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Panorama of ancient metazoan macromolecular complexes

View Article: PubMed Central - PubMed

ABSTRACT

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we then generated a draft conservation map consisting of >1 million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering revealed a spectrum of conservation, ranging from ancient Eukaryal assemblies likely serving cellular housekeeping roles for at least 1 billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, by affinity-purification and by functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic significance and adaptive value to animal cell systems.

No MeSH data available.


Functional validation of complexesa, Morpholino knockdown of COMMD2 (n = 55 animals, 2 clutches, 1 eye each) or COMMD3 (n = 64) in X. laevis embryos causes defective head and eye development (control n = 57; Extended Data Fig. 9f, h). ***, p < 0.0001, 2-sided Mann-Whitney test. b, COMMD2/3 knockdown animals (5 embryos per treatment examined) show altered neural patterning, including posterior shift or loss of expression of mid-brain marker EN2 and KROX20(EGR1), the latter in rhombomeres R3/R5 (compare to Extended Data Fig. 9g, h). c, Enhanced embryonic lethality (i.e., epistasis) following RNAi knockdown in C. elegans of B0035.1 (ZNF207) and bub-3 together (eggs laid: HT115, 1308; B0035.1, 1096; bub-3, 445; bub-3 + B0035.1, 341). d, Enhanced sensitivity (mean +/− s.d. across four cell culture experiments) of two independent CCDC97-knockout lines to the SF3b inhibitor pladienolide B (PB) relative to control HEK293 cells. e, Enrichment (permutation test p-value) for interactions among sequential pathway components and metabolic enzymes relative to shuffled controls (n refers to enzyme index, where n,n+1 denotes sequential enzymes, n,n+2 sequential-but-one, etc, as described in SI (“Analysis of consecutively acting signal transduction and metabolic enzyme interactions”). f, Metabolic channeling as opposed to traditional (typical) two-step cascade model. g, Conserved interactions among consecutively acting enzymes involved in purine biosynthesis (2 representative co-fractionation profiles of the 69 total generated are shown).
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Figure 5: Functional validation of complexesa, Morpholino knockdown of COMMD2 (n = 55 animals, 2 clutches, 1 eye each) or COMMD3 (n = 64) in X. laevis embryos causes defective head and eye development (control n = 57; Extended Data Fig. 9f, h). ***, p < 0.0001, 2-sided Mann-Whitney test. b, COMMD2/3 knockdown animals (5 embryos per treatment examined) show altered neural patterning, including posterior shift or loss of expression of mid-brain marker EN2 and KROX20(EGR1), the latter in rhombomeres R3/R5 (compare to Extended Data Fig. 9g, h). c, Enhanced embryonic lethality (i.e., epistasis) following RNAi knockdown in C. elegans of B0035.1 (ZNF207) and bub-3 together (eggs laid: HT115, 1308; B0035.1, 1096; bub-3, 445; bub-3 + B0035.1, 341). d, Enhanced sensitivity (mean +/− s.d. across four cell culture experiments) of two independent CCDC97-knockout lines to the SF3b inhibitor pladienolide B (PB) relative to control HEK293 cells. e, Enrichment (permutation test p-value) for interactions among sequential pathway components and metabolic enzymes relative to shuffled controls (n refers to enzyme index, where n,n+1 denotes sequential enzymes, n,n+2 sequential-but-one, etc, as described in SI (“Analysis of consecutively acting signal transduction and metabolic enzyme interactions”). f, Metabolic channeling as opposed to traditional (typical) two-step cascade model. g, Conserved interactions among consecutively acting enzymes involved in purine biosynthesis (2 representative co-fractionation profiles of the 69 total generated are shown).

Mentions: We used multiple approaches to assess the accuracy (Fig. 4) and functional significance (Fig. 5) of the predicted complexes. First, we performed affinity purification-mass spectrometry (AP/MS) experiments on select novel complexes from the ‘new’, ‘old’ and ‘mixed’ age clusters, validating most associations in both worm and human (Fig. 4a, Extended Data Fig. 6a). We next performed a global validation by comparing our derived complexes to a newly reported large-scale AP/MS study of 23,756 putative human protein interactions detected in cell culture (BioGrid pre-publication 166968, Huttlin EL et al., downloaded Feb. 10, 2015), and observed a partial, but exceptionally significant, overlap to a degree comparable to literature-derived complexes (Fig. 4b, Extended Data Fig. 6b).


