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PSD-95 interactome in presence and absence of Xph20 intrabody

ABSTRACT: In the context of intrabodies development for the synaptic protein PSD-95, we use proteomic approaches to evaluate the possible impact of one of our evolved PSD-95 binders on the endogenous PSD-95 function. More precisely, we compare the interactome of PSD-95 in presence or absence of the Xph20 binder in primary rat neuron culture infected with the Xph20-derived probe or a control protein, EGFP.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Stephane Claverol

LAB HEAD: Stephane Claverol

PROVIDER: PXD045002 | Pride | 2024-01-26

REPOSITORIES: Pride

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Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Rimbault Charlotte C Breillat Christelle C Compans Benjamin B Toulmé Estelle E Vicente Filipe Nunes FN Fernandez-Monreal Monica M Mascalchi Patrice P Genuer Camille C Puente-Muñoz Virginia V Gauthereau Isabel I Hosy Eric E Claverol Stéphane S Giannone Gregory G Chamma Ingrid I Mackereth Cameron D CD Poujol Christel C Choquet Daniel D Sainlos Matthieu M

eLife 20240103

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop end ...[more]

PMID: 38167295

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MS analysis of photocrosslink adducts for the mapping of interaction sites of selective engineered PSD-95 PDZ domain ligands

Project description: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 mapping by photocrosslinking and LC/MS/MS analysis of the interaction sites of engineered selective PSD-95 PDZ domain ligands, Xph20-ETWV, Xph20-Stg and Xph20-ETMA.

2019-11-12 | PXD015366 | Pride

Correlation profiling of synaptic sub-fraction proteomes of the mouse brain hippocampus, cortex and cerebellum

Project description:Synaptic transmission forms the main mode of signaling and information integration in the nervous system. Here, we identified and quantified proteins from three brain regions and five biochemical synapse sub-fractions. We identified around 4500 proteins in total, of which about 2000 proteins each were quantified from microsome, P2 fraction, synaptosome, synaptic membrane, and postsynaptic density (PSD). Correlation profiling using these data sets identified synaptic functional groups and showed enrichment of canonical presynaptic proteins in synaptosome, and canonical postsynaptic proteins in PSD. In addition, we detected profound brain region specific differences in the extent of enrichment for some functionally associated proteins, exemplified by different AMPAR subunits and the large differences in spatial distribution of their potential interactors. Furthermore, we revealed novel PSD proteins PLEKHA5 and ADGRA1, which using super-resolution microscopy on primary neuronal culture were confirmed to reside in the PSD.

2017-09-25 | PXD005634 | Pride

Targeting the Post-Synaptic Proteome in Alzheimer Disease with Psychosis

Project description:Individuals with Alzheimer Disease who develop psychotic symptoms (AD+P) experience more rapid cognitive decline and have reduced indices of synaptic integrity relative to those without psychosis (AD-P). We sought to determine whether the postsynaptic density (PSD) proteome is altered in AD+P relative to AD-P, analyzing PSDs from dorsolateral prefrontal cortex of AD+P, AD-P, and a reference group of cognitively normal elderly subjects. The PSD proteome of AD+P showed a global shift towards lower levels of all proteins relative to AD-P, enriched for kinases, proteins regulating Rho GTPases, and other regulators of the actin cytoskeleton. We computationally identified potential novel therapies predicted to reverse the PSD protein signature of AD+P. Five days of administration of one of these drugs, the C-C Motif Chemokine Receptor 5 inhibitor, maraviroc, led to a net reversal of the PSD protein signature in adult mice, nominating it as a novel potential treatment for AD+P.

2023-06-19 | PXD042025 | Pride

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

Project description: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).

2019-11-12 | PXD015313 | Pride

Developmental profile of core postsynaptic density protein complexes integrates signaling components of brain disorders

Project description:The postsynaptic density (PSD) contains a collection of scaffold proteins used for the assembly of synaptic signaling complexes and disruption of this signaling machinery might be implicated in a variety of brain disorders. However, it is not known how the core-scaffold machinery associates this collection of proteins through development and how proteins coding for genes involved in psychiatric and other brain disorders are distributed through spatio-temporal protein complexes. Here, using immunopurification, proteomics, bioinformatics and mouse genetics, we isolated 2876 proteins across 41 in-vivo protein complexes and determined their protein domain composition, correlation to gene expression levels, and developmental integration to the PSD. We defined major protein clusters for enrichment of schizophrenia (SCZ), autism spectrum disorders (ASD), developmental delay (DD), and intellectual disability (ID) risk factors at embryonic day 14 and the adult PSD. These protein complexes contained a discrete number of protein domains defining molecular functions. Mutations in highly-connected nodes alter protein-protein interactions that modulate the assembly of macromolecular complexes enriched in disease risk candidates. These results were integrated into a software platform: Synaptic Protein/Pathways Resource (SyPPRes), enabling the prioritization of brain disease risk factors and their placement within synaptic protein interaction networks.

