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Quantitative mass spectrometric analysis of UBE2S auto-ubiquitination throughout the cell cycle.

ABSTRACT: Immunoprecipitated UBE2S from HeLa K cells synchronized in pro metaphase, anaphase and G1-phase, were analyzed by tryptic digest and LC-MS/MS.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD012643 | Pride | 2022-03-23

REPOSITORIES: Pride

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Proteomic analysis of Dscc1 knockout mouse cells

Project description:In this project we aimed to explore the effect of Dscc1 gene loss on the proteome. Embyros from wildtype and aged matched Dssc1 null embryos were profiled.

2023-11-17 | PXD034902 | Pride

Cross-talk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii

Project description:Toxoplasma gondii encodes three Protein Kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with dually acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 results in a block in parasite division. In contrast, conditional expression of a dominant negative PKAr isoform unable to bind cAMP, triggers premature egress of parasites from infected cells. This untimely egress critically depends on parasite density and host cell acidification. A comparative phosphoproteome analysis reveals that PKA genetic inhibition significantly changed the phosphorylation profile of a putative cGMP-phosphodiesterase, PDE2. Consistently, the phenotype of PKA genetic inhibition is alleviated by chemical inhibition of the cGMP-dependent protein kinase G (PKG). A phosphodiesterase inhibitor is able to circumvent egress repression by PKA or pH neutralisation, indicating that environmental acidification and PKA signalling act as balancing regulators of cGMP degradation to control PKG-mediated egress. Collectively, these results reveal a cross-talk between PKA and PKG pathways to govern egress in T. gondii.

2017-10-12 | PXD006045 | Pride

E2~dID: A versatile approach to identify substrates modified with ubiquitin and ubiquitin-like molecules by specific E3 enzymes

Project description:Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to most, if not all biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we have developed an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2 conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and identification of modified proteins by mass spectrometry under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of the APC/C in human cells. Finally, performing E2~dID with SUMO in S. cerevisiae we show that E2-dID can be easily adapted to other ubiquitin-like modifiers and experimental models.

2019-04-03 | PXD008624 | Pride

Multifactorial remodeling of the cancer immunopeptidome by interferon gamma

Project description:In this study we combined previous transcriptomics and immunopeptidomics data with new proteomics data from untreated and IFNg-treated CRC PDOs to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNg treatment (this is from the paper introduction, let me know if it needs any stylistic changes). · Experiment description: The cells were grown in DMEM/F12 media with 20% fetal bovine serum, 1X Glutamax, 100 units/ml penicillin/streptomycin and 2% matrigel, and treated with 600ng/mL IFNg for 48 hours before harvesting. Cells were washed twice with ice-cold PBS then snap-frozen.

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Cyclin B1-Cdk1 binds MAD1 and facilitates its release from the nuclear pore complex to ensure a robust Spindle Assembly Checkpoint

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2020-06-29 | PXD017202 | Pride

Genetic determinants of genomic stability identified by comprehensive in vivo analyses of micronucleus formation

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2023-11-17 | PXD030499 | Pride

Differential proteomic profiling between Cancer Associated Fibroblasts and KRAS mutated cancer cell lines

Project description:Purpose Cancer-associated fibroblasts (CAFs) are a key component of tumors. We aimed to profile the proteome of cancer cell lines representing three common cancer types (lung, colorectal and pancreatic) and a representative CAF cell line from each tumor type to gain insight into CAF function and identify new CAF biomarkers. Experimental Design We used isobaric labeling, liquid chromatography and mass spectrometry to evaluate the proteome of 9 cancer and 3 CAF cell lines. Functions of selected differentially expressed proteins in CAF cell lines were further investigated using siRNA and the expression levels of candidate novel CAF biomarkers were evaluated by immunohistochemistry in 30 human tumor specimens. Results Of the 9460 proteins evaluated, functional enrichment analysis revealed N-glycan biosynthesis and extracellular membrane proteins to be upregulated in CAFs and not in cancer cells. 85 proteins had 16-fold higher expression in CAFs compared to cancer cells. These included previously known CAF markers like ACTA2, FAP and SDC2. Novel biomarkers overexpressed in CAFs were noted, including HSPB6 and PTGS1. SiRNA knockdown of these genes encoding these proteins did not reduce contractility in lung CAFs, suggesting they were not crucial to this function despite high expression. Immunohistochemical analysis of 30 tumor samples (10 lung, 10 colorectal and 10 pancreatic) showed HSPB6 and PTGS1 expression were restricted to the stroma. Conclusions An unbiased analysis of the differences in the proteome of cancer cells compared to CAFs revealed increased expression of HSPB6 and PTGS1 in CAFs.

2023-08-21 | PXD040360 | Pride

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2023-03-11 | PXD024659 | Pride

Differentially secreted proteins in KMT5B deficient-cells compared to their isogenic clone KMT5B wild-type assessed by mass spectrometry

Project description:In patient-derived DIPG models from multiple patients, with both H3K27M mutation and EZHIP overexpression, were generated CRISPR/Cas9-engineered and subclonal isogenic cells with KMT5B and/or KMT5C deficiency.

2021-07-20 | PXD026670 | Pride

Multi-omics Analysis of Salmonella enterica Serovar Typhimurium Infection of Wild Type C57BL/6 and Immunodeficient gp91-/-phox Mice

Project description:Three groups of sex- and aged-matched 9-12 week old C57BL/6 wild type mice and gp91-/-phox mice were infected by i.v. injection of bacterial suspensions: Group 1 represents wild type C57BL/6 mice infected with virulent S. Typhimurium SL1344 grown in vitro. (Raw files: OTNCS_AGSL1344#3C#9C#10C-XX) Group 2 represents wild type C57BL/6 mice infected with virulent S. Typhimurium SL1344 grown in vivo for 72 h in the Group 1 C57BL/6 mice. (Raw files: OTNCS_AGSL1344#9F#10F-XX) Group 3 represents immunodeficient gp91-/-phox mice infected with virulent S. Typhimurium SL1344 grown in vitro. (Raw files: OTNCS_AGSL1344#11C#12C#13C-XX) And Input (Raw files: OTNCS_AGSL1344mixedAa-XX and OTNCS_AGSL1344mixedAb-XX)

2018-10-26 | PXD005855 | Pride

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