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Temporal profiling of redox-dependent heterogeneity in single cells

ABSTRACT: The cellular redox status plays an essential role in diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus even cells with identical genetic background but different redox status behave differently. Here, we propose a non-laborious and robust methodology to quantify cell-to- cell redox-dependent variability within thousands of cells, detect oxidation levels in cytosol, mitochondria, and peroxisome, and sort cells based on their redox status. We report that cells of the same age have a bi-modular distribution of reduced and oxidized cells starting in the late logarithmic phase. Although the ratio between the oxidized and reduced subpopulations changes during chronological aging, their growth, proteomic and transcriptomic features are conserved during their first days of chronological aging. A comparative proteomic analysis identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE112997 | GEO | 2018/06/17

REPOSITORIES: GEO

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Temporal profiling of redox-dependent heterogeneity in single cells

Project description:Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe roGFP. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach we defined redox-dependent heterogeneity of yeast cells, and characterized growth, as well as proteomic and transcriptomic profiles of subpopulations of cells that differ in their redox status, but are similar in age. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

2018-06-18 | PXD009443 | Pride

Genome-Wide Study Based On Abasic Site Revealed Selective RNA Oxidation In Neuro 2a Cells

Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale. Neuro 2a cells received hydrogen peroxide treatment or no treatment as a control. Samples were applied for RNA extraction and ARP labeling, which could bind with apurinic/apyrimidinic sites, and then a pull-down process to isolate oxidized RNA. Total RNA and oxidized RNA were used for subsequent transcriptomic profiling. 4 types of samples were analyzed: Basal-total: untreated N2a cells labeled with ARP, but not processed for the pull-down assay. Ox-total: hydrogen peroxide-treated N2a cells labeled with ARP, but not processed for the pull-down assay. Basal-ARP: untreated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis. Ox-ARP: hydrogen peroxide-treated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis.

2012-10-01 | E-GEOD-39641 | biostudies-arrayexpress

Genome-Wide Study Based On Abasic Site Revealed Selective RNA Oxidation In Neuro 2a Cells

2012-10-01 | GSE39641 | GEO

Genome-Wide Study Based On Abasic Site Revealed Selective RNA Oxidation In Neuro 2a Cells

2012-10-01 | GSE39641 | GEO

CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Project description:Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function, urging the need for accurate cysteine oxidation quantification to decode protein redox regulation. Here, we present a novel approach called CysQuant that enables simultaneous quantification of cysteine oxidation degrees and protein abundancies. Reduced and reversibly oxidized cysteines are differentially labeled with light and heavy iodoacetamide isotopologues and analyzed using data-dependent acquisition (DDA) or data-independent acquisition (DIA) mass spectrometry. Using in silico predicted spectral libraries, plexDIA quantified on average 18% oxidized in the model plant Arabidopsis thaliana, though revealed a subset of highly oxidized cysteines part of disulfide bridges in AlphaFold2 predicted protein structures. Studying protein redox regulation of plant seedlings in response to excessive light, CysQuant successfully identified the well-established increased reduction of Calvin-Benson cycle enzymes, in addition to discovery of other, yet uncharacterized redox-sensitive disulfides in plastidial enzymes. CysQuant is widely applicable to diverse mass spectrometry platforms studying the cysteine modification status.

2023-07-21 | PXD043895 | Pride

Quantifying changes in the bacterial thiol redox proteome during host-pathogen interaction

Project description:In this study, we used a quantitative redox proteomic method (OxICAT) to assess the in vivo thiol oxidation status of phagocytized E. coli. The majority (65.5%) of identified proteins harbored thiols that were significantly oxidized (>30%) upon phagocytosis. A substantial number of these proteins are from major metabolic pathways or are involved in cell detoxification and stress response, suggesting a systemic breakdown of the bacterial cysteine proteome in phagocytized bacteria.

2019-01-07 | PXD011386 | Pride

Oxicam-type nonsteroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway

Project description:Nonsteroidal anti-inflammatory drugs (NSAIDs), including salicylic acid (SA), target mammalian cyclooxygenases. In plants, SA is a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identify that the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent plant immune response. TNX treatment decreases NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. A cysteine labeling assay reveals that cysteine residues in NPR1 can be oxidized in vitro, leading to disulfide-bridged oligomerization of NPR1, but not in vivo regardless of SA or TNX treatment. Therefore, this study indicates that oxicam inhibits NPR1-mediated SA signaling without affecting the redox status of NPR1.

2021-11-09 | GSE154593 | GEO

Project description:yeast chronological aging

| PRJNA544860 | ENA

SZ 95 sebocytes - OLR1 knockdown - treated with UV-oxidized PAPC

Project description:Human SZ95 sebocytes were transfected by lipofection with either scramble siRNA or OLR1 siRNA and after 24h were treated with 25µg/ml of UV-oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphocholine (UVPAPC) for 7 hours. Phospholipid oxidation products (OxPL) are versatile stress signaling mediators in the skin. These lipid signaling molecules can be generated non-enzymatically or enzymatically by ultraviolet light, the major extrinsic skin aging factor. OxPL regulate cytoprotective, immunological and metabolic adaptation of the skin to oxidant stress. We here investigated whether the scavenger receptor Oxidized Low Density Lipoprotein Receptor 1 (OLR1, LOX-1) would have a function in cutaneous oxPL signaling. We found that OLR1 is expressed in several cutaneous cell types, most prominently in cells of the sebaceous gland and in keratinocytes. We repressed OLR1 expression with siRNA in SZ95 sebocytes, exposed cells to oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatiylcholine (PAPC) and performed transcriptomic profiling. Bioinformatic analysis revealed that OxPL exposure induced the Nrf2 antioxidant stress response and aldosterone signaling. The analysis also revealed that OLR1 is not required for the transcriptional regulation induced by oxidized PAPC, but interestingly, OLR1 knockdown affected expression of CNN2, HMRR, ITGB6 and KIF20A, all genes governing cell proliferation and motility. We identify sebocytes as cutaneous cells responsive to lipid mediated redox stress which is not dependent on the scavenger receptor OLR1.

2020-04-15 | GSE98335 | GEO

SIRT1 prevents R-loops during chronological aging by modulating DNA replication at rDNA loci

Project description:In metazoans, the largest sirtuin, SIRT1, is a nuclear protein implicated in epigenetic modifications, circadian signaling, DNA recombination, replication and repair. Our previous studies have demonstrated that SIRT1 binds replication origins and inhibits replication initiation from a group of potential initiation sites (dormant origins). We studied the effects of aging and SIRT1 activity on replication origin usage and the incidence of transcription-replication collisions (creating R-loop structures) in adult human cells obtained at different time points during chronological aging and in cancer cells. In primary, untransformed cells, SIRT1 activity declined, and the prevalence of R-loops rose with chronological aging. Both the reduction of SIRT1 activity and the increased abundance of R-loops were also observed during the passage of primary cells in culture. All cells, regardless of donor age or transformation status, reacted to short-term, acute chemical inhibition of SIRT1 with the activation of excessive replication initiation events coincident with an increased prevalence of R-loops. However, only cancer cells showed genome-wide activation of dormant origins during long-term proliferation with mutated or depleted SIRT1, whereas in primary cells, aging-associated SIRT1-mediated activation of dormant origins was restricted to rDNA loci. These observations suggest that chronological aging and the associated decline in SIRT1 activity relaxes the regulatory networks that protect cells against excess replication and that the mechanisms protecting from replication-transcription collisions at the rDNA loci manifest as a differentially enhanced sensitivity to SIRT1 decline and chronological aging.

2023-11-27 | GSE247469 | GEO

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