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Effect of POR knockdown on the proteome of HepaRG cells

ABSTRACT: Cytochrome P450 oxidoreductase (POR) is involved in the oxidative metabolism of xenobiotics and endogenous compounds like fatty acid and cholesterol. While effects of diminished Por were extensively studied in mouse models, studies in human models are still missing. We used previously established CRISPR/Cas9-mediated POR knockdown in HepaRG cells as a human hepatic cell model to investigate the effects of POR knockdown on global protein expression levels.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Cell Culture

SUBMITTER: Thierry Schmidlin

LAB HEAD: Ute Hofmann

PROVIDER: PXD028577 | Pride | 2021-11-04

REPOSITORIES: Pride

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Proteomic analysis of human follicular fluid in poor ovarian responders during in vitro fertilization

Project description:Poor ovarian response (POR) observed in controlled ovarian stimulation (COS) is often observed during in vitro fertilization and embryo transfer (IVF-ET) cycles and is a major problem. A POR has been found to be related to several factors, including advanced age, high body mass index, and history of ovarian or pelvic surgery. However, it is difficult to predict POR, as there are no specific biomarkers known. In this study, we used quantitative proteomic analyses to investigate potential biomarkers that can predict poor response during IVF based on follicular fluid samples. A total of 1,079 proteins were identified using a high-resolution orbitrap mass spectrometer coupled online to a nano flow liquid chromatography system. It is notable that 65 up-regulated and 66 down-regulated proteins were found to be functionally enriched in poor responders. We also validated these differentially expressed proteins (DEPs) using a triple-quadrupole (QQQ) mass spectrometer for quantification of targeted proteins. Of the DEPs, three proteins (pregnancy zone protein, renin, and sushi repeat-containing protein SRPX) were regarded as statistically significant (p < 0.05). These proteins (one known as an inhibitor of cell growth and another as a regulator of angiogenesis) are potential biomarkers candidates of POR in IVF.

2018-07-05 | PXD005436 | Pride

The identification of bacterial protein interaction partners pointed out to new intracellular functions of Francisella tularensis glyceraldehyde-3-phosphate dehydrogenase

Project description:Glyceraldehyde-3-phopshate dehydrogenase (GAPDH) is well-known for its involvement in numerous non-metabolic processes inside the mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization and the ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there is so far only one study dealing with this issue. We previously reported several evidence that the GAPDH homologue of Francisella tularensis, GapA, might also exert additional non-enzymatic functions. To follow up our former observation, here we decided to find out GapA’s interacting partners within the bacterial proteome to explore its new roles at the intracellular level. The quantitative proteomics approach based on stable isotope labelling of amino acids in cell culture (SILAC) in combination with affinity purification and mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA, six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with the quantitative label-free comparative analysis of proteomes isolated from the wild-type strain and strain with deleted gapA gene suggests that the GapA is implicated in DNA repair processes. The absence of GapA promotes secretion of its most potent interaction partner, the hypothetical protein with peptidase propeptide domain (PepSY) indicating its impact on subcellular distribution of some proteins.

2020-08-31 | PXD019739 | Pride

Transcription profiling of colon, ileum, jejunum and liver from wild type and hepatic cytochrome P-450 reductase null mice treated with quercetin to identify differential transcriptional responses to quercetin.

Project description:This SuperSeries is composed of the following subset Series: GSE4256: Colon transcriptional response to quercetin in WT and POR-null mice; GSE4257: Ileum transcriptional response to quercetin in WT and POR-null mice; GSE4258: Jejunum transcriptional response to quercetin in WT and POR-null mice; GSE4259: Liver transcriptional response to quercetin in WT and POR-null mice Experiment Overall Design: Refer to individual Series

2007-11-06 | E-GEOD-4262 | biostudies-arrayexpress

Effects of Cellular Senescence on Human Mesenchymal Stem Cells

Project description:In the present study we analyzed the effect of cellular senescence on the transcriptome of human mesenchymal stem cells (hMSC; alternatively named mesenchymal stromal cells) from bone marrow. Human MSC were isolated from femoral heads of non-osteoporotic donors after total hip arthroplasty. Cells were isolated from human bone marrow according to the previously described protocol (Noth et al., 2002, J Orthop Res, 20/5, 1060-1069) under agreement of the local Ethics Committee of the University of Würzburg. Bone marrow was obtained of femoral heads after total hip arthroplasty due to osteoarthritis and/or hip dysplasia. MSC were replated after reaching 70-90% confluence until they entered state of cellular senescence. RNA samples of control cells were taken from passage 1 or passage 2.