Panorama of ancient metazoan macromolecular complexes
Functional validation of complexesa, Morpholino knockdown of COMMD2 (n = 55 animals, 2 clutches, 1 eye each) or COMMD3 (n = 64) in X. laevis embryos causes defective head and eye development (control n = 57; Extended Data Fig. 9f, h). ***, p < 0.0001, 2-sided Mann-Whitney test. b, COMMD2/3 knockdown animals (5 embryos per treatment examined) show altered neural patterning, including posterior shift or loss of expression of mid-brain marker EN2 and KROX20(EGR1), the latter in rhombomeres R3/R5 (compare to Extended Data Fig. 9g, h). c, Enhanced embryonic lethality (i.e., epistasis) following RNAi knockdown in C. elegans of B0035.1 (ZNF207) and bub-3 together (eggs laid: HT115, 1308; B0035.1, 1096; bub-3, 445; bub-3 + B0035.1, 341). d, Enhanced sensitivity (mean +/− s.d. across four cell culture experiments) of two independent CCDC97-knockout lines to the SF3b inhibitor pladienolide B (PB) relative to control HEK293 cells. e, Enrichment (permutation test p-value) for interactions among sequential pathway components and metabolic enzymes relative to shuffled controls (n refers to enzyme index, where n,n+1 denotes sequential enzymes, n,n+2 sequential-but-one, etc, as described in SI (“Analysis of consecutively acting signal transduction and metabolic enzyme interactions”). f, Metabolic channeling as opposed to traditional (typical) two-step cascade model. g, Conserved interactions among consecutively acting enzymes involved in purine biosynthesis (2 representative co-fractionation profiles of the 69 total generated are shown).
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Figure 5: Functional validation of complexesa, Morpholino knockdown of COMMD2 (n = 55 animals, 2 clutches, 1 eye each) or COMMD3 (n = 64) in X. laevis embryos causes defective head and eye development (control n = 57; Extended Data Fig. 9f, h). ***, p < 0.0001, 2-sided Mann-Whitney test. b, COMMD2/3 knockdown animals (5 embryos per treatment examined) show altered neural patterning, including posterior shift or loss of expression of mid-brain marker EN2 and KROX20(EGR1), the latter in rhombomeres R3/R5 (compare to Extended Data Fig. 9g, h). c, Enhanced embryonic lethality (i.e., epistasis) following RNAi knockdown in C. elegans of B0035.1 (ZNF207) and bub-3 together (eggs laid: HT115, 1308; B0035.1, 1096; bub-3, 445; bub-3 + B0035.1, 341). d, Enhanced sensitivity (mean +/− s.d. across four cell culture experiments) of two independent CCDC97-knockout lines to the SF3b inhibitor pladienolide B (PB) relative to control HEK293 cells. e, Enrichment (permutation test p-value) for interactions among sequential pathway components and metabolic enzymes relative to shuffled controls (n refers to enzyme index, where n,n+1 denotes sequential enzymes, n,n+2 sequential-but-one, etc, as described in SI (“Analysis of consecutively acting signal transduction and metabolic enzyme interactions”). f, Metabolic channeling as opposed to traditional (typical) two-step cascade model. g, Conserved interactions among consecutively acting enzymes involved in purine biosynthesis (2 representative co-fractionation profiles of the 69 total generated are shown).
Mentions: We used multiple approaches to assess the accuracy (Fig. 4) and functional significance (Fig. 5) of the predicted complexes. First, we performed affinity purification-mass spectrometry (AP/MS) experiments on select novel complexes from the ‘new’, ‘old’ and ‘mixed’ age clusters, validating most associations in both worm and human (Fig. 4a, Extended Data Fig. 6a). We next performed a global validation by comparing our derived complexes to a newly reported large-scale AP/MS study of 23,756 putative human protein interactions detected in cell culture (BioGrid pre-publication 166968, Huttlin EL et al., downloaded Feb. 10, 2015), and observed a partial, but exceptionally significant, overlap to a degree comparable to literature-derived complexes (Fig. 4b, Extended Data Fig. 6b).

View Article: PubMed Central - PubMed

ABSTRACT

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we then generated a draft conservation map consisting of &gt;1 million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering revealed a spectrum of conservation, ranging from ancient Eukaryal assemblies likely serving cellular housekeeping roles for at least 1 billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, by affinity-purification and by functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic significance and adaptive value to animal cell systems.

No MeSH data available.