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Proximity proteomics of C9orf72 dipeptide repeat proteins

ABSTRACT: Profiling of proximity proteomes of C9orf72-derived dipeptide repeat proteins (poly-GA, poly-GR, poly-PR) in HEK293 cells using BioID assay

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Wilfried Rossoll

PROVIDER: MSV000088581 | MassIVE | Fri Dec 17 09:15:00 GMT 2021

SECONDARY ACCESSION(S): PXD030490

REPOSITORIES: MassIVE

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Proximity proteomics of C9orf72 dipeptide repeat proteins identifies molecular chaperones as modifiers of poly-GA aggregation.

Liu Feilin F Morderer Dmytro D Wren Melissa C MC Vettleson-Trutza Sara A SA Wang Yanzhe Y Rabichow Benjamin E BE Salemi Michelle R MR Phinney Brett S BS Oskarsson Björn B Dickson Dennis W DW Rossoll Wilfried W

Acta neuropathologica communications 20220214 1

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusio ...[more]

PMID: 35164882

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Arginine valency in C9ORF72 dipolypeptides mediates promiscuous proteome binding that transmits multiple modes of toxicity

Project description:Motor Neuron Disease patients with the C9ORF72 hexanucleotide expansion mutations feature abnormal expression of 5 different dipeptide repeat polymers (DPRs). Two of these, poly-GR and poly-PR, have proven highly toxic to cell and animal models. To investigate the mechanisms, we defined the interactomes of the DPRs in 10× and 101× repeat lengths as fusions to GFP in Neuro2a cells using quantitative proteomics. Relative to the more inert poly-GA and poly-PA peptides, poly-PR and poly-GR of both repeat lengths were promiscuous binders to the proteome and especially ribosomal proteins, translation initiation factors and translation elongation factors including ribosome recycling factor ABCE1, suggesting a role in ribosome stalling. The PR101 and GR101 DPRs robustly stalled the ribosome compared to the other DPRs and an unrelated mutant protein (Huntingtin) that causes neurodegeneration. Poly-GR also bound to arginine methylases and led to hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in the GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

2020-02-25 | PXD015177 | Pride

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

Project description:An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR) and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces phase-separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

2023-07-10 | GSE212761 | GEO

Translation deficits drive C9orf72 associated poly-GR/PR toxicity

Project description:Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients with the C9orf72 mutation show predominantly cytoplasmic aggregates of poly-GR and poly-PR proteins that are acutely toxic in various model systems. To identify the molecular mediators of neurotoxicity of poly-GR/PR, we analyzed their interactomes in primary neurons. GFP-(GR)149 and (PR)175-GFP preferentially interacted with RNA-binding proteins, including stress granule-associated and nucleolar proteins, as well as ribosomes. Overexpression of the poly-GR/PR interactors Staufen 1/2 (STAU1/2) and YBX1 led to cytoplasmic aggregation of poly-GR/PR into large stress granule-like inclusions, while the poly-GR/PR interactor nucleophosmin (NPM1) recruited poly-GR into the nucleolus. In addition, poly-PR expression reduced ribosome levels and translation, which is consistent with the widespread reduction of synaptic proteins detected by proteomics. Surprisingly, only GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation in neurons, suggesting impaired ribosome biogenesis is driving the acute toxicity commonly observed in vitro. In C9orf72 patient brains, we detected co-aggregation of poly-GR/PR inclusions with ribosomes, but not stress granules. Partial sequestration of ribosomes may chronically impair protein synthesis and contribute to C9orf72 ALS/FTD pathogenesis.

2018-06-27 | PXD008691 | Pride

Interactome screening of C9orf72 dipeptide repeats reveals sequestration and functional impairment of VCP by polyGA

Project description:Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation to produce unnatural dipeptide repeat (DPR) proteins that accumulate in aggregates and contribute to the pathology. There are 5 different DPRs poly(GA), poly(GR), poly(PR), poly(PA), poly(GP) which are neurotoxic in vitro. In our study, we identified the interactome of all five DPR proteins, consisting of 125 repeats, overexpressed in HEK293T cells using BioID2 proximity labeling. We identified 140 interacting partners for polyGR, 130 for polyGA, 138 for polyPR, 62 for polyPA, and 38 for polyGP. Gene ontology enrichment analysis of proteomic data identified interaction candidates involved in protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes, and RNA-binding. Through further analyses, we evaluated the interaction between polyGA and Valosin-containing protein (VCP) in HEK293T cells as well as in brains of C9orf72 patients. Furthermore, we are showing that polyGA sequester VCP in polyGA aggregates and hinders its function. Additionally, we analyzed the effect of decreased level of VCP on autophagic markers p62 and LC3.

2022-05-19 | PXD024120 | Pride

Arginine valency in C9ORF72 dipolypeptides mediates promiscuous proteome binding that transmits multiple modes of toxicity

Project description:C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the valency of Arg in the most toxic DPRS, PR and GR, drives promiscuous binding to the proteome, compared to a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats robustly stalled the ribosome suggesting high-valency Arg electrostatically jams the ribosome exit tunnel during synthesis. Poly-GR also bound to arginine methylases and induced hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

2020-02-25 | PXD015180 | Pride

Toxic C9orf72 poly(PR) binds heterochromatin, disrupts HP1α and causes dsRNA accumulation

Project description:How G4C2 repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. Here, we report the first mouse model to express poly(PR), a dipeptide repeat protein synthesized from expanded G4C2 repeats. Expression of GFP-(PR)50 throughout the mouse brain yielded progressive brain atrophy, neuron5 loss, loss of poly(PR)-positive cells and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused abnormal histone methylation, lamin invaginations, decreases in HP1α expression, and disruptions of HP1α liquid phases. These aberrations of histone methylation, lamins and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element10 expression and double-stranded RNA accumulation. Thus, we uncover new mechanisms by which poly(PR) contributes to c9FTD/ALS pathogenesis.

2019-05-29 | GSE124834 | GEO

Meta-organization of Translation Centers Revealed by Proximity Mapping of Endoplasmic Reticulum Ribosome Interactors

Project description:We report a method to use BioID proximity labeling to map the sub-organization of translating mRNAs on the endoplasmic reticulum. This method first isolates membrane bound polysomes using a Sephacryl 400 resin-based enrichment followed by isolation of biotin labeled polysomes by streptavidin beads and high-throughput sequencing of the bound polysomes. We find that, for the two pools of polysomes labeled by either LRRC59 or Sec61beta BioID reporters, there is an interesting divergence in specific mRNAs that are entering into the secretory pathway such that secreted proteins are much more enriched towards the LRRC59 reporter with Sec61beta serving a more varied role in mRNA translation.

2019-04-18 | GSE118873 | GEO

C9orf72 arginine-rich dipeptide repeat proteins disrupt importin beta-mediated nuclear import

Project description:Disruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin beta cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. Importin beta and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin beta, disrupt its cargo loading, and inhibit nuclear import in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with nuclear transport receptors in the vicinity of the nuclear envelope.

2020-05-20 | PXD015656 | Pride

Identifying the BACE1 interactome through BioID proximity assay

Project description:This study used the BioID proximity assay to establish the BACE1 interactome in healthy neuronal cells and identified interactions involved in BACE1 trafficking, post-translational modification and substrates.

2021-12-30 | PXD021464 | Pride

Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila

Project description:G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/Igf signalling is reduced in fly models of C9orf72 repeat expansion using RNA-sequencing of adult brain. We further demonstrate that activation of insulin/Igf signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/Igf signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/Igf signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

2021-03-20 | GSE151826 | GEO

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