FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2994893002> ?p ?o ?g. }

• W2994893002 endingPage "191" @default.
• W2994893002 startingPage "178" @default.
• W2994893002 abstract "Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25−/− versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH. Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25−/− versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH. Nonalcoholic fatty liver disease (NAFLD), defined as fatty infiltration in >5% of hepatocytes (steatosis) in the absence of excessive alcohol consumption, is emerging as a leading cause of liver disease worldwide (1Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2385) Google Scholar). Subjects with NAFLD have an increased risk of progressing into nonalcoholic steatohepatitis (NASH) as well as developing type 2 diabetes, cardiovascular disease, and liver cancer (2Younossi Z. Stepanova M. Ong J.P. Jacobson I.M. Bugianesi E. Duseja A. Eguchi Y. Wong V.W. Negro F. Yilmaz Y. Global Nonalcoholic Steatohepatitis Council et al.Nonalcoholic steatohepatitis is the fastest growing cause of hepatocellular carcinoma in liver transplant candidates.Clin. Gastroenterol. Hepatol. 2019; 17: 748-755.e3Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 3Anstee Q.M. Targher G. Day C.P. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis.Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 330-344Crossref PubMed Scopus (1098) Google Scholar, 4Younes R. Bugianesi E. A spotlight on pathogenesis, interactions and novel therapeutic options in NAFLD.Nat. Rev. Gastroenterol. Hepatol. 2019; 16: 80-82Crossref PubMed Scopus (20) Google Scholar). Thus, understanding the pathobiology of NAFLD is of high medical relevance for the efficient prevention and treatment of a range of complex metabolic diseases. In NAFLD, excess lipids accumulate within intrahepatocellular lipid droplets (LDs), which are composed of a neutral lipid core of mainly triacylglycerol (TAG) and cholesterol esters surrounded by a phospholipid monolayer that harbors specific proteins. Once thought to be only inert energy storage depots, hepatic LDs are increasingly recognized as organelles that not only orchestrate liver lipid partitioning but also play a critical role in protein quality control and storage, cell signaling, and viral replication (5Mashek D.G. Khan S.A. Sathyanarayan A. Ploeger J.M. Franklin M.P. Hepatic lipid droplet biology: Getting to the root of fatty liver.Hepatology. 2015; 62: 964-967Crossref PubMed Scopus (79) Google Scholar). Notably, LDs dynamically interact with a variety of cellular organelles including the ER, mitochondria, peroxisomes, and endosomes (6Barbosa A.D. Siniossoglou S. Function of lipid droplet-organelle interactions in lipid homeostasis.Biochim. Biophys. Acta Mol. Cell Res. 2017; 1864: 1459-1468Crossref PubMed Scopus (64) Google Scholar). Liver LD surface-associated proteins regulate the functional properties of LDs, and the composition of hepatic LD proteins is dynamically influenced by dietary fat content as well as liver metabolic status (7Khan S.A. Wollaston-Hayden E.E. Markowski T.W. Higgins L. Mashek D.G. Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis.J. Lipid Res. 2015; 56: 2260-2272Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Crunk A.E. Monks J. Murakami A. Jackman M. Maclean P.S. Ladinsky M. Bales E.S. Cain S. Orlicky D.J. McManaman J.L. Dynamic regulation of hepatic lipid droplet properties by diet.PLoS One. 2013; 8: e67631Crossref PubMed Scopus (56) Google Scholar). Consequently, exploring how LD-coating proteins affect hepatic lipid homeostasis is important for understanding the molecular pathophysiology of NAFLD. In the search for novel targets that regulate ectopic lipid accumulation in the context of nutritional stress and obesity, we identified serine/threonine kinase (STK)25, a member of the Ste20 kinase superfamily (9Thompson B.J. Sahai E. MST kinases in development and disease.J. Cell Biol. 2015; 210: 871-882Crossref PubMed Scopus (103) Google Scholar), as a hepatic LD-associated protein, which critically controls the development and progression of NAFLD (10Amrutkar M. Cansby E. Chursa U. Nunez-Duran E. Chanclon B. Stahlman M. Friden V. Manneras-Holm L. Wickman A. Smith U. et al.Genetic disruption of protein kinase STK25 ameliorates metabolic defects in a diet-induced type 2 diabetes model.Diabetes. 