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Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus

IL1RAcP:Arg286 peptide and mAb abolish interleukin signalling.(a) Synthetic antagonist peptide Arg286, identified with protein painting, inhibited SAPK/JNK signalling downstream from IL1RI as effectively as IL1RAcP recombinant protein. In lane 8, scrambled peptide obtained by randomly shuffling Arg286 sequence does not inhibit the signalling downstream from IL1RI. Data are representative of three independent experiments. (b) Arg286 peptide inhibition of ligand pull-down within the receptor complex by His-tagged IL1RAcP. Schematic representation of the complex is depicted in insert. IL1RAcP in the absence of IL1RI does not pull down IL1β, lane 8. (c) Arg286 peptide mAb specific for IL1RAcP peptide extinguishes complex formation depicted in insert.
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f6: IL1RAcP:Arg286 peptide and mAb abolish interleukin signalling.(a) Synthetic antagonist peptide Arg286, identified with protein painting, inhibited SAPK/JNK signalling downstream from IL1RI as effectively as IL1RAcP recombinant protein. In lane 8, scrambled peptide obtained by randomly shuffling Arg286 sequence does not inhibit the signalling downstream from IL1RI. Data are representative of three independent experiments. (b) Arg286 peptide inhibition of ligand pull-down within the receptor complex by His-tagged IL1RAcP. Schematic representation of the complex is depicted in insert. IL1RAcP in the absence of IL1RI does not pull down IL1β, lane 8. (c) Arg286 peptide mAb specific for IL1RAcP peptide extinguishes complex formation depicted in insert.

Mentions: To test the mechanistic significance of the sequence data (Tables 1 and 2; Fig. 5b), we treated IL1β-stimulated cells (NCI-ADR-RES32) with synthetic beta loop peptides corresponding to the three-way interface (Fig. 6a; Supplementary Figs 19 and 20). Synthetic peptides corresponding to the narrow IL1RAcP contact point Arg286 completely blocked IL1β ligand-stimulated signalling in a dose-dependent manner (Fig. 6a). To verify that this region was functionally active, we tested whether the peptide blocked the formation of the three-protein complex in vitro (Fig. 6b), by pulling down IL1β in the complex through a His-tagged IL1RAcP partner. We found that the Arg286 peptide completely blocked IL1RAcP three-way binding to the receptor–ligand complex (Fig. 6b). Finally, to further verify the importance of the IL1RAcP sequence identified by protein painting, we raised a mAb against this region. The mAb blocked the three-protein complex formation in a dose-dependent manner (Fig. 6b). Since the Arg286 peptide, or the mAb, acts on opposite faces of the binding site, and can both block pull-down of the ligand–receptor complex, and the peptide itself can substitute for the entire soluble IL1RAcP protein as a competitive inhibitor13 (Fig. 6b), this sequence may be necessary for the interaction of the accessory protein with the other two partners. The Arg286 peptide and/or the mAbs directed against this region constitute novel therapies to potentially block interleukin signalling in vivo.


Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Luchini A, Espina V, Liotta LA - Nat Commun (2014)

IL1RAcP:Arg286 peptide and mAb abolish interleukin signalling.(a) Synthetic antagonist peptide Arg286, identified with protein painting, inhibited SAPK/JNK signalling downstream from IL1RI as effectively as IL1RAcP recombinant protein. In lane 8, scrambled peptide obtained by randomly shuffling Arg286 sequence does not inhibit the signalling downstream from IL1RI. Data are representative of three independent experiments. (b) Arg286 peptide inhibition of ligand pull-down within the receptor complex by His-tagged IL1RAcP. Schematic representation of the complex is depicted in insert. IL1RAcP in the absence of IL1RI does not pull down IL1β, lane 8. (c) Arg286 peptide mAb specific for IL1RAcP peptide extinguishes complex formation depicted in insert.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109009&req=5

f6: IL1RAcP:Arg286 peptide and mAb abolish interleukin signalling.(a) Synthetic antagonist peptide Arg286, identified with protein painting, inhibited SAPK/JNK signalling downstream from IL1RI as effectively as IL1RAcP recombinant protein. In lane 8, scrambled peptide obtained by randomly shuffling Arg286 sequence does not inhibit the signalling downstream from IL1RI. Data are representative of three independent experiments. (b) Arg286 peptide inhibition of ligand pull-down within the receptor complex by His-tagged IL1RAcP. Schematic representation of the complex is depicted in insert. IL1RAcP in the absence of IL1RI does not pull down IL1β, lane 8. (c) Arg286 peptide mAb specific for IL1RAcP peptide extinguishes complex formation depicted in insert.
Mentions: To test the mechanistic significance of the sequence data (Tables 1 and 2; Fig. 5b), we treated IL1β-stimulated cells (NCI-ADR-RES32) with synthetic beta loop peptides corresponding to the three-way interface (Fig. 6a; Supplementary Figs 19 and 20). Synthetic peptides corresponding to the narrow IL1RAcP contact point Arg286 completely blocked IL1β ligand-stimulated signalling in a dose-dependent manner (Fig. 6a). To verify that this region was functionally active, we tested whether the peptide blocked the formation of the three-protein complex in vitro (Fig. 6b), by pulling down IL1β in the complex through a His-tagged IL1RAcP partner. We found that the Arg286 peptide completely blocked IL1RAcP three-way binding to the receptor–ligand complex (Fig. 6b). Finally, to further verify the importance of the IL1RAcP sequence identified by protein painting, we raised a mAb against this region. The mAb blocked the three-protein complex formation in a dose-dependent manner (Fig. 6b). Since the Arg286 peptide, or the mAb, acts on opposite faces of the binding site, and can both block pull-down of the ligand–receptor complex, and the peptide itself can substitute for the entire soluble IL1RAcP protein as a competitive inhibitor13 (Fig. 6b), this sequence may be necessary for the interaction of the accessory protein with the other two partners. The Arg286 peptide and/or the mAbs directed against this region constitute novel therapies to potentially block interleukin signalling in vivo.

Bottom Line: The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface.We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP.We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling.

View Article: PubMed Central - PubMed

Affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Boulevard, Manassas, Virginia 20110, USA.

ABSTRACT
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein-protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

No MeSH data available.


Related in: MedlinePlus