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Purification of ubiquitylated proteins from HCT116 cells

ABSTRACT: By hyperosmotic stimuli, a slight increase in the level of ubiquitylated proteins was observed. So we tried to identify the di-Gly peptides.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

SUBMITTER: Hikaru Tsuchiya

LAB HEAD: Yasushi Saeki

PROVIDER: PXD016369 | Pride | 2020-02-18

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	EX_1Di-GlyproteomeSucrose0min20171226.mgf	Mgf
	EX_1Di-GlyproteomeSucrose0min20171226.raw	Raw
	EX_1Di-GlyproteomeSucrose15min20171226.mgf	Mgf
	EX_1Di-GlyproteomeSucrose15min20171226.raw	Raw
	EX_1Di-GlyproteomeSucrose30min.mgf	Mgf

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1 - 5 of 14

Publications

Stress- and ubiquitylation-dependent phase separation of the proteasome.

Yasuda Sayaka S Tsuchiya Hikaru H Kaiho Ai A Guo Qiang Q Ikeuchi Ken K Endo Akinori A Arai Naoko N Ohtake Fumiaki F Murata Shigeo S Inada Toshifumi T Baumeister Wolfgang W Fernández-Busnadiego Rubén R Tanaka Keiji K Saeki Yasushi Y

Nature 20200205 7794

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins<sup>1,2</sup>. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes<sup>3-8</sup>, but it remains unclear whether the proteas ...[more]

PMID: 32025036

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Improvement of ubiquitination site detection by Orbitrap mass spectrometry.

Project description:We describe a modified workflow to enrich for and detect diGly peptides originating from ubiquitinated proteins using immunopurification. Using a combination of several relatively simple modifications in the sample preparation and mass spectrometric detection protocols, we now routinely detect over 24,000 diGly modified peptides in a single sample. We show the efficacy of this strategy for cell lysates from both non-labeled and metabolically labeled (SILAC) mammalian cells. Furthermore, we demonstrate that this optimized strategy is also useful for the in-depth identification of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. As such, this study presents an addition to the toolbox of the ubiquitination site analysis for the identification of the deep ubiquitinome.

2017-11-14 | PXD007101 | Pride

Phosphoproteome of Francicella Tularensis

Project description:The pathogenic bacterium Francisella tularensis possesses a non-canonical T6SS encoded on the Francisella pathogenicity island (FPI) that is essential for efficient phagosomal escape and access to the cytosolic compartment of infected host cells. Using a global and site-specific phosphoproteomic analysis of F. novicida, we identified a phosphorylated form of IglB, which constitutes with iglA the outer sheath of the T6SS. We show here that substitution of the unique phosphorylated tyrosine (Y139) of IglB to alanine or to the non-phosphorylatable analogue phenylalanine prevents the formation of the sheath-like structures and impairs normal bacterial phagosomal escape. We propose that IglB phosphorylation is involved in the dynamics of assembly-disassembly of the sheath.

2021-03-29 | PXD009225 | Pride

Data-independent acquisition method for ubiquitinome analysis reveals non-degrative regulation of circadian biology

Project description:Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent (DIA) acquisition with regards to sensitivity in single run analysis and data completeness have not yet been explored. We developed a sensitive workflow combining diGly antibody-based enrichment, optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identified 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells â€“ double the number and quantitative accuracy of data dependent acquisition. Applied to TNF-alpha signaling, the workflow comprehensively captured known sites while adding many novel ones. A first systems-wide investigation of ubiquitination of the circadian cycle uncovered hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting novel connections between metabolism and circadian regulation.