2015; 64: 2791-2804Crossref PubMed Scopus (42) Google Scholar, 11Amrutkar M. Cansby E. Nunez-Duran E. Pirazzi C. Stahlman M. Stenfeldt E. Smith U. Boren J. Mahlapuu M. Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH.FASEB J. 2015; 29: 1564-1576Crossref PubMed Scopus (56) Google Scholar, 12Amrutkar M. Chursa U. Kern M. Nunez-Duran E. Stahlman M. Sutt S. Boren J. Johansson B.R. Marschall H.U. Bluher M. et al.STK25 is a critical determinant in nonalcoholic steatohepatitis.FASEB J. 2016; 30: 3628-3643Crossref PubMed Scopus (36) Google Scholar, 13Amrutkar M. Kern M. Nunez-Duran E. Stahlman M. Cansby E. Chursa U. Stenfeldt E. Boren J. Bluher M. Mahlapuu M. Protein kinase STK25 controls lipid partitioning in hepatocytes and correlates with liver fat content in humans.Diabetologia. 2016; 59: 341-353Crossref PubMed Scopus (33) Google Scholar, 14Nuñez-Durán E. Aghajan M. Amrutkar M. Sutt S. Cansby E. Booten S.L. Watt A. Stahlman M. Stefan N. Haring H.U. et al.Serine/threonine protein kinase 25 antisense oligonucleotide treatment reverses glucose intolerance, insulin resistance, and nonalcoholic fatty liver disease in mice.Hepatol. Commun. 2017; 2: 69-83Crossref PubMed Scopus (27) Google Scholar, 15Cansby E. Nunez-Duran E. Magnusson E. Amrutkar M. Booten S.L. Kulkarni N.M. Svensson T.L. Boren J. Marschall H.U. Aghajan M. et al.Targeted delivery of Stk25 antisense oligonucleotides to hepatocytes protects mice against nonalcoholic fatty liver disease.Cell. Mol. Gastroenterol. Hepatol. 2019; 7: 597-618Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We found that STK25 knockdown attenuates lipid deposition in human hepatocytes and mouse liver by repressing lipid synthesis and enhancing β-oxidation and VLDL-TAG secretion, with the reciprocal phenotype seen when STK25 protein is overexpressed (10Amrutkar M. Cansby E. Chursa U. Nunez-Duran E. Chanclon B. Stahlman M. Friden V. Manneras-Holm L. Wickman A. Smith U. et al.Genetic disruption of protein kinase STK25 ameliorates metabolic defects in a diet-induced type 2 diabetes model.Diabetes. 2015; 64: 2791-2804Crossref PubMed Scopus (42) Google Scholar, 11Amrutkar M. Cansby E. Nunez-Duran E. Pirazzi C. Stahlman M. Stenfeldt E. Smith U. Boren J. Mahlapuu M. Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH.FASEB J. 2015; 29: 1564-1576Crossref PubMed Scopus (56) Google Scholar, 12Amrutkar M. Chursa U. Kern M. Nunez-Duran E. Stahlman M. Sutt S. Boren J. Johansson B.R. Marschall H.U. Bluher M. et al.STK25 is a critical determinant in nonalcoholic steatohepatitis.FASEB J. 2016; 30: 3628-3643Crossref PubMed Scopus (36) Google Scholar, 13Amrutkar M. Kern M. Nunez-Duran E. Stahlman M. Cansby E. Chursa U. Stenfeldt E. Boren J. Bluher M. Mahlapuu M. Protein kinase STK25 controls lipid partitioning in hepatocytes and correlates with liver fat content in humans.Diabetologia. 2016; 59: 341-353Crossref PubMed Scopus (33) Google Scholar, 14Nuñez-Durán E. Aghajan M. Amrutkar M. Sutt S. Cansby E. Booten S.L. Watt A. Stahlman M. Stefan N. Haring H.U. et al.Serine/threonine protein kinase 25 antisense oligonucleotide treatment reverses glucose intolerance, insulin resistance, and nonalcoholic fatty liver disease in mice.Hepatol. Commun. 2017; 2: 69-83Crossref PubMed Scopus (27) Google Scholar). Notably, administration of hepatocyte-targeting GalNAc-conjugated Stk25 antisense oligonucleotide (ASO) in obese mice effectively ameliorates high-fat diet-induced hepatic steatosis as well as NASH features (i.e., liver inflammation, hepatocellular ballooning, and fibrosis), providing in vivo nonclinical proof-of-principle for the metabolic benefit of pharmacological STK25 inhibitors (15Cansby E. Nunez-Duran E. Magnusson E. Amrutkar M. Booten S.L. Kulkarni N.M. Svensson T.L. Boren J. Marschall H.U. Aghajan M. et al.Targeted delivery of Stk25 antisense oligonucleotides to hepatocytes protects mice against nonalcoholic fatty liver disease.Cell. Mol. Gastroenterol. Hepatol. 