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Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin - PubMed

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin

Sarah Garrido-Urbani et al. FEBS Lett. 2016 Jan.

Abstract

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.

Keywords: PDZ domain; peptide interaction; phage display; protein-protein interaction; short linear motif.

© 2015 Federation of European Biochemical Societies.

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Figures

Figure 1
Figure 1

Analysis of the ProP‐PD selection data. (A) Histogram of the next‐generation sequencing data of the phage pools after the fourth and fifth round of selections, with the number of sequencing counts on the x‐axis and the number of peptides receiving a certain number of counts on the y‐axis. (B) PWMs representing the peptide binding specificity of syntenin PDZ1‐2. The numbers on the x‐axis indicate the position in the peptide following the typical PDZ ligand nomenclature, with p0 typically being the C‐terminal residue. The relative sized of the letters indicate their occurrences among the aligned sequences. The high of the stacked letters indicate the information content at the position given in bits. (C) Pie chart of the binding motifs assigned among syntenin ligands identified through ProP‐PD.

Figure 2
Figure 2

Analysis of the nectin‐1‐syntenin interaction through SPR, Y2H and colocalization experiments. (A) Double reference subtracted sensograms of full‐length syntenin injected over the biotinylated C‐terminus of nectin‐1 (left, representative example). The apparent K d value of the interaction was determined to be 14 ± 3 μ

m

(n = 3) by fitting a 1 : 1 binding isotherm to the plot of the RUmax against protein concentration (right). (B) Co‐immunoprecipitation of syntenin and nectin‐1 in MCF‐7 cells. Nectin‐1 was immunoprecipitated using a specific antibody (R1.302) and detected using another antibody (R1.54) (left). Coprecipitated syntenin was revealed using a rabbit anti‐syntenin antibody. Syntenin was immunoprecipitated using an anti‐syntenin antibody and detected using the same antibody (right). Coprecipitated Nectin‐1 was revealed using a rabbit anti‐nectin‐1 antibody (R1.54). (C) Colocalization of endogenous nectin‐1 and syntenin. MCF‐7 cells were grown for 5 h on coverslips precoated with fibronectin and processed for immunofluorescence using anti‐nectin‐1 antibody (green in merge) and anti‐syntenin antibody (red in merge). Inserts are enlarged at the bottom. Scale bar: 4 μm.

Figure 3
Figure 3

Characterization of the interaction between syntenin and nectin‐1. (A) Co‐IPs of overexpressed wild‐type and truncated (deleted PDZ binding motif, ΔPDZBM) nectin‐1 and myc‐tagged syntenin in Cos cells. Syntenin was immunoprecipitated using anti‐syntenin1 antibody, and detected with the same antibody, and bound nectin was detected with an anti‐nectin antibody. Truncation of the PDZBM of nectin‐1 confers a loss of interaction. (B) Top: Schematic representation of C‐terminally Myc‐tagged full‐length syntenin and truncated constructs thereof that were used for co‐IPs of overexpressed nectin‐1. ‘Nt’ and ‘Ct’ indicate ‘N‐terminal region and C‐terminal region’. ‘+’ indicates that the co‐IPs of nectin‐1 and syntenin was positive with the indicated construct. Bottom: Co‐IP of nectin‐1 in and truncated C‐terminally Myc‐tagged syntenin constructs.

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References

    1. Beekman JM and Coffer PJ (2008) The ins and outs of syntenin, a multifunctional intracellular adaptor protein. J Cell Sci 121, 1349–1355. - PubMed
    1. Zimmermann P, Zhang Z, Degeest G, Mortier E, Leenaerts I, Coomans C, Schulz J, N'Kuli F, Courtoy PJ and David G (2005) Syndecan recycling [corrected] is controlled by syntenin‐PIP2 interaction and Arf6. Dev Cell 9, 377–388. - PubMed
    1. Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E et al (2012) Syndecan‐syntenin‐ALIX regulates the biogenesis of exosomes. Nat Cell Biol 14, 677–685. - PubMed
    1. Ghossoub R, Lembo F, Rubio A, Gaillard CB, Bouchet J, Vitale N, Slavik J, Machala M and Zimmermann P (2014) Syntenin‐ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2. Nat Commun 5, 3477. - PubMed
    1. Helmke BM, Polychronidis M, Benner A, Thome M, Arribas J and Deichmann M (2004) Melanoma metastasis is associated with enhanced expression of the syntenin gene. Oncol Rep 12, 221–228. - PubMed

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