of partner-specific edgetic alleles for each CED-9 partner even though the CED-9 interaction surfaces seem intricate, with partly overlapping sites.


'Edgetic' perturbation of a C. elegans BCL2 ortholog.

Dreze M, Charloteaux B, Milstein S, Vidalain PO, Yildirim MA, Zhong Q, Svrzikapa N, Romero V, Laloux G, Brasseur R, Vandenhaute J, Boxem M, Cusick ME, Hill DE, Vidal M - Nat. Methods (2009)

Positioning edgetic residues in CED-9 structures. (a) Positions of edgetic residues in the CED-9 sequence. The portion of CED-9 present in the crystal (PDB ID code 1OHU)19 and the α-helices observed in the corresponding structure are indicated above the sequence; BCL2 Homology (BH) domains19 are indicated under the sequence. Edgetic and non-edgetic residues are in bold font, edgetic residues are colored as indicated. (b) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left); residues mutated in edgetic alleles defective for CED-4 and/or SPD-5 interaction are shown as sticks. Helix α4 (the region undergoing EGL-1-induced conformational changes) is indicated18. Van der Waals surface of the same structure in identical orientation (right). The CED-4 binding site and the hypothetical SPD-5 binding site are shown. (c) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left) at opposite orientation with respect to (b). Residues mutated in edgetic alleles defective for SPD-5 and/or F25F8.1 interactions are shown as sticks. Van der Waals surface of the same structure in identical orientation (right). The EGL-1 binding site is shown.
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Related In: Results  -  Collection

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Figure 5: Positioning edgetic residues in CED-9 structures. (a) Positions of edgetic residues in the CED-9 sequence. The portion of CED-9 present in the crystal (PDB ID code 1OHU)19 and the α-helices observed in the corresponding structure are indicated above the sequence; BCL2 Homology (BH) domains19 are indicated under the sequence. Edgetic and non-edgetic residues are in bold font, edgetic residues are colored as indicated. (b) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left); residues mutated in edgetic alleles defective for CED-4 and/or SPD-5 interaction are shown as sticks. Helix α4 (the region undergoing EGL-1-induced conformational changes) is indicated18. Van der Waals surface of the same structure in identical orientation (right). The CED-4 binding site and the hypothetical SPD-5 binding site are shown. (c) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y)16 (left) at opposite orientation with respect to (b). Residues mutated in edgetic alleles defective for SPD-5 and/or F25F8.1 interactions are shown as sticks. Van der Waals surface of the same structure in identical orientation (right). The EGL-1 binding site is shown.
Mentions: Since there is no obvious clustering of edgetic residues for any specific partner on the CED-9 primary sequence (Fig. 5a), suggesting that the binding sites for SPD-5, F25F8.1 and CED-4 are conformational, we used sets of edgetic residues to map the putative binding sites for SPD-5 and F25F8.1 (Fig. 5b,c, Supplementary Figs. 9 and 10; see Supplementary Data 4 for detail). Our edgetic strategy enabled the isolation of partner-specific edgetic alleles for each CED-9 partner even though the CED-9 interaction surfaces seem intricate, with partly overlapping sites.

Bottom Line: Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively.Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein.This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

ABSTRACT
Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

Show MeSH