Dataset Information

Chemoproteomic Profiling of Reactive Cysteines in Response to Oxidative Stress Induced by 6-Hydroxydopamine

ABSTRACT: Oxidative stress is a well-known driver of neurodegeneration, yet it remains poorly understood how the global cysteine (Cys) proteome is remodeled in such conditions. Proteins with aberrantly modified Cys in response to oxidative stress can induce and exacerbate neurodegeneration. In this study, we induced oxidative stress by treating SH-SY5Y neuronal cells with the neurotoxin 6-hydroxydopamine (6-OHDA). To identify proteins with Cys that had altered oxidation or post-translational modification statuses, we utilized a desthiobiotin iodoacetamide (DBIA) probe, which selectively labels Cys with unmodified and preserved thiols. We identified numerous proteins that exhibited reduced or enhanced Cys reactivity to DBIA in response to 6-OHDA-induced oxidative stress, many of which are critically involved in biological processes linked to cell stress responses (e.g., mitochondrial oxidative stress and apoptosis). Our study defines the remodeling of the Cys proteome and suggests potential Cys-mediated regulatory mechanisms under 6-OHDA-induced oxidative stress conditions. In the context of neurodegeneration and under these conditions, our findings provide a valuable resource for further exploration of the Cys proteome.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Rayan Murtada

LAB HEAD: Yonghao Yu

PROVIDER: PXD060980 | Pride | 2025-10-20

REPOSITORIES: Pride

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Chemoproteomic Profiling of Reactive Cysteines in Response to Oxidative Stress Induced by 6-Hydroxydopamine.

Murtada Rayan R Kim Chiho C Wang Xu-Dong XD Yang Youkai Y Yu Yonghao Y

Proteomics 20250925

Although oxidative stress is a well-established driver of neurodegeneration, it remains poorly understood as to how the global cysteine (Cys) proteome is remodeled under oxidative stress conditions. Proteins with aberrantly modified cysteines in response to oxidative stress can induce and exacerbate neurodegeneration, contributing to disorders like Alzheimer's, Parkinson's, frontotemporal dementia, and amyotrophic lateral sclerosis. In this study, we induced oxidative stress in SH-SY5Y neuronal ...[more]

PMID: 40999799

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Project description:Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis as well as to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. To assess the response to chemicals at different stages of development, zebrafish (Danio rerio) embryos at 1, 2, 3, 4, 5, and 6 days post-fertilization (dpf) were exposed to 0.1% dimethyl sulfoxide (DMSO), 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 10 uM tert-butylhydroquinone (tBHQ) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray (Agilent 22k). By microarray, 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated embryos at 4 dpf. Of these, 220 were 2-fold or greater up-regulated and 108 were 2-fold or greater down-regulated by tBHQ. For TCDD, 17 probes were 2-fold or greater up-regulated and 6 were 2-fold or greater down-regulated. Genes induced by tBHQ, included genes involved in glutathione synthesis and utilization, signal transduction, and DNA damage / stress response. Keywords: Danio rerio, TCDD, tBHQ, oxidative stress, gene expression Separate groups of 30 embryos generated from TL adults were placed in 15 ml egg water in a 10-cm glass petri and at 1, 2, 3, 4, 5, and 6-dpf were exposed for 6 hr to 0.1% dimethyl sulfoxide (DMSO), 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 10 uM tert-butylhydroquinone (tBHQ) (4 replicates per compound per time point). Embyros were frozen immediately after the 6 hr exposure and RNA isolated for microarray analysis. A Universal RNA Reference Sample was created by mixing equal amounts of total RNA from 2 replicates each from all toxicants (TCDD, tBHQ, DMSO) and timepoints (1, 2, 3, 4, 5, 6-dpf). Based on qRT-PCR results, the 4 dpf timepoint was selected for microarray analysis, in which the RNA samples (4 replicates per compound) were Cy3 labeled and co-hybridized with Cy5 labeled Universal RNA Reference Sample to generate data for analysis of variance.

Project description:Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in Cys biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidposis thaliana. Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favour of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to Cd. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under non-stress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. To further elucidate the specific function(s) of the OAS-A1 isoform in the adaptation response to cadmium we extended the trasncriptome experiment to the wild type and oas-a1.1 mutant plants exposed to Cd. The comparison of transcriptomic profiles showed a higher proportion of genes with altered expression in the mutant than in the wild type, highlighting up-regulated genes identified as of the general oxidative stress response rather than metal-responsive genes. Wild type and oas-a1.1 mutant plants were grown hydroponically and, after a two-week acclimation period, the roots and shoots were harvested separately. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. We made two different comparisons to classify the differently expressed genes in the mutant plant: oas-a1.1 roots versus wild-type roots and oas-a1.1 shoots versus wild-type shoots. Hydroponically-grown wild type and oas-a1.1 mutant plants were further treated with 50µM CdCl2 and 18h-treated-roots and 24h-treated-shoots were harvested. Total RNA was then prepared and analyzed using the Affymetrix-Arabidopsis ATH1GeneChip array. Three biological replicates were performed for each sample. Different comparisons were performed as follows: 18h Cd-treated wild type roots versus untreated wild type roots; 24h Cd-treated wild type shoots versus untreated wild type shoots; 18h Cd-treated oas-a1.1 roots versus untreated oas-a1.1 roots; 24h Cd-treated oas-a1.1 shoots versus untreated oas-a1.1 shoots; 18h Cd-treated oas-a1.1 roots versus 18h Cd-treated wild type roots; 24h Cd-treated oas-a1.1 shoots versus 24h Cd-treated wild type shoots

			Action	DRS
	6OHDA_wcl.xlsx	Xlsx
	6OHdopa.xlsx	Xlsx
	EC00824.raw	Raw
	EC00825.raw	Raw
	EC00826.raw	Raw