2019; 7: 597-618Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Furthermore, we observed that STK25 mRNA and protein levels in human liver biopsies correlate with the severity of NASH, and several common nonlinked SNPs in the human STK25 gene are associated with altered liver fat content (12Amrutkar M. Chursa U. Kern M. Nunez-Duran E. Stahlman M. Sutt S. Boren J. Johansson B.R. Marschall H.U. Bluher M. et al.STK25 is a critical determinant in nonalcoholic steatohepatitis.FASEB J. 2016; 30: 3628-3643Crossref PubMed Scopus (36) Google Scholar, 13Amrutkar M. Kern M. Nunez-Duran E. Stahlman M. Cansby E. Chursa U. Stenfeldt E. Boren J. Bluher M. Mahlapuu M. Protein kinase STK25 controls lipid partitioning in hepatocytes and correlates with liver fat content in humans.Diabetologia. 2016; 59: 341-353Crossref PubMed Scopus (33) Google Scholar, 14Nuñez-Durán E. Aghajan M. Amrutkar M. Sutt S. Cansby E. Booten S.L. Watt A. Stahlman M. Stefan N. Haring H.U. et al.Serine/threonine protein kinase 25 antisense oligonucleotide treatment reverses glucose intolerance, insulin resistance, and nonalcoholic fatty liver disease in mice.Hepatol. Commun. 2017; 2: 69-83Crossref PubMed Scopus (27) Google Scholar). Although these studies suggest a key role for STK25 in the control of liver LD dynamics, the mechanism-of-action of STK25 in regulation of hepatic lipid partitioning has remained elusive. To provide novel insights into the biological function of STK25 in the liver under steatotic conditions, we here characterized the hepatic LD-associated phosphoproteome in Stk25 knockout mice and their wild-type littermates following high-fat diet feeding using a nonbiased approach by isobaric tagging for relative quantification. Of the 4,515 proteins and 982 phosphoproteins identified with a 1% false discovery rate, we report a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in the livers from high-fat diet-fed Stk25−/− mice compared with wild-type controls. Stk25 knockout mice were generated by deletion of exons 4 and 5 and genotyped as previously described (16Matsuki T. Matthews R.T. Cooper J.A. van der Brug M.P. Cookson M.R. Hardy J.A. Olson E.C. Howell B.W. Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment.Cell. 2010; 143: 826-836Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Heterozygous mice were backcrossed to a C57BL6/J genetic background, and the heterozygous mice were then intercrossed to obtain the homozygotes used in all experiments. From the age of 6 weeks, male knockout mice and their wild-type littermates were fed a pelleted high-fat diet (45% kilocalories from fat; D12451; Research Diets, New Brunswick, NJ) for 18 weeks. At the age of 24 weeks, the mice were euthanized after 4 h of food withdrawal and livers were collected for preparation of LDs. Histological evaluation performed in our previous study revealed that wild-type male mice fed an identical high-fat diet for 20 weeks developed pronounced macro- and microvesicular steatosis in the liver, whereas markedly less hepatic lipid accumulation was observed in high-fat-fed Stk25−/− mice (about 2-fold reduction in the total lipid area) (10Amrutkar M. Cansby E. Chursa U. Nunez-Duran E. Chanclon B. Stahlman M. Friden V. Manneras-Holm L. Wickman A. Smith U. et al.Genetic disruption of protein kinase STK25 ameliorates metabolic defects in a diet-induced type 2 diabetes model.Diabetes. 2015; 64: 2791-2804Crossref PubMed Scopus (42) Google Scholar). However, as whole livers were used for LD preparation in this study, it was not possible to perform histological or biochemical characterization of the extent of diet-induced liver damage in these mice. All animal experiments were performed after approval from the local Ethics Committee for Animal Studies at the Administrative Court of Appeals in Gothenburg, Sweden, and followed appropriate guidelines. LC-MS analysis included five biological replicates per genotype and no technical replicates due to the small sample amount. The livers were minced, resuspended in 4 ml of buffer