2012-02-22 | E-GEOD-35957 | biostudies-arrayexpress

Effects of aging, primary osteoporosis, and cellular senescence on Human Mesenchymal Stem Cells

Project description:This SuperSeries is composed of the following subset Series: GSE35955: Effects of aging on Human Mesenchymal Stem Cells GSE35956: Effects of Primary Osteoporosis and Advanced Age on Human Mesenchymal Stem Cells GSE35957: Effects of Cellular Senescence on Human Mesenchymal Stem Cells GSE35958: Effects of Primary Osteoporosis on Human Mesenchymal Stem Cells Refer to individual Series

2012-02-22 | E-GEOD-35959 | biostudies-arrayexpress

Endothelial deletion of the cytochrome P450 reductase leads to cardiac remodelling

Project description:The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR-/-). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR-/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR-/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling.

2022-12-21 | GSE214776 | GEO

Expression data of embryonic kidney from MWF E15.5 and SHR 16.0 rats

Project description:Defects in nephrogenesis can have detrimental effects on cardiovascular and renal health in adult life. This is confirmed by observations in the Munich Wistar Frömter (MWF) rat that exhibits a congenital nephron deficit and renal failure with age. We performed genome-wide transcriptome analysis in embryonic kidneys to identify candidate genes for the reduced nephron number in MWF. We compared MWF E15.5 with stage-matched spontaneously hypertensive rats (SHR) at E16. Microarray analysis revealed 311 transcripts representing 253 known genes with differential expression between MWF and SHR (FC >+1.5 or <-1.5, FDR<0.05). 8 samples were analyzed, n=4 per group

2015-07-01 | E-GEOD-66079 | biostudies-arrayexpress

Synthesis of chlorophyll-binding proteins in a fully-segregated ∆ycf54 strain of the cyanobacterium Synechocystis PCC 6803

Project description:The MS analysis is part of a wider study into chlorophyll biosynthesis. There is previous evidence, from both pulldown and CN-PAGE/immunoblot analyses that the enzyme Mg-protoporphyrin IX methylester cyclase (Cyc1), which catalyses the formation of the the fifth (E) ring in chlorophyll, associates with a protein of unknown function called Ycf54. There is also evidence for the association of Cyc1 with other enzymes in the chlorophyll biosynthetic pathway, eg. POR, ChlP and DVR. In the MS part of this study, FLAG-tagged Cyc1 was used to capture proteins from a Synechocystis cell lysate, with a comparison of FLAG-Cyc1 strains against wild-type and Ycf54 deletion backgrounds. MS-based quantification of the amounts of POR, ChlP and DVR relative to the Cyc1 bait supported evidence by immunoblotting of a significant reduction in these co-isolated proteins in the Ycf54 deletion strain.

2016-04-01 | PXD003149 | Pride

Novel targets for the transcription factors MEF2 in MA-10 Leydig cells

Project description:Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) is a transcription factor recently identified in somatic cells of the male gonad. In other tissues, MEF2 is an essential regulator of organogenesis and cell differentiation. So far in the testis, MEF2 was found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MA-10 Leydig cells depleted in MEF2 and results were analyzed using the Partek and IPA softwares. Several genes were differentially expressed in MEF2-depleted Leydig cells and 15 were validated by qPCR. A large number of these genes are known to be involved in fertility, gonad morphology and steroidogenesis and include Pde8a, Por, Ahr, Bmal1, Cyp1a1, Cyp1b1, Map2k1, Tsc22d3, Nr0b2, Smad4, and Star, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2 binding sites within the first 2 kb upstream the transcription start site of the Por, Bmal1, and Nr0b2 promoters, which suggests a direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, siRNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1 and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction. MA-10 Leydig cells were treated with siRNA MEF2A/2D (siRNA MEF2) or scrambled siRNA as control (siRNA Ctrl) 48h before total RNA extraction.

2015-04-12 | E-GEOD-64128 | biostudies-arrayexpress

Genome-wide analysis of gene expression response upon infection with Toxoplasma gondii

Project description:The in vitro effect of infection with different strains of Toxoplasma gondii was tested 24 hours after infection of Human Foreskin Fibroblasts (HFF) The strains tested include RH, VEG, and transgenic strains of RH overexpressing ROP38 or ROP21 Total RNA of Toxoplasma gondii infected HFF cell was compared to uninfected cells

2010-07-31 | E-GEOD-22402 | biostudies-arrayexpress

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