Dataset Information

MS analysis of cellular targets of an engineered selective PSD-95 PDZ domain ligand

ABSTRACT: Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces and PPI domains. We report here a strategy that addresses this challenge by using a semi-rational approach that separates the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by employing a selection by phage display coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to bind against the target interface. We have applied this approach to PSD-95, an essential multi-PDZ domain-containing synaptic scaffold protein that belongs to a larger family of paralogs. We show here that with this strategy we could specifically target a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides the first paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules. This archive contains the comparative LC/MS/MS analysis of cellular targets of an engineered selective PSD-95 PDZ domain ligand, Xph20-ETWV, against the fusion with a naïve clone, Xph0-ETWV and a control non-binding protein (mSacrlet-i).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Stephane Claverol

LAB HEAD: Marc Bonneu

PROVIDER: PXD015313 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Rimbault Charlotte C Maruthi Kashyap K Breillat Christelle C Genuer Camille C Crespillo Sara S Puente-Muñoz Virginia V Chamma Ingrid I Gauthereau Isabel I Antoine Ségolène S Thibaut Coraline C Tai Fabienne Wong Jun FWJ Dartigues Benjamin B Grillo-Bosch Dolors D Claverol Stéphane S Poujol Christel C Choquet Daniel D Mackereth Cameron D CD Sainlos Matthieu M

Nature communications 20191004 1

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outsi ...[more]

PMID: 31586061

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Project description:PDZ (Post-synaptic density, PSD-95)/ Disc large, Dlg/ Zonula occludens, ZO-1) proteins are central in the assembly of multiprotein signalling complexes that are formed in distinctive, specialized cell regions. In these complexes different signals are orchestrated and traduced into quick and efficient responses controlling cell polarization and cell communication. Viral pathogens target host PDZ proteins by expressing viral proteins containing a PDZ binding motif (PBM). SARS-CoV-1 and -2 harbour the protein E, a viroporin with a conserved PBM in its carboxyl-terminal region. SARS-CoV-1 E protein is a pathogenicity factor that in epithelia interacts with the PDZ protein PALS1, which promote disruption of cell junctions. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes, dendritic cells, and alveolar macrophages. Identification of targets of SARS-CoV-2 E protein in immune cells might offer valuable clues to understand how SARS-CoV-2 alters immune response. Here we generated a SARS-CoV-2 E protein fused to a GFP-tag at the amino terminal. This recombinant protein was used as bait to identify associated proteins in THP-1 cells, a human monocytic cell line that can be differentiated to macrophages and dendritic cells. Analysis of the GFP-E protein interactome provided 372 proteins that fall into different functional groups. Only eight of these proteins harbor PDZ domains, including the cell polarity protein ZO-2 and the chemoattractant IL-16. Syntenin, a PDZ protein previously identified as interactor of SARS-CoV-1 E protein in epithelia, was also found in our analysis. Little is known about these PDZ proteins in immune cells, so we addressed their expression in monocytes and along the differentiation towards macrophages and dendritic cells. Our findings support the notion that viral targeting of PDZ proteins alters inflammatory response and highlight the importance of identifying the functions of PDZ proteins into maintenance of immune fitness.

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