A [25 mmol/l tricene (Sigma-Aldrich, St. Louis, MO), 250 mmol/l sucrose (Sigma-Aldrich), and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA)] and dounce homogenized on ice, followed by further homogenization using a 25 gauge needle and a syringe. After addition of 4 ml of buffer A, the postnuclear supernatant was divided into two ultracentrifuge tubes, overlaid with 3 ml of buffer B [20 mmol/l HEPES, 100 mmol/l KCl, and 2 mmol/l MgCl2 (Sigma-Aldrich)] and centrifuged at 300,000 g for 1 h at 4°C. The LD-containing band was transferred into a fresh tube and centrifuged at 20,000 g for 20 min at 4°C. The underlying liquid was carefully removed and the LD fraction was washed four times with buffer B to remove copurifying membranes (17Ding Y. Zhang S. Yang L. Na H. Zhang P. Zhang H. Wang Y. Chen Y. Yu J. Huo C. et al.Isolating lipid droplets from multiple species.Nat. Protoc. 2013; 8: 43-51Crossref PubMed Scopus (115) Google Scholar, 18Zhang H. Wang Y. Li J. Yu J. Pu J. Li L. Zhang H. Zhang S. Peng G. Yang F. et al.Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I.J. Proteome Res. 2011; 10: 4757-4768Crossref PubMed Scopus (142) Google Scholar). The isolated LDs were then stored at –80°C until further analysis. For LC-MS analysis, the liver LD preparations were mixed at 4°C with the Pierce phosphatase inhibitor ×2 solution (Invitrogen, Carlsbad, CA) and 16% sodium dodecyl sulfate (SDS)/200 mmol/l triethylammonium bicarbonate (TEAB) to a final concentration of approximately 1.5% SDS. The solution was centrifuged at 13,500 g for 20 min and the colorless protein extract layer was transferred to a separate vial. The remaining white top layer was then washed with 110 μl of the solution containing the phosphatase inhibitor at ×0.5 concentration, 1.5% SDS, and 20 mmol/l TEAB, and centrifuged at 13,500 g for 20 min; the colorless aqueous layer was collected and combined with the protein extract from the previous step. Protein concentrations in the combined protein extracts were determined using the Pierce BCA protein assay (Pierce Biotechnology, Rockford, IL) and the Benchmark Plus microplate reader (Bio-Rad, Hercules, CA). Aliquots containing 180 μg of protein were digested with trypsin using the filter-aided sample preparation method (19Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5042) Google Scholar). In brief, the samples were incubated with 100 mmol/l dithiothreitol at 60°C for 30 min, transferred onto the 30 kDa MWCO Nanosep centrifugal filters (Pall Life Sciences, Ann Arbor, MI), washed with 8 M urea solution, and alkylated with 10 mmol/l methyl methanethiosulfonate in 50 mmol/l TEAB and 1% sodium deoxycholate. Digestion was performed in 50 mmol/l TEAB/1% sodium deoxycholate at 37°C in two stages: the samples were incubated with 1.8 μg of Pierce MS-grade trypsin (trypsin to protein ratio 1:100; Pierce Biotechnology) overnight; then 1.8 μg of trypsin were added, and the digestion was performed for three additional hours. The peptides were collected by centrifugation and labeled using tandem mass tag (TMT) 10-plex isobaric mass tagging reagents (Pierce Biotechnology) according to the manufacturer's instructions. The labeled samples were mixed and sodium deoxycholate was removed by acidification with 10% trifluoroacetic acid and subsequent centrifugation at 13,500 g. An aliquot containing approximately 90 μg of the pooled labeled peptide mixture (1/20 of the total amount by volume) was fractionated using the Pierce high pH reversed-phase peptide fractionation kit (Pierce Biotechnology) according to the manufacturer's recommendations. Eight fractions were collected using the elution solvents containing 0.1% of triethylamine and 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 22.5, and 50.0% of acetonitrile. The fractions were dried on Speedvac and reconstituted in 3% acetonitrile/0.2% formic acid for analysis. For the phosphopeptide enrichment, the remaining part (19/20 of the total volume) of the pooled TMT-labeled peptide sample (approximately 1,700 μg) was divided into two equal aliquots and both aliquots were evaporated on Speedvac to dryness. One