Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

• Klíčová slova
• Cellular senescence, Interleukin 6, Peroxiredoxin 6, Redox proteomics, SILAC-iodoTMT, Senescence-associated secretory phenotype,
• MeSH
• cytokiny * metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• peroxiredoxin VI * genetika   metabolismus   MeSH
• stárnutí buněk fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny * MeSH
• peroxiredoxin VI * MeSH

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
• Názvy látek
• 9-hydroxystearic acid MeSH Prohlížeč
• biologické markery MeSH
• estery MeSH
• faktor 2 související s NF-E2 MeSH
• kyselina palmitová MeSH
• kyseliny stearové MeSH
• Nfe2l2 protein, mouse MeSH Prohlížeč
• Nfe2l2 protein, rat MeSH Prohlížeč
• peroxiredoxin VI MeSH
• Prdx6 protein, mouse MeSH Prohlížeč

Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.

• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• hlístice mikrobiologie   MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• otevřené čtecí rámce MeSH
• peroxiredoxiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Pseudomonas fluorescens chemie   enzymologie   genetika   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• peroxiredoxiny MeSH
• rekombinantní proteiny MeSH