Refine
Document Type
- Article (5)
Language
- English (5)
Has Fulltext
- yes (5)
Is part of the Bibliography
- no (5)
Keywords
- NADPH oxidase (2)
- Reactive oxygen species (2)
- Apoptosis (1)
- BIAM switch assay (1)
- Cardiology (1)
- Chemiluminescence (1)
- Long non-coding RNAs (1)
- Lucigenin (1)
- Mass spectrometry (1)
- Membrane assays (1)
Institute
- Medizin (5) (remove)
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 acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases. Using a combination of native protein electrophoresis, NADPH-stimulated assays and mass spectrometry, mitochondrial proteins and cytochrome P450 were identified as possible source of the assay signal. Cells lacking functional mitochondrial complexes, however, displayed a normal activity in NADPH-stimulated membrane assays suggesting that mitochondrial oxidoreductases are unlikely sources of the signal. Microsomes overexpressing P450 reductase, cytochromes b5 and P450 generated a NADPH-dependent signal in assays utilizing lucigenin, L-012 and dihydroethidium (DHE). Knockout of the cytochrome P450 reductase by CRISPR/Cas9 technology (POR-/-) in HEK293 cells overexpressing Nox4 or Nox5 did not interfere with ROS production in intact cells. However, POR-/- abolished the signal in NADPH-stimulated assays using membrane fractions from the very same cells. Moreover, membranes of rat smooth muscle cells treated with angiotensin II showed an increased NADPH-dependent signal with lucigenin which was abolished by the knockout of POR but not by knockout of p22phox. In conclusion: the cytochrome P450 system accounts for the majority of the signal of Nox activity chemiluminescence based assays.
Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs.
Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.
Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.
Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.
BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4
(2019)
Aim: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.
In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins.
Results: TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1.
Innovation & conclusion: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.