aliquot was resuspended in the buffer and processed using the Pierce TiO2 phosphopeptide enrichment and clean up kit (Pierce Biotechnology) according to the manufacturer's protocol. Another aliquot was resuspended in the buffer and processed using the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific) according to the manufacturer's protocol. In both cases, the enriched samples were purified using the Pierce C18 spin columns (8 mg of the C18 material; Pierce Biotechnology), and the purified samples were then dried on Speedvac. TMT-labeled phosphopeptides from both TiO2 and Fe-NTA enrichment were reconstituted in 3% acetonitrile/0.2% formic acid and analyzed separately. All the samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) interfaced with an Easy-nLC 1200 nanoflow LC system (Thermo Fisher Scientific). Peptides were trapped on the Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm; Thermo Fisher Scientific) and separated on the in-house packed C18 analytical column (packing material 75 μm × 30 cm, particle size 3 μm; Dr. Maisch, Ammerbuch, Germany) using 0.2% formic acid as eluent A and 80% acetonitrile/0.2% formic acid as eluent B. For the whole proteome fractions, the gradient was from 5% to 33% B for 105 min, from 33% to 100% B for 5 min, and 100% B for 10 min. The above-mentioned gradient was also used for both phosphopeptide-enriched samples, and the TiO2-enriched samples were additionally analyzed using the gradient from 3% to 25% B for 105 min, from 25% to 100% B for 5 min, and 100% B for 10 min. The precursor ion spectra were recorded at 120,000 resolution, the most intense precursor ions were selected (“top speed” setting with a duty cycle of 3 s), fragmented using collision-induced dissociation at collision energy setting of 35, and the MS2 spectra were recorded in ion trap with the maximum injection time of 50 ms and the isolation window of 0.7 Da. Charge states 2–7 were selected for fragmentation and dynamic exclusion was set to 45 s with 10 ppm tolerance. MS3 spectra for reporter ion quantitation were recorded at 50,000 resolution with higher-energy collisional dissociation fragmentation at collision energy of 60 using the synchronous precursor selection of the seven most abundant MS2 fragments, with the maximum injection time of 100 ms. In order to increase the depth of the phosphopeptide analysis, the Fe-NTA-enriched samples were additionally analyzed using the neutral loss-triggered TMT MS3 method with the neutral loss of phosphoric acid. Furthermore, the lists of the identified peptides for the first data-dependent acquisitions of both phosphopeptide samples were used to create the exclusion mass lists, and both samples were reanalyzed using the data-dependent TMT MS3 method that excluded the masses of the previously identified peptides from the data-dependent selection. Data analysis was performed using Proteome Discoverer version 2.2 (Thermo Fisher Scientific). The database search was performed against the Swissprot Mus musculus database (October 2017), Mascot 2.5.1 (Matrix Science, London, UK) was used as a search engine with precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.6 Da; tryptic peptides with one missed cleavage were accepted, mono-oxidation on methionine was set as a variable modification, methylthiolation on cysteine and TMT-6 reagent modification on lysine and peptide N terminus were set as a fixed modification for all files; phosphorylation on serine, threonine, and tyrosine was set as a variable modification for the processing of the phosphopeptide-enriched samples. Trypsin cleaves at the carboxylic side of lysine and arginine when the next amino acid is not proline. Percolator (Matrix Science) was used for the validation of identification results with the strict target false discovery rate of 1%. Reporter ion intensities were quantified in MS3 spectra at 0.003 Da mass tolerance and normalized on the total protein abundance within the Proteome Discoverer 2.2 (Thermo Fisher Scientific). Only the quantification values from the unique peptides were used for quantification. We estimated the uncertainty