2020-11-20 | PXD019854 | Pride

Phosphoproteome analysis of TgPP1 depleted Toxoplasma gondii tachyzoites

Project description:Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell-cycle is therefore key to their pathogenesis. Phosphorylation is a crucial post-transcriptional modification that regulates many aspects of the cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the production of the daughter cells inner membrane complex (IMC), the segregation of organelles and the nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is normally found in the latent bradyzoite form of the parasite. Using transcriptomics and phosphoproteomics, we show that TgPP1 mainly acts through post-transcriptional mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylate a protein bearing a starch binding domain. Mutagenesis analysis reveals that the targeted phosphor-sites are linked to the ability of the parasite to produce amylopectin in conditions inducing bradyzoite differentiation. Therefore, we show that TgPP1 have pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

2024-08-08 | PXD043539 | Pride

Phosphorylation Francisella

Project description:Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. Using a global and site-specific phosphoproteomic analysis of Francisella we identified here a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. Phosphorylation of the sheath may constitute a previously unrecognized mechanism contributing to the dynamics of assembly-disassembly of the T6SS.

2023-12-20 | PXD012507 | Pride

Phosphoproteomic analysis of Francisella

2019-10-10 | PXD013619 | Pride

Subclass I SnRK2-interacting protein, LC-MS/MS

Project description:To reveal subclass I SnRK2-interacting proteins, we conducted co-immunoprecipitation using anti-GFP antibody from subclass I SnRK2-GFP expressing plants. Immunoprecipitates were identified by LC-MS/MS analyses.

2020-04-27 | PXD017371 | Pride

TRIM21 innate immune detection of GBP1-positive Toxoplasma gondii is essential for host survival

Project description:Interferon gamma (IFN) is the major pro-inflammatory cytokine conferring resistance to the intracellular vacuolar pathogen Toxoplasma gondii. Autophagy along with immunity-related GTPases (IRGs), guanylate binding proteins (GBPs) and the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) mediate clearance of Toxoplasma in IFN-stimulated cells. With the exception of inflammasomes, no host innate immune mediators impacting host survival have been identified at disrupted parasitophorous vacuoles. We show that IFN drives recruitment of the E3 ubiquitin ligase TRIM21 to GBP1-positive avirulent Toxoplasma vacuoles. This led to Lys63-linked ubiquitination of the vacuole and secretion of pro-inflammatory cytokines. GBPs were ubiquitinated during infection and TRIM21 controlled their expression levels and the efficient recruitment of GBP1 to the vacuole. TRIM21 deficiency led to an enhanced early replication of Toxoplasma without interfering with vacuolar disruption. TRIM21-/- mice were highly susceptible to Toxoplasma infection, exhibiting decreased levels of pro-inflammatory cytokines and higher parasite burden in the brain. This study identifies TRIM21 as a previously unknown modulator of Toxoplasma gondii resistance thereby extending host innate immune recognition of eukaryotic pathogens to include E3 ubiquitin ligases.

2018-03-12 | PXD003217 | Pride

Role of ITCH in the endolysosomal damage response

Project description:Ubiquitin remnant (diGly) profiling of parental and ITCH knockout (KO) cells under endolysosomal damage conditions (LLOMe).

2024-10-18 | PXD039088 | Pride

Ubiquitination in treated T. cruzi parasites

Project description:T. cruzi epimastigotes in the logarithmic growth phase (3×106 cells mL-1) were incubated for 12h with bortezomib (1.8 µM), GNF6702 (2.9 µM) or compound 1 (24 µM), equivalent to 8× the EC50 values of each compound. Controls were incubated in the presence of diluent (DMSO). Cells were harvested by centrifugation (1912g, 15 min, 4 °C) and washed with ice-cold PBS (1912g, 5 min, 4 °C), and finally, the cell pellets were resuspended in 1.5 mL of ice-cold lysis buffer (1 mM EDTA, 1 mM DTT, 100 μM TLCK, and 1× Roche EDTA-free cOmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer, pH 7.4). Cell suspensions were submitted to 3 freeze–thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and then lysed using the One ShotTM Cell disruptor (Constant Systems, UK) at 30 kpsi.

2022-02-17 | PXD027524 | Pride

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