Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme, a unique member of the peroxiredoxin family, with an important role in antioxidant defense. Moreover, it has also been linked with the biosynthesis of anti-inflammatory and anti-diabetic lipids called fatty acid esters of hydroxy fatty acids (FAHFAs) and many diseases, including cancer, inflammation, and metabolic disorders. Here, we performed metabolomic and lipidomic profiling of subcutaneous adipose tissue from mouse models with genetically modified Prdx6. Deletion of Prdx6 resulted in reduced levels of FAHFAs containing 13-hydroxylinoleic acid (13-HLA). Mutation of Prdx6 C47S impaired the glutathione peroxidase activity and reduced FAHFA levels, while D140A mutation, responsible for phospholipase A2 activity, showed only minor effects. Targeted analysis of oxidized phospholipids and triacylglycerols in adipocytes highlighted a correlation between FAHFA and hydroxy fatty acid production by Prdx6 or glutathione peroxidase 4. FAHFA regioisomer abundance was negatively affected by the Prdx6 deletion, and this effect was more pronounced in longer and more unsaturated FAHFAs. The predicted protein model of Prdx6 suggested that the monomer-dimer transition mechanism might be involved in the repair of longer-chain peroxidized phospholipids bound over two monomers and that the role of Prdx6 in FAHFA synthesis might be restricted to branching positions further from carbon 9. In conclusion, our work linked the peroxidase activity of Prdx6 with the levels of FAHFAs in adipose tissue.
- MeSH
- antioxidancia MeSH
- fosfolipidy MeSH
- mastné kyseliny MeSH
- metabolomika * MeSH
- myši MeSH
- peroxiredoxin VI * genetika MeSH
- peroxiredoxiny MeSH
- tukové buňky MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
- MeSH
- buněčný cyklus fyziologie MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace MeSH
- glutamáty metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy metabolismus MeSH
- peroxiredoxin VI chemie metabolismus MeSH
- proteolýza * MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- sestřihové faktory chemie metabolismus MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The patatin-like phospholipase domain containing 3 (PNPLA3) gene (viz. its I148M variant) is one of the key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We have identified a novel insertion/deletion variant of 1114 bp, localized in the second intron of the PNPLA3 gene, which corresponds to the 3' terminal sequence of the long-interspersed element (LINE-1). DNA analysis of 122 NAFLD patients and 167 control subjects as well as RNA analysis of 19 liver biopsies revealed that the novel variant is very common (frequency = 0.41), fully linked to the clinically important I148M variant, and clinically silent. Although the LINE-1 insertion does not seem to have any biological effect, it can impede genotyping of the I148M variant. If insertion prevents the attachment of the diagnostic primer, then the non-insertion allele will be selectively amplified; and thus the frequency of the 148M "risk" allele will be significantly overestimated due to the complete linkage of the LINE-1 insertion and the 148I allele of the PNPLA3 gene. Therefore, our findings underline the importance of careful design and consistent documentation of the methodology, including primer sequences. Critical revisions of the results of some studies that have already been reported may therefore be needed.
- MeSH
- acyltransferasy genetika MeSH
- alely MeSH
- dlouhé rozptýlené jaderné elementy genetika MeSH
- fosfolipasy A2 nezávislé na vápníku genetika MeSH
- genetická predispozice k nemoci genetika MeSH
- genotyp MeSH
- játra patologie MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- nealkoholová steatóza jater genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The human iPSC cell lines, PLANFiPS1-Sv4F-1 (RCPFi004-A), PLANFiPS2-Sv4F-1 (RCPFi005-A), PLANFiPS3-Sv4F-1 RCPFi006-A), derived from dermal fibroblast from three patients suffering PLAN (PLA2G6-associated neurodegeneration; MIM 256600) caused by mutations in the PLA2G6 gene, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors. The pluripotency was assessed by immunocytochemistry and RT-PCR. Differentiation capacity was verified in vitro. This iPSC line can be further differentiated toward affected cells to better understand molecular mechanisms of disease and pathophysiology.
Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with promising antidiabetic and anti-inflammatory properties that are formed in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, quantitative trait locus mapping, and correlation analyses in rat BXH/HXB recombinant inbred strains, as well as response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs. Comprehensive analysis of WAT samples identified ∼160 regioisomers, documenting the complexity of this lipid class. The linkage analysis highlighted several members of the nuclear factor, erythroid 2 like 2 (Nrf2)-mediated antioxidant defense system (Prdx6, Mgst1, Mgst3), lipid-handling proteins (Cd36, Scd6, Acnat1, Acnat2, Baat), and the family of flavin containing monooxygenases (Fmo) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically. Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein-driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues.
- MeSH
- bílá tuková tkáň enzymologie metabolismus MeSH
- biologické markery metabolismus MeSH
- estery chemie metabolismus MeSH
- faktor 2 související s NF-E2 genetika metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina palmitová chemie metabolismus MeSH
- kyseliny stearové chemie metabolismus MeSH
- metabolomika metody MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- náhodné rozdělení MeSH
- oxidační stres * MeSH
- peroxiredoxin VI genetika metabolismus MeSH
- potkani inbrední BN MeSH
- potkani inbrední SHR MeSH
- potkani transgenní MeSH
- regulace genové exprese enzymů * MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondrial uncoupling protein-2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. Here we tested the hypothesis that UCP2 provides feedback downregulation of oxidative stress in vivo via synergy with an H2O2-activated mitochondrial calcium-independent phospholipase A2 (mt-iPLA2). Tert-butylhydroperoxide or H2O2 induced free fatty acid release from mitochondrial membranes as detected by gas chromatography/mass spectrometry, which was inhibited by r-bromoenol lactone (r-BEL) but not by its stereoisomer s-BEL, suggesting participation of mt-iPLA2γ isoform. Tert-butylhydroperoxide or H2O2 also induced increase in respiration and decrease in mitochondrial membrane potential in lung and spleen mitochondria from control but not UCP2-knockout mice. These data suggest that mt-iPLA2γ-dependent release of free fatty acids promotes UCP2-dependent uncoupling. Upon such uncoupling, mitochondrial superoxide formation decreased instantly also in the s-BEL presence, but not when mt-iPLA2 was blocked by R-BEL and not in mitochondria from UCP2-knockout mice. Mt-iPLA2γ was alternatively activated by H2O2 produced probably in conjunction with the electron-transferring flavoprotein:ubiquinone oxidoreductase (ETFQOR), acting in fatty acid β-oxidation. Palmitoyl-d,l-carnitine addition to mouse lung mitochondria, respiring with succinate plus rotenone, caused a respiration increase that was sensitive to r-BEL and insensitive to s-BEL. We thus demonstrate for the first time that UCP2, functional due to fatty acids released by redox-activated mt-iPLA2γ, suppresses mitochondrial superoxide production by its uncoupling action. In conclusion, H2O2-activated mt-iPLA2γ and UCP2 act in concert to protect against oxidative stress.
- MeSH
- antioxidancia metabolismus MeSH
- buněčné dýchání účinky léků MeSH
- down regulace účinky léků MeSH
- fosfolipasy A2, skupina VI metabolismus MeSH
- iontové kanály metabolismus MeSH
- játra cytologie MeSH
- mastné kyseliny sekrece MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie účinky léků enzymologie metabolismus MeSH
- myši MeSH
- oxidace-redukce MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku farmakologie MeSH
- slezina cytologie MeSH
- superoxidy metabolismus MeSH
- terc-butylhydroperoxid farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Železo hraje významnou úlohu v lidském organizmu. Neurodegenerace s akumulací železa (NBIA) charakterizuje heterogenní skupinu neurodegenerativních onemocnění spojenou s hromaděním železa v centrálních oblastech mozku. Nejčastějším onemocněním této skupiny je neurodegenerace asociovaná s pantotenátkinázou (PKAN), autozomálně recesivní onemocnění charakterizované mutací PANK2 genu a postižením metabolizmu koenzymu A. Dále sem patří neurodegenerace spojená s fosfolipázou A2 s mutací PLA2G6 genu a onemocnění, jako je neuroferitinopatie, aceruloplazminémie a další, které se v současné době řadí ke skupině NBIA. S metabolizmem železa souvisí i další systémová onemocnění, např. hemochromatóza. Článek uvádí charakteristiku, klinické projevy společně s možnostmi diagnostiky a diferenciální diagnostiky těchto chorob.