for correct peptide sequence to be 1%, as we filtered for false discovery rate 1% toward a decoy database, and the uncertainty in quantification to be less than 20% based on previous validation of this method. KEGG pathway analysis was performed using STRING (v10) (20Szklarczyk D. Franceschini A. Wyder S. Forslund K. Heller D. Huerta-Cepas J. Simonovic M. Roth A. Santos A. Tsafou K.P. et al.STRING v10: protein-protein interaction networks, integrated over the tree of life.Nucleic Acids Res. 2015; 43: D447-D452Crossref PubMed Scopus (6709) Google Scholar) (with a confidence score of 0.700) and Cytoscape (v3.1.1) (21Smoot M.E. Ono K. Ruscheinski J. Wang P.L. Ideker T. Cytoscape 2.8: new features for data integration and network visualization.Bioinformatics. 2011; 27: 431-432Crossref PubMed Scopus (3477) Google Scholar). Liver samples were fixed with 4% (vol/vol) phosphate-buffered formaldehyde (Histolab Products, Gothenburg, Sweden), embedded in paraffin, and sectioned. Liver sections were incubated with primary antibodies, followed by incubation with fluorescent-dye-conjugated secondary antibodies (see supplemental Table S1 for antibody information). The PEX5-positive area was quantified in six randomly selected microscopic fields (×40) per mouse using the ImageJ software (1.47v; National Institutes of Health, Bethesda, MD). Liver lipids were extracted using the BUME method and an aliquot of the total lipid extract was further treated with methanol and heptane to remove the neutral lipids (TAGs and cholesteryl esters) and purify the phospholipid fraction (22Löfgren L. Stahlman M. Forsberg G.B. Saarinen S. Nilsson R. Hansson G.I. The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma.J. Lipid Res. 2012; 53: 1690-1700Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). From this fraction, the plasmalogens were analyzed using direct infusion MS as previously described (23Ekroos K. Ejsing C.S. Bahr U. Karas M. Simons K. Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.J. Lipid Res. 2003; 44: 2181-2192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 24Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (247) Google Scholar). For this a robotic nanoflow ion source, TriVersa NanoMate (Advion BioSciences, Ithaca, NJ) was used together with a QTRAP 5500 mass spectrometer (Sciex, Concord, Ontario, Canada). For analysis of very long chain fatty acids (VLCFAs), an aliquot of the phospholipid fraction was hydrolyzed by incubating at 50°C overnight in 300 μl of acetonitrile:concentrated HCl (4:1). After cooling to room temperature, the free fatty acids were extracted using 600 μl of iso-hexane. The hexane phase was evaporated and the fatty acids were reconstituted in 100 μl of chloroform:methanol (1:2) with 5 mmol/l ammonium acetate. Analysis and detection of the VLCFAs were made using direct infusion MS as previously reported (25Valianpour F. Selhorst J.J. van Lint L.E. van Gennip A.H. Wanders R.J. Kemp S. Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry.Mol. Genet. Metab. 2003; 79: 189-196Crossref PubMed Scopus (118) Google Scholar). Immortalized human hepatocytes [IHHs; a gift from B. Staels, Pasteur Institute of Lille, University of Lille Nord de France, Lille, France (26Samanez C.H. Caron S. Briand O. Dehondt H. Duplan I. Kuipers F. Hennuyer N. Clavey V. Staels B. The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism.Arch. Physiol. Biochem. 2012; 118: 102-111Crossref PubMed Scopus (42) Google Scholar)] were maintained in William's E medium (GlutaMAX supplemented; Gibco, Paisley, UK) supplemented with human insulin (20 U/l; Actrapid Penfill; Novo Nordisk, Bagsværd, Denmark), dexamethasone (50 nmol/l; Sigma-Aldrich), 10% (vol/vol) FBS (Gibco), and 1% (vol/vol) penicillin/streptomycin (Gibco). Cells were demonstrated to be free of mycoplasma infection by use of the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). IHHs were transfected with STK25 siRNA (s20570; Ambion, Austin, TX) or scrambled siRNA (SIC001; Sigma-Aldrich) using Lipofectamine RNAiMax (Invitrogen). Before the experiments, cells were exposed to 50 μmol/l oleic acid (Sigma-Aldrich