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Affinity purification of HA-tagged Chromodomain helicase DNA-binding protein 6 in HEK293T cells

ABSTRACT: Affinity purification and mass spectrometry analysis of HA-tagged CHD6 protein overexpressed in HEK293T cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Luc Provencher

LAB HEAD: Aaron Goodarzi

PROVIDER: PXD057410 | Pride | 2025-05-07

REPOSITORIES: Pride

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			Action	DRS
	F007539.mgf	Mgf
	F007540.mgf	Mgf
	F007543.mgf	Mgf
	F007544.mgf	Mgf
	F007547.mgf	Mgf

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1 - 5 of 26

Publications

CHD6 has poly(ADP-ribose)- and DNA-binding domains and regulates PARP1/2-trapping inhibitor sensitivity via abasic site repair.

Provencher Luc L Nartey Wilson W Brownlee Peter M PM Atkins Austin W AW Gagné Jean-Philippe JP Baudrier Lou L Ting Nicholas S Y NSY Piett Cortt G CG Fang Shujuan S Pearson Dustin D DD Moore Shaun S Billon Pierre P Nagel Zachary D ZD Poirier Guy G GG Williams Gareth J GJ Goodarzi Aaron A AA

Nature communications 20250125 1

To tolerate oxidative stress, cells enable DNA repair responses often sensitive to poly(ADP-ribose) (PAR) polymerase 1 and 2 (PARP1/2) inhibition-an intervention effective against cancers lacking BRCA1/2. Here, we demonstrate that mutating the CHD6 chromatin remodeler sensitizes cells to PARP1/2 inhibitors in a manner distinct from BRCA1, and that CHD6 recruitment to DNA damage requires cooperation between PAR- and DNA-binding domains essential for nucleosome sliding activity. CHD6 displays dire ...[more]

PMID: 39863586

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Pseudomonas aeruginosa PA3904 pull down coupled to LC-MS/MS

Project description:pETDUET His-PAAR4 and pETDUET Flag-PAAR4 were transformed into BL21 E. coli DE3 and expressed as described above. An equal volume of PAO1 retS was grown to an OD600 of 2.0. E. coli expressing His-PAAR4 or Flag-PAAR4 and Pseudomonas cells were harvested by centrifugation and lysed as described above. Lysates of both species were combined and His-PAAR4 or Flag-PAAR4 were purified as before. Eluted sample was mixed with SDS-loading buffer, boiled and run on a 12% SDS-PAGE for 0.6 cm. After coomassie staining and de-staining, the resulting band was excised and used for LC-MS/MS.

2018-03-09 | PXD008829 | Pride

Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography-Tandem Mass Spectrometry

Project description:We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography-tandem mass spectrometry. Using a next-generation proteomic pipeline with a <1.0% false discovery rate at the peptide and protein levels, we identified 3,731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2,000 additional single amino acid variants.

2018-08-17 | PXD010025 | Pride

Identification of oxidized cysteine residues in signaling proteins in U937 cells at basal levels and upon diamide and hypochlorous acid stimulations.

Project description:Here, we report a detection of oxidative post-translational modifications (ox-PTM) of Cys residues in the U937 cell line either non-stimulated or stimulated with diamide, a thiol-oxidizing agent at 0.5mM for 20min or HOCl at 100µM for 10min. Reversible Cys ox-PTMs were labelled using a bioswitch method using a Maleimide-(Polyethylene Glycol)2-Biotin (Mal-PEG2-Bio) probe independently of the oxidation type. Labeled proteins were analyzed by Mass spectrometry. We identified Cys ox-PTMs encompassing 1473 proteins containing at least ox-PTM-modified Cys. These sites include ox-PTM sites previously described in vitro validating the approach. Numerous novel Cys susceptible to ox-PTMs were identified including signaling proteins, including kinases, phosphatases and transcription and translation factors and proteins relevant to virus-host interactions.

