Dataset Information

PRODH regultes naive pluripotency by influencing mitochondrial oxidative phosphorylation (Untargeted metabolomics)

ABSTRACT:

Naïve human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naïve hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naïve hESCs, indicating that PRODH maintains naïve pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naïve pluripotency of hESCs.

Untargeted metabolomics - H9N PLKO.1 vs H9P PLKO.1 / H9P shPRODH vs H9P PLKO.1 / H9N shPRODH vs H9N PLKO.1 - is reported in the current study MTBLS7840.

Targeted metabolomics (amino acids and derivatives) - H9P (Primed) VS H9N (Naïve) - is reported in MTBLS7832.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Liquid Chromatography MS - positive - reverse phase

SUBMITTER: cheng chen

PROVIDER: MTBLS7840 | MetaboLights | 2024-02-12

REPOSITORIES: MetaboLights

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Publications

PRODH safeguards human naive pluripotency by limiting mitochondrial oxidative phosphorylation and reactive oxygen species production.

Chen Cheng C Liu Qianyu Q Chen Wenjie W Gong Zhiyuan Z Kang Bo B Sui Meihua M Huang Liming L Wang Ying-Jie YJ

EMBO reports 20240313 4

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent ...[more]

PMID: 38480845

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Project description:Pluripotency can be maintained in the naïve state through manipulation of ERK and WNT signalling (2i), shielding embryonic stem cells (ESCs) from inductive cues. Alternatively, inhibiting CDK8/19 (CDK8/19i), a repressor of the Mediator co-activator complex, directly stimulates super-enhancer activity, and was recently shown to stabilize cells in a functional state that resembles naïve pluripotency. Naïve ESCs exhibit important epigenetic, transcriptional and metabolic features. However, our understanding on how these regulatory layers are inter-connected to promote the naive state is in progress. To fill this gap, here we used mass spectrometry to describe the dynamic molecular events (i.e. phosphoproteome, proteome and metabolome) executed by 2i and CDK8/19i, as they transition cell identity into naïve pluripotency. We observed rapid proteomic reprogramming, revealing widespread commonalities, and some important differences, between these two approaches, suggesting a largely over-lapping mechanism. CDK8/19i acts directly on the control of the transcriptional machinery, which elicits a rapid and direct activation of key identity genes including those that maintain the naïve program. Additional molecular changes in 2i are achieved by phosphorylation of critical downstream effectors that reinforce the naïve transcriptional circuitry while repressing factors from the more-differentiated formative and primed states. Comparing transcriptomic and proteomic changes, we found that post-transcriptional de-repression is a major feature of naïve pluripotency conferred by both 2i and CDK8/19i, and this may support the enhanced mitochondrial capacity of naive cells. Furthermore, at the level of metabolome, while 2i- and CDK8/19i-treated cells share similar aspects in one-carbon metabolism and beta-oxidation, in other regards they are divergent, a feature which may explain their differences in DNA methylation. These datasets provide a valuable resource for exploring the molecular mechanisms underlying pluripotency and cell identity transitions.

Project description:PtomitAPX, which is a newly discovered ascorbate peroxidase (APX) specifically localized in the mitochondria of Populus tomentosa. Our antisense and overexpression studies are based on PtomitAPX. [Abstract] Plant mitochondria are major organelles of reactive oxygen species (ROS) production, are also targeted by ROS, which reduces mitochondrial efficiency and increases ROS production in a self-destructive cycle. Ascorbate peroxidase have pivotal roles in ROS-scavenging because even very low concentrations are sufficient for H2O2 decomposition We identified two ascorbate peroxidases, PtomitAPX and PtosAPX, are targeted to mitochondria in Populus tomentosa Carr. PtosAPX which is the homologous gene of arabidopsis thaliana sAPX, is also dual targeted to both chloroplast and mitochondria but is closer to chloroplastic isoform by ELISA and qPCR analysis. And PtomitAPX which is specifically targeted to mitochondria, is primary APX in mitochondria. The expression of PtomitAPX was significantly different from that of PtosAPX. PtomitAPX plays an important role in controlling the ROS balance and maintaining mitochondrial structure and function, thus regulating cell PCD and plant development in P opulus tomentosa Carr. The structure and function of mitochondria, particularly the efficiency of oxidative phosphorylation (OXPHOS), were impaired, cell PCD were increased and plant growth was retarded and partially restored by exogenous ASA in antisense PtomitAPX transgenic Populus,. The PtomitAPX expression level showed a strong relationship with ROS balance, mitochondrial structure, and plant growth. Our findings provide valuable insight into the unexplored mechanism of independent ROS balance by the ascorbate-glutathione cycle in mitochondria. Moreover, the data enhance understanding of the central role of mitochondria during plant growth, particularly in non-photosynthetic tissues of woody trees. Methods: Total RNA was isolated from wild type (WT), antisense (anti-3) and overexpression (OX) cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). The samples were sequenced using an Illumina Genome Analyzer (HiSeq™ 2000; Illumina, San Diego, CA). The raw reads were data-filtered to obtain high-quality clean reads. The clean reads were mapped to the P. trichocarpa reference genome and reference genes using SOAPaligner/SOAP2. No more than two mismatches were allowed in the alignment. Gene expression levels were calculated as reads per kilobase per million reads (RPKM). Differential expression analysis among the WT, anti-3 and OX was performed using the DEGseq R package based on normalized read counts. A corrected P value of < 0.001 and |log2Ratio| > 1.5 were set as the threshold for significantly differential expression. Results: Compared with the WT, 3571 genes with significantly different expression levels (fold change > 1.5 or < −1.5, and corrected P < 0.001) were detected in anti-3 and 95.58% (3413) of them reverted or partial reverted to WT in anti-3 treated with 1 mM AsA. RNA-seq analysis of WT, overexpression, and overexpression cells treated with 10 mM H2O2, were also employed. Compared with the WT, 5542 genes with significantly different expression levels(fold change > 1.5 or < −1.5, and corrected P < 0.001) were detected in OX and 66.47% (3684) of them were reverted or partial reverted to WT in OX treated with 10 mM H2O2.

Dataset Information

PRODH regultes naive pluripotency by influencing mitochondrial oxidative phosphorylation (Untargeted metabolomics)

Publications

PRODH safeguards human naive pluripotency by limiting mitochondrial oxidative phosphorylation and reactive oxygen species production.

Similar Datasets

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