Iron plays a significant role in the human body. Neurodegeneration with brain iron accumulation (NBIA) characterizes a heterogeneous group of neurodegenerative diseases associated with iron accumulation in the central areas of the brain. The most common disease of this group is pantothenate kinase-associated neurodegeneration (PKAN), an autosomal recessive disease characterized by a mutation in the PANK2 gene and impaired metabolism of coenzyme A. Also included here are phospholipase A2-associated neurodegeneration caused by a mutation in the PLA2G6 gene and conditions, such as neuroferritinopathy, aceruloplasminaemia, and others, that currently belong to the NBIA group. Iron metabolism is also linked to other systemic conditions, e.g. haemochromatosis. The paper presents the characteristic features, clinical manifestations as well as the options for diagnosis and differential diagnosis of these conditions. aceruloplasminaemia, haemochromatosis. The paper presents the characteristic features, clinical manifestations as well as the options for diagnosis and differential diagnosis of these conditions.
- Klíčová slova
- neurodegenerace spojená s akumulací železa, neurodegenerace spojená s pantotenátkinásou, neuroferitinopatie, aceruloplazminémie,
- MeSH
- ceruloplasmin metabolismus MeSH
- ferritin genetika metabolismus MeSH
- fosfolipasy A2, skupina VI genetika MeSH
- genetická predispozice k nemoci MeSH
- Hallervordenův-Spatzův syndrom diagnóza etiologie genetika MeSH
- hemochromatóza diagnóza genetika terapie MeSH
- kyselina pantothenová metabolismus MeSH
- lidé MeSH
- lidské chromozomy, pár 19 genetika MeSH
- lidské chromozomy, pár 6 genetika MeSH
- magnetická rezonanční tomografie MeSH
- metabolické nemoci mozku metabolismus patofyziologie MeSH
- mozek metabolismus patologie radiografie MeSH
- mutace MeSH
- nemoci bazálních ganglií diagnóza genetika MeSH
- neurodegenerativní nemoci genetika patofyziologie patologie MeSH
- parkinsonské poruchy etiologie genetika patofyziologie MeSH
- poruchy metabolismu železa patofyziologie MeSH
- rodokmen MeSH
- železo metabolismus MeSH
- Check Tag
- lidé MeSH
Homeostasis of reactive oxygen species (ROS) in cardiomyocytes is critical for elucidation of normal heart physiology and pathology. Mitochondrial phospholipases A2 (mt-PLA2) have been previously suggested to be activated by ROS. Therefore, we have attempted to elucidate physiological role of such activation. We have found that function of a specific i-isoform of mitochondrial phospholipase A2 (mt-iPLA2) is activated by tert-butylhydroperoxide in isolated rat heart mitochondria. Isoform specificity was judged from the inhibition by bromoenol lactone (BEL), a specific iPLA2 inhibitor. Concomitant uncoupling has been caused by free fatty acids, since it was inhibited by bovine serum albumin. The uncoupling was manifested as a respiration burst accompanied by a slight decrease in mitochondrial inner membrane potential. Since this uncoupling was sensitive to carboxyatractyloside and purine nucleotide di- and triphosphates, we conclude that it originated from the onset of fatty acid cycling mediated by the adenine nucleotide translocase (major contribution) and mitochondrial uncoupling protein(s) (minor contribution), respectively. Such a mild uncoupling may provide a feedback downregulation of oxidative stress, since it can further attenuate mitochondrial production of ROS. In conclusion, ROS-induced function of cardiac mt-iPLA2 may stand on a pro-survival side of ischemia-reperfusion injury.
- MeSH
- aktivace enzymů fyziologie účinky léků MeSH
- financování organizované MeSH
- fosfolipasy A2, skupina VI metabolismus MeSH
- iontové kanály metabolismus MeSH
- krysa rodu rattus MeSH
- mastné kyseliny metabolismus MeSH
- mitochondriální ADP/ATP-translokasy metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- mitochondrie enzymologie účinky záření MeSH
- myokard enzymologie patologie MeSH
- oxidační stres fyziologie účinky léků MeSH
- oxidativní fosforylace MeSH
- potkani Wistar MeSH
- reaktivní formy kyslíku metabolismus MeSH
- reperfuzní poškození myokardu metabolismus patologie MeSH
- terc-butylhydroperoxid farmakologie MeSH
- viabilita buněk fyziologie MeSH
- zpětná vazba fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
