Prevalence, Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments

Ivarsson, Ylva; Wawrzyniak, Anna Anna Maria; Kashyap, Rudra; Polanowska, Jolanta; Betzi, S.; Lembo, Frédérique; Vermeiren, Elke; Chiheb, Driss; Lenfant, Nicolas; Morelli, Xavier; Borg, Jean-Paul; Reboul, Jérôme; Zimmermann, Pascale

doi:10.1371/journal.pone.0054581

Cited by 23 publications

(20 citation statements)

References 60 publications

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“…This versatile format is thus amenable to other potential uses, in particular the expression of each individual PDZ domain fused to GFP or to GST in mammalian cells and bacteria (32). As an example, the PDZome has been used to screen novel PDZ–lipid interactions (86). This array is thus adapted for the screen of PDZ interactors and, beyond, should facilitate the understanding of their functions.…”

Section: Discussionmentioning

confidence: 99%

The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions

Belotti

Polanowska

Daulat

et al. 2013

Molecular & Cellular Proteomics

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Protein–protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.

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Section: Discussionmentioning

confidence: 99%

The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions

Belotti

Polanowska

Daulat

et al. 2013

Molecular & Cellular Proteomics

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“…Overlay assays were performed as described in ref. 51. 100 pmol lipids were immobilized on the nitrocellulose membrane.…”

Section: Methodsmentioning

confidence: 99%

Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2

et al. 2014

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“…The PDZ family is one of the largest domain families in the human proteome, with about 270 members that typically interact with C-terminal peptides (class I binding motif: x-S/T-x-Φ-COO-, class II: x-Φ-x-Φ-COO-) (17) but also with internal peptide stretches and phosphoinositides (18,19). PDZ-peptide interactions have been extensively analyzed by distinct experimental efforts, such as peptide-phage display (7,20), peptide arrays (9,21,22), and split-ubiquitin membrane Y2H (23), as well as by computational approaches (24)(25)(26)(27)(28).…”

Section: Significancementioning

confidence: 99%

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

Ivarsson

Arnold

McLaughlin

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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133

153

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The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/ Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1-to 40-μM range. Furthermore, we identified several well-established host-virus proteinprotein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.T here are an estimated 650,000 protein-protein interactions in a human cell (1). These interactions are integral to cellular function and mediate signaling pathways that are often misregulated in cancer (2) and may be hijacked by viral proteins (3). Commonly, signaling pathways involve moderate affinity interactions between modular domains and short linear motifs (SLiMs; conserved 2-to 10-aa stretches in disordered regions) (4) that are difficult to capture using high-throughput methods, such as yeast two-hybrid (Y2H) or affinity-purification mass spectrometry (AP/MS) but can be identified using peptide arrays, splitprotein systems (5, 6), or peptide-phage display (7-10). A major limitation of peptide arrays is coverage, because the number of potential binding peptides in the proteome is orders of magnitude larger than what can be printed on an array. Conventional phage libraries display combinatorially generated peptide sequences that can identify biophysically optimal ligands of modular domains but this approach can exhibit a hydrophobic bias and may not be ideal for detecting natural binders (11). Thus, there is a need for alternative approaches for identification of relevant domain-SLiMs interactions.Here, we report an appr...

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Prevalence, Specificity and Determinants of Lipid-Interacting PDZ Domains from an In-Cell Screen and In Vitro Binding Experiments

Cited by 23 publications

References 60 publications

The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions

The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions

Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

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