2017-08-22 | PXD006277 | Pride

Transcription profiling of mouse SAP102 and PSD-95 knockouts compared to their wild-type littermates

Project description:Effect of ablation of SAP102 or PSD-95 genes in mice compared to wild-types littermates

2007-09-01 | E-MEXP-1159 | biostudies-arrayexpress

Experience-induced remodeling of the hippocampal post-synaptic proteome and phosphoproteome

Project description:The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following 4 different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3,938 proteins and 2,761 phosphoproteins in the sequential strategy covering a total of 4,968 unique protein groups (at least 2 peptides including an unique peptide). On the phosphoproteins, we identified a total of 6,188 unambiguous phosphosites (75% site localization probability)

2024-01-26 | PXD045496 | Pride

Targeting the Post-Synaptic Proteome in Alzheimer Disease with Psychosis

Project description:Individuals with Alzheimer Disease with psychotic symptoms (AD+P) experience more rapid cognitive and functional decline, and have reduced indices of synaptic integrity, relative to those without psychosis (AD-P). We hypothesized the postsynaptic density (PSD) proteome is altered in AD+P relative to AD-P, and that this proteomic signature could be used to nominate novel pharmacotherapies. METHODS: Liquid-Chromatography/Mass Spectrometry analysis of PSDs from dorsolateral prefrontal cortex of AD+P, AD-P and cognitively normal elderly subjects. RESULTS: The PSD proteome signature of AD+P was characterized by lower levels of a network of kinases, proteins regulating Rho GTPases, and proteins regulating the actin cytoskeleton. Potential novel therapies identified included the C-C Motif Chemokine Receptor 5 inhibitor, maraviroc. DISCUSSION: AD+P is characterized by broad changes in the PSD proteome in prefrontal cortex. Further testing of potential therapies for their ability to reverse these changes and protect against psychotic-like behaviors in model systems is warranted.

2023-06-19 | PXD042026 | Pride

Targeting postsynaptic glutamate receptor scaffolding proteins PSD-95 and PICK1 for obesity treatment [2]

Project description:Human genome-wide association studies (GWAS) suggest a functional role for central glutamate receptor signaling and plasticity in body weight regulation. Here, we utilize UK Biobank GWAS summary statistics of body mass index (BMI) and body fat percentage (BF%) to identify genes encoding for proteins known to interact with postsynaptic AMPA and NMDA receptors. Loci in/near Discs Large Homolog 4 (DLG4) and protein interacting with C kinase 1 (PICK1) reached genome-wide significance (P<5x10-8) for BF% and/or BMI. To further evaluate the functional role of PSD-95 (gene name: DLG4) and PICK1 in energy homeostasis, we used dimeric PDZ-domain-targeting peptides of PSD-95 and PICK1 to demonstrate that pharmacological inhibition of PSD-95 and PICK1 induces prolonged weight-lowering effects in obese mice. Collectively, these data demonstrate that the glutamate receptor scaffolding proteins, PICK1 and PSD-95, are genetically linked to obesity and that pharmacological targeting of their PDZ domains represents a promising new therapeutic avenue for sustained weight loss.

2024-01-04 | GSE251836 | GEO

Unbiased proteomic analysis of postsynaptic density fractions from Shank3 mutant mice reveals brain region specific changes highly relevant to autism spectrum disorder

Project description:Disruption of the human SHANK3 gene can cause several neuropsychiatric disease entities including Phelan-McDermid syndrome (PMS), autism spectrum disorders (ASDs) and intellectual disability (ID). Although a wide array of neurobiological studies strongly supports a major role for SHANK3 in organizing the postsynaptic protein scaffold, the molecular processes at synapses of individuals harboring SHANK3 mutations are still far from being understood. In this study, we biochemically isolated the postsynaptic density (PSD) fraction from striatum and hippocampus of Shank3Δ11-/- mutant mice and performed ion-mobility enhanced data-independent label-free LC-MS/MS to obtain the corresponding PSD proteomes. This unbiased approach to identify molecular disturbances at PSDs devoid of major Shank3 isoforms revealed largely distinct molecular alterations in striatum and hippocampus. Being the first comprehensive analysis of brain region specific PSD proteomes from a Shank3 mutant line, our study provides crucial information on molecular alterations that could foster translational treatment studies for SHANK3 mutation-associated synaptopathies and possibly also ASD in general.

2017-03-08 | PXD005192 | Pride

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