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Heat shock chaperone HSPB1 mitigates poly-glycine-induced neurodegeneration via restoration of autophagic flux

ABSTRACT: HEK293T cells were transfected with GFP-poly-G-TurboID or GFP-TurboID as a control. Following biotin labelling, the cells were lysed in RIPA buffer to separate the soluble and insoluble fractions. The insoluble proteins in the pellet containing poly-G aggregates were further lysed in the lysis buffer containing 8 M urea to ensure effective solubilization

ORGANISM(S): Homo Sapiens

SUBMITTER: Lei Li

PROVIDER: PXD059955 | iProX | Mon Jan 20 00:00:00 GMT 2025

REPOSITORIES: iProX

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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

TMT analysis of detergent-soluble and insoluble fractions from HEK293T cells in normal conditions and upon heat shock

Project description:HEK293T cells were cultured according to standard protocols and heat shocked at 42oC for 6 hours. Detergent-soluble and insoluble fractions from HEK293T cells were obtained following the procedures described before. Specifically, HEK293T cells were lysed in NP40 lysis buffer (Invitrogen #FNN0021) with protease inhibitors by gentle pipetting. The cell homogenate was centrifuged at 14000 rpm and the supernatant was retrieved (detergent-soluble fraction). The remaining pellet was washed twice in ice-cold NP40 lysis buffer and then resuspended in urea buffer (RIPA buffer with 8M urea). The samples were then centrifuged at 14000 rpm for 10 min to pellet any remaining cell debris. The resulting supernatant (detergent-insoluble fraction) was retrieved and analyzed as explained below.

2025-08-01 | PXD046967 | Pride

CLIP-Seq Analysis Enables the Design of Ribosomal RNA Bait Oligonucleotides That Protect Against C9ORF72 ALS/FTD-Associated Poly-GR Pathophysiology

Project description:We report CLIP-Seq results for GFP tagged poly-GR in order to define poly-GR interacting RNAs in vivo.

2023-11-13 | GSE182008 | GEO

Transcriptome-wide profiling of poly(A)-tail length, translation efficiency and mRNA stability using TED-seq, mRNA-seq, Ribo-seq and PRO-seq in ER stress conditions

Project description:We report systematical profiling of translation efficiency and mRNA stability dependent on the dynamics of poly(A)-tail length in stress conditions of human cells. In this study, we developed a new feasible method measuring poly(A)-tail length called TED-seq and applied it to investigate the change of mRNA's poly(A)-tail lengths in ER stress pharmacologically induced by thapsigargin (THAP). Combined with other global RNA analyses such as RNA-seq, Ribo-seq and PRO-seq, we observed that ER stress induced lenthening poly(A)-tail length, in particular of ER-stress-regulators, upon ER stress. More specifically, these mRNAs are translationally de-repressed and more stabilized based on increase in poly(A)-tail length. We also identified that insoluble fractions which include stress-induced RNA-granules have overall shorter length of poly(A) tail. Taken together, our data suggest that poly(A)-tail lengths are dynamically regulated and influence both translation efficiency and mRNA stability in ER stress.

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

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2023-07-10 | GSE212761 | GEO

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Explored the underlying mechanism by which ILF3 exerted carcinogenesis and its potential targets in MM

Project description:An RIP experiment was performed in ARP1 cell following the instructions of the article. ~5-20 × 10^6 cells were harvested and lysed. Protein A/G MagBeads pre-coated with 5μg of the antibody of interest (HNRNPA2B1/ILF3, Proteintech) and incubated with cell lysate supernatant overnight at 4°C.The beads containing immunoprecipitated RNA-protein complex were treated with 150μL of Proteinase K buffer to digest the proteins. Specific binding RNAs were isolated by using TRIzol.

2021-11-30 | GSE162303 | GEO

Pianalto KM et al "Characterization of novel components of the environmental pH-sensing complex in Cryptococcus neoformans"

Project description:Three analyses performed by the Duke Proteomics and Metabolomics Shared Resource in support of the project entitled "". Sample ID21519 was called Rra1-GFP, a 1D coomassie-stained gel band from a GFP-Trap pulldown of Rra1-GFP from the membrane protein fraction of Cryptococcus neoformans. Sample ID21570 was called no-GFP control, and consisted a GFP-Trap pulldown from the insoluble fraction of C. neoformans H99 lysate (non GFP expressing). ID21571 was called Rra1-GFP, and consisted of a GFP-Trap pulldown from the insoluble fraction of C. neoformans H99 lysate containing Rra1-GFP.

2018-03-06 | MSV000082127 | MassIVE

Effect of heat-shock on total, poly-ubiquitylated, and insoluble proteomes of human IMR-90 fibroblasts in different proliferation states

Project description:Cellular stress responses are crucial for survival in sub-optimal conditions, and almost invar-iably involve proteome-wide alterations at the levels of protein abundance, post-translational modification with poly-ubiquitin chains (poly-ubiquitylation), and solubility. However, most of our understanding of human cell stress responses at the protein level stems from experi-ments in rapidly-dividing cell culture models. Here, we used primary human diploid lung fibro-blasts (IMR-90) in proliferating, contact-inhibited quiescent, or doxorubicin-induced senes-cent states, to explore their responses to a severe heat-shock (44 degrees Celsius for 2 hours) at the total, poly-ubiquitylated, and insoluble proteome levels.

2025-10-08 | PXD067225 | Pride

Ectopic expression of URT1 remodels poly(A) tail profiles in Nicotiana benthamiana

Project description:3’RACE-seq experiments were conducted to determine the impact of URT1 ectopic expression on the poly(A) tail length profile of both a GFP reporter mRNA co-expressed in Nicotiana benthamiana leaves and an endogenous mRNA encoding PATHOGENESIS-RELATED PROTEIN 2 (PR2)

2020-05-28 | GSE148409 | GEO

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