2023-03-10 | PXD020270 | Pride

The chromatin modifier CHD8 targets autism risk genes during human neurodevelopmentM-BM-

Project description:Whole-exome sequencing studies have implicated chromatin modifiers and transcriptional regulators in autism spectrum disorder (ASD) through the identification of de novo loss of function mutations in affected individuals. Many of these genes are co-expressed in mid-fetal human cortex, suggesting ASD risk genes converge in regulatory networks that are perturbed in ASD during neurodevelopment. To elucidate such networks we mapped promoters and enhancers bound by the chromodomain helicase CHD8, which is strongly enriched in ASD-associated de novo loss of function mutations, using ChIP-seq in mid-fetal human brain, human neural stem cells (hNSCs), and embryonic mouse cortex. We find that CHD8 targets are strongly enriched for ASD risk genes that converge in ASD-associated co-expression networks in human midfetal cortex. CHD8 knockdown in hNSCs results in significant dysregulation of ASD risk genes targeted by CHD8, as well as additional genes important for neurodevelopment, including members of the Wnt/M-NM-2-catenin signaling pathway. Integration of CHD8 binding data with genetic and gene co-expression data in ASD risk models provides support for additional ASD risk genes. Together, our results suggest that loss of CHD8 function contributes to ASD through regulatory perturbation of other ASD risk genes during human cortical development. Two biological replicates for each ChIP with appropriate Input control Four biological replicates for each condition in knockdown experiments (Ctrl construct, Chd8 target C, and Chd8 target G)

2015-01-21 | E-GEOD-57369 | biostudies-arrayexpress

SARS-CoV-2 Evades immune detection in Alveolar Macrophages

Project description:Respiratory infections, like the current pandemic SARS-CoV-2 virus, target the epithelial cells in the respiratory tract. However, alveolar macrophages (AMs) are tissue-resident macrophages located within the alveoli of the lung and they play a key role in the early phases of an immune response to respiratory infections. We expect that AMs are the first immune cells to encounter the SARS-CoV-2 and therefore their reaction to SARS-CoV-2 infection will have a profound impact upon the outcome of the infection. Interferons (IFNs) are antiviral cytokines and the first cytokine produced upon viral infection. Here, we challenge AMs with SARS-CoV-2 and to our surprise find that the AMs are incapable of recognising SARS-CoV-2 and produce IFN. This is in contrast to respiratory pathogens, such as influenza A virus and Sendai virus. Callenge of AMs with those viruses resulted in a robust IFN response. The absence of IFN production by AMs upon challenge could explain the initial asymptotic phase of SARS-CoV-2 infections and argues against the AMs as the source of proinflamatory cytokines later in infection.

2020-10-20 | PXD021685 | Pride

C-terminal glycosylation of type IX secretion system cargo proteins in Prevotella intermedia bearing both short and long secretion signals

Project description:Prevotella intermedia is a Gram-negative bacterium that is notably linked to periodontitis and acute necrotizing ulcerative gingivitis. P. intermedia is known to utilise the T9SS to secrete and anchor virulence factors to the cell surface, presumably via C-terminal modification of the cargo protein with a cell surface polysaccharide. The identity of the linking sugar and the sites of modification on the cargo have yet to be determined. Here, we first employed Hidden Markov Models to predict cargo proteins in P. intermedia, and then conducted LC-MS/MS analyses of partially deglycosylated fractions to characterise the C-terminal glycosylation. A total of 80 cargo proteins were predicted based on the presence of a T9SS C-terminal domain signal (CTD), and these were divided into 48 short CTDs predicted by AlphaFold to adopt a single 3-stranded β-sheet and 32 long CTDs predicted to form the more typical larger β-sandwich structures. Cleavage sites for five short CTDs and four long CTDs were experimentally determined, and glycosylation was observed at the mature C-terminus of six cargo. Two glycans were identified of delta masses 419.198 and 433.185 Da, corresponding to novel linkages to N-alanyl dHex-HexNAc and N-alanyl (Me-dHex)-HexNAc, respectively, with the alanine amide-linked to the protein C-terminus. This indicated that both short and long CTDs were cleaved and glycosylated. AlphaFold multimer modelling predicted that both kinds of CTDs could bind to the PorV shuttle protein in the same manner, with the conserved CTD motifs interacting with the same sites in PorV.

2025-05-07 | PXD057272 | Pride

Differential RNA aptamer affinity profiling on plasma as a potential diagnostic tool for bladder cancer

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Willaertia magna c2c maky global proteome

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2021-09-08 | PXD016724 | Pride

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

Project description:LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective and closely related PPM1J/M exhibit virtually no activity toward substrates such as Rab8a phosphorylated at T72 (pT72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a conserved catalytic domain that adopts a β-sandwich fold. The striking difference is that PPM1H has evolved a 110-residue flap domain that punctuates the catalytic domain. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pT72 of Rab8a with a higher specific activity than PPM1H. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

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2012-05-28 | E-GEOD-16646 | biostudies-arrayexpress

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