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Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.

Rothé B, Leal-Esteban L, Bernet F, Urfer S, Doerr N, Weimbs T, Iwaszkiewicz J, Constam DB - Mol. Cell. Biol. (2015)

Bottom Line: In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway.Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects.We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.

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Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A. Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.
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Figure 4: Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A. Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.

Mentions: To test the predictive power of our structural model of dimeric Bicc1 SAM, we individually replaced six surface-exposed patches of electrostatic amino acids within or outside the SAM-SAM interface by alanines (Fig. 4A). Four of these groups of mutations (those in mutB, mutC, mutD, and mutE) should affect intermolecular H bonds, while two others (those in mutA and mutF) were deliberately introduced outside the predicted dimerization interface (Fig. 4B to D). To screen for polymerization defects, we transfected HA-tagged Bicc1 mutants A to F into HEK293T cells and assessed their retention on glutathione-Sepharose beads coated with recombinant GST-SAM fusion protein as a bait. We found that wild-type HA-Bicc1 efficiently bound to GST-SAM-coated beads but not to beads coated with GST alone (Fig. 4E). In contrast, the Bicc1 mutant lacking SAM (HA-Bicc1ΔSAM) failed to bind GST-SAM beads, suggesting that GST-SAM specifically interacts with the SAM domain of full-length Bicc1. Moreover, a comparison with HA-Bicc1 mutants showed that while the mutations in mutants C, D, and E abolished the SAM-SAM interaction, the mutations in mutants A and F did not, a result that concurs with that of our structure model. The mutation in mutant B exhibited an intermediate effect. The mutation in mutant B affected three amino acids at the periphery of the EH surface, and only one of them (K891) is predicted to contribute to the SAM-SAM interaction (Fig. 3C). According to theoretical predictions, this electrostatic patch is involved in only 1.1 intermolecular H bonds, on average, explaining why its mutation is insufficient to fully disrupt dimerization (Fig. 4D). Altogether, these results strongly corroborate our structure model and provide new tools to specifically probe the consequences of the loss of SAM-SAM interactions in vivo.


Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.

Rothé B, Leal-Esteban L, Bernet F, Urfer S, Doerr N, Weimbs T, Iwaszkiewicz J, Constam DB - Mol. Cell. Biol. (2015)

Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A. Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Screen for Bicc1 SAM polymer mutants. (A) Bicc1 SAM mutant collection. Shading is as for Fig, 3A. Individual electrostatic patches (patches A to F) at the protein surface were replaced by alanines. (B, C) Positions of mutations in the Bicc1 SAM dimer model (B) and on the ML surface and EH surface (C). (D) Table summarizing, for each amino acid patch, the average number of H bonds per time frame during the MD. (E and F) Pulldown of the WT, point mutants, or 8-fold excess bpk mutant HA-tagged Bicc1 from HEK293T cell extracts by glutathione-Sepharose beads coated with a recombinant GST control or GST-Bicc1 SAM. Five percent of total cell extracts were loaded as input.
Mentions: To test the predictive power of our structural model of dimeric Bicc1 SAM, we individually replaced six surface-exposed patches of electrostatic amino acids within or outside the SAM-SAM interface by alanines (Fig. 4A). Four of these groups of mutations (those in mutB, mutC, mutD, and mutE) should affect intermolecular H bonds, while two others (those in mutA and mutF) were deliberately introduced outside the predicted dimerization interface (Fig. 4B to D). To screen for polymerization defects, we transfected HA-tagged Bicc1 mutants A to F into HEK293T cells and assessed their retention on glutathione-Sepharose beads coated with recombinant GST-SAM fusion protein as a bait. We found that wild-type HA-Bicc1 efficiently bound to GST-SAM-coated beads but not to beads coated with GST alone (Fig. 4E). In contrast, the Bicc1 mutant lacking SAM (HA-Bicc1ΔSAM) failed to bind GST-SAM beads, suggesting that GST-SAM specifically interacts with the SAM domain of full-length Bicc1. Moreover, a comparison with HA-Bicc1 mutants showed that while the mutations in mutants C, D, and E abolished the SAM-SAM interaction, the mutations in mutants A and F did not, a result that concurs with that of our structure model. The mutation in mutant B exhibited an intermediate effect. The mutation in mutant B affected three amino acids at the periphery of the EH surface, and only one of them (K891) is predicted to contribute to the SAM-SAM interaction (Fig. 3C). According to theoretical predictions, this electrostatic patch is involved in only 1.1 intermolecular H bonds, on average, explaining why its mutation is insufficient to fully disrupt dimerization (Fig. 4D). Altogether, these results strongly corroborate our structure model and provide new tools to specifically probe the consequences of the loss of SAM-SAM interactions in vivo.

Bottom Line: In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway.Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects.We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.

Show MeSH
Related in: MedlinePlus