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SILAC-based quantitative Nox4-Co-immunoprecipitation

ABSTRACT: Within the family of NADPH oxidases, Nox4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity and generates H2O2. We hypothesize that these features are consequences of a so far unidentified Nox4-interacting protein. Interacting proteins were screened by quantitative SILAC-Co-immunoprecipitation in HEK293 cells stably overexpressing Nox4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ralf Brandes

PROVIDER: MSV000080772 | MassIVE | Wed Mar 29 12:50:00 BST 2017

SECONDARY ACCESSION(S): PXD003514

REPOSITORIES: MassIVE

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Publications

The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein.

Prior Kim-Kristin KK Wittig Ilka I Leisegang Matthias S MS Groenendyk Jody J Weissmann Norbert N Michalak Marek M Jansen-Dürr Pidder P Shah Ajay M AM Brandes Ralf P RP

The Journal of biological chemistry 20160209 13

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino a ...[more]

PMID: 26861875

Similar Datasets

SILAC-based quantitative Nox4-Co-immunoprecipitation

Project description:Within the family of NADPH oxidases, Nox4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity and generates H2O2. We hypothesize that these features are consequences of a so far unidentified Nox4-interacting protein. Interacting proteins were screened by quantitative SILAC-Co-immunoprecipitation in HEK293 cells stably overexpressing Nox4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction.

2016-02-17 | PXD003514 | Pride

Complexome profiling of membrane fraction from Nox4 overexpressing HEK293 cells

Project description:Within the family of NADPH oxidases, Nox4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity and generates H2O2. We hypothesize that these features are consequences of a so far unidentified Nox4-interacting protein. Blue native gel electrophoresis of solubilized membranes from HEK293 cells (overexpressing Nox4) separated macromolecular complexes containing Nox4. The combination of native gel electrophoresis and quantitative mass spectrometry was applied to identify proteins that co-migrate with Nox4. In addition, the data set contains information about macromolecular protein complexes in human cellular membranes that are stable to the solubilization with digitonin.

2017-04-07 | PXD003509 | Pride

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4

Project description:PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H2O2 metabolized by Peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. As such, this study has identified NOX4 as a protein which paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.

2021-05-25 | GSE166165 | GEO

The reducing equivalent NADPH dictates histone acetylation via direct inactivation of HDAC3

Project description:NADPH has been long well-recognized as a key cofactor for antioxidant defense and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We found that reduction of cellular NADPH levels by silencing malic enzyme (ME) or G6PD impairs global histone acetylation and transcription in both adipocytes and tumor cells. These effects can be reversed by supplementation of exogenous NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH or inhibition of histone deacetylase 3 (HDAC3). Mechanistically,NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator Ncor2 (SMRT) or Ncor1, thereby impairs HDAC3 activation. Interestingly, it appears that NADPH and Ins(1,4,5,6)P4 bind to the same domains on HDAC3, and NADPH has relatively higher affinity towards HDAC3. Thus, while Ins(1,4,5,6)P4 acts as an ‘intermolecular glue’, NADPH may function as a HDAC3-Ncor assembly inhibitor. Collectively, our findings uncovered a previous unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

2020-11-27 | GSE137694 | GEO

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis

Project description:Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions.

2021-07-30 | MTBLS533 | MetaboLights

The role of the OX1 protein kinase in H2O2 signal transduction

Project description:Expression of the OX1 gene coding for a putative protein kinase was shown to be induced in Arabidopsis thaliana ecotypes Col-0, WS and RLD seedlings by treatment with H2O2 superoxidecolddroughtsalt and cellulase (elicitor). All of these treatments will ultimately lead to production of H2O2 either as a secondary effect of the applied stress through disturbance of electron transport processes or as a direct product of the plant NADPH oxidases (oxidative burst). However the sets of protective genes switched on (end-responses) will differ depending on the original stress. The induction characteristics of OX1 point to a role of this kinase in signal transduction downstream of H2O2; however it is not clear which primary signal is relayed. OX1 may function in the perception of oxidative stress caused by all/any one of the environmental stresses and/or it may be important in signalling downstream of the oxidative burst triggered by pathogen infection and wounding. An OX1 knock-out was isolated from the Wisconsin T-DNA lines with an insertion between the first and second conserved kinase domains rendering the protein product non-functional. Aim of the microarray experiment is to compare gene induction in the homozygous knock-out line to that in the wild-type (ecotype WS) following H2O2 treatment. To this end 7 day-old seedlings will be immersed in 10 mM solution for 1 h. Analysis of differences in expression of genes belonging to a certain end-response will disclose the signalling pathway which OX1 functions in. This is necessary for further planned experiments e.g. analysis of inducible OX1 antisense and constitutive lines as well as kinase activity assays.

2003-10-21 | E-NASC-5 | biostudies-arrayexpress

Hyperthermophilic archaeon Pyrococcus furiosus response to hydrogen peroxide

Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs.

2010-02-25 | GSE20470 | GEO

Transcription profiling of mouse Hexose-6-phosphate dehydrogenase knockout muscle at 4 weeks

Project description:Hexose-6-phosphate dehydrogenase (H6PD)is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver, H6PD is required for the 11-oxoreductase activity of 11ss-hydroxysteroid dehydrogenase type 1 (11ss-HSD1), which converts inactive 11-oxo glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in Type II (fast) fibers which have increased glycogen content. They also display a progressive vacuolar myopathy evident after 4 weeks of age. We carried out microarray analysis on TA and soleus muscles from 4 week old WT and KO mice to determine an expression profile predicting myopathy. Experiment Overall Design: 4 week old mice are weaned and do not display overt histological evidence of myopathy. Soleus and tibialis anterior are used as comparison groups as they have distinct fibre type content and differing metabolic properties. 3 biological replicates are used for each genotpye and sample type.

2008-06-11 | E-GEOD-10347 | biostudies-arrayexpress

Gene expression data from Hexose-6-phosphate dehydrogenase knockout mouse muscle at 4 weeks

Project description:Hexose-6-phosphate dehydrogenase (H6PD)is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver, H6PD is required for the 11-oxoreductase activity of 11ss-hydroxysteroid dehydrogenase type 1 (11ss-HSD1), which converts inactive 11-oxo glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in Type II (fast) fibers which have increased glycogen content. They also display a progressive vacuolar myopathy evident after 4 weeks of age. We carried out microarray analysis on TA and soleus muscles from 4 week old WT and KO mice to determine an expression profile predicting myopathy. Keywords: Age dependant disease state phenotype comparison

2008-02-01 | GSE10347 | GEO

Transcriptome analysis in HT29 and SW480 cells depleted of Prdx2

Project description:Prdx2 is the thioredoxin-dependent peroxidase that reduces H2O2 using reducing power NADPH in the presence of thioredoxin and thioredoxin reductase. Prdx2 plays an important role in growth. factor signaling in mammlian cells. Therefore, we examined the gene expression in colon adenocarcinoma cell line HT29 after Prdx2 depletion. Prdx2 depletion resulted in a significant alteration on gene expression, including protein synthesis, metabolisms, and cell cycle.

2017-05-13 | GSE81429 | GEO

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