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

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