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Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-nonenal Modifications in Cells

ABSTRACT: 4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we de-scribe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analysis reveals four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adducts to cysteine. ONE-derived adducts co-localize and crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. In summary, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jing Yang

LAB HEAD: Jing Yang

PROVIDER: PXD006680 | Pride | 2022-02-24

REPOSITORIES: Pride

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4-Oxo-2-nonenal Adductions in Cells

Project description:4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we de-scribe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analysis reveals four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adducts to cysteine. ONE-derived adducts co-localize and crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. In summary, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile.

2017-08-25 | PXD007149 | Pride

Detection of Dichlorvos Aducts in Hepatocyte Cell Line LC-MS/MS

Project description:Toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP causes cellular perturbation by modifying non-cholinesterase targets. Here, we identify novel DDVP-modified targets in lysates of a human hepatocyte-like cell line (HepaRG) using a direct LC-MS assay of DDVP exposed lysates or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. Using these strategies, we show that DDVP forms adduct to several proteins important to the cellular metabolic pathways and cellular differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues.

2014-07-31 | PXD001107 | Pride

Identification of mRNAs modulated by knockdown of CD24

Project description:We endeavored to identify factors and pathways regulated by CD24 and to obtain leads regarding the mechanism by which CD24 inhibited the invasiveness and metastasis of pancreatic cancer cells by performing cDNA microarray analysis on S2-013 CD24 RNAi clones and control clones. The group of genes showing increased expression was mainly comprised of genes for molecules with functions related to RNA metabolism, transcription factors, proteases, and molecules involved in embryonic development and cell growth. The set of genes downregulated by knockdown of CD24 included the target mRNAs of a phosphorylation-dependent endoribonuclease G3BP that interacted with CD24. The target mRNAs of G3BP were identified in GSE17056. These results suggested that CD24 inhibited the RNase activity of G3BP, and that a function of CD24 was to inhibit the degradation of specific mRNAs. Transcripts upregulated or downregulated by knockdown of CD24 were identified through expression profiling of a total of 12,135 genes in S2-013 CD24 RNAi clones compared to control clones. Before labeling with Cy5 or Cy3, total RNA from two S2-013 CD24 RNAi clones were mixed and total RNA from two control clones were mixed. The supplementary files 'GSE17055_higher_siCD24.txt' and 'GSE17055_higher_control.txt' list the differentially expressed genes.

2010-06-24 | E-GEOD-17055 | biostudies-arrayexpress

Dissecting the antibacterial activity of Oxadiazolone-core derivatives against Mycobacterium abscessus

Project description:Mycobacterium abscessus is nowadays under the spotlight of the scientific community. This pathogenic mycobacteria is indeed responsible for a wide spectrum of infections involving mostly pulmonary infections in patients with cystic fibrosis. M. abscessus is intrinsically resistant to a broad range of antibiotics, including most antitubercular drugs, and is considered the most pathogenic and chemotherapy-resistant rapidly growing mycobacterium. Consequently, with very limited treatment options, the development of new therapeutic approaches to fight this pathogen are urgently needed. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs were active against extracellular M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., iBpPPOX via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis.

2020-08-14 | PXD015680 | Pride

Cyclipostins and Cyclophostin analogs impair growth of Mycobacterium abscessus by inhibiting enzymes involved in many physiological processes

Project description:Mycobacterium abscessus is nowadays under the spotlight of the scientific community. This pathogenic mycobacteria is indeed responsible for a wide spectrum of infections involving mostly pulmonary infections in patients with cystic fibrosis. M. abscessus is intrinsically resistant to a broad range of antibiotics, including most antitubercular drugs, and is considered the most pathogenic and chemotherapy-resistant rapidly growing mycobacterium. Consequently, with very limited treatment options, the development of new therapeutic approaches to fight this pathogen are urgently needed. 38 new analogs of Cyclipostins & Cyclophostin (CyC), compounds naturally produced by Streptomyces species, have been synthesized. Their antibacterial activities against clinical isolates belonging to the M. chelonae-abscessus clade, as well as Gram-negative and Gram-positive bacteria have been evaluated by the REMA method. The intracellular activities of the CyC against intramacrophagic M. abscessus have also been investigated and compared to those of imipenem. The CyCs displayed very low toxicity towards host cells and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate, CyC17, showed a high selectivity for mycobacteria with MIC values (<2 up to 40 µg/mL) comparable to those of most classical antibiotics used to treat M. abscessus infections. Of importance, several CyCs were active against extracellular M. abscessus growth (i.e., CyC17 / CyC18β / CyC25 / CyC26) or against intracellular mycobacteria inside macrophages (i.e., CyC7α,β / CyC8α,β) with MIC values similar to or better than those of standard antibiotics. Based on these results, we intended to identify the potential target enzymes of CyC17/CyC26 in M. abscessus by activity-based protein profiling (ABPP) approach coupled with mass spectrometry differential analysis.

2019-10-08 | PXD014255 | Pride

Identification of differential gene expression modulated by knockdown of MUC1 in MKN45 gastric carcinoma cells

Project description:Transcripts upregulated or downregulated by knockdown of MUC1 were identified through expression profiling of a total of 12,135 genes in comparison with MKN45- MUC1 RNAi clones and control clones. We endeavored to identify genes which expression is affected by MUC1 by performing cDNA microarray analysis on two MKN45 MUC1 RNAi clones and one control clone.

2011-11-15 | E-GEOD-28975 | biostudies-arrayexpress

Identification of mRNAs modulated by overexpression of N-terminal G3BP

Project description:Transcripts upregulated or downregulated by overexpression of N-terminal G3BP were identified through expression profiling of a total of 12,135 genes in comparison with S2-013 clones in which N-terminal G3BP is overexpressed and control clones. We endeavored to identify factors and pathways regulated by CD24 and to obtain leads regarding the mechanism underpinning the observed phenotypic changes by performing cDNA microarray analysis on two S2-013 CD24 RNAi clones and two control clones. Total RNAs from two clones of S2-013 CD24 RNAi or control cells were mixed and used for cDNA microarray analysis.

2010-07-31 | E-GEOD-23342 | biostudies-arrayexpress

Analysis of Spt16 depletion

Project description:Transcription rate analysis in Yeast by means of GRO, mRNA amount and ChIP-on-chip during the depletion of Spt16p. Keywords: Genomic run-on GRO ChIP-chip Transcription rate analysis by means of GRO and mRNA amount (RA) of three independent replicates during the depletion of Spt16p (Control and 5 & 7 hours after the depletion). Each time point replicate has been hybridized on a different macroarray (F14-F16). ChIP-on-chip analysis of Spt16 were done during exponential grow in YPD.

2010-06-24 | E-GEOD-14082 | biostudies-arrayexpress

Peniophora incarnata T-7 LC-MSMS

Project description:To explore the mechanism of selective degradation of lignocellulose components by P. incarnate T-7, TMT labeling quantitative proteomic analysis was performed to explore the differential expression of secretory proteins of P. incarnate T-7 by submerged fermentation on poplar wood substrate relative to glucose substrates

2021-09-10 | PXD025248 | Pride

GRO analysis of rap1-sil, tpk1 & tpk2.

Project description:GRO (Genomic run-on) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: Genomic run-on GRO There are 4 different strains: rap1-sil (without the silencing domain), RAP1(both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). For each experiment there are GRO data (Transcription Rate) and gDNA data used for normalizing the GRO signals. The last number of the GRO filters correspond to the number of gDNA filters.There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.

2010-06-24 | E-GEOD-14080 | biostudies-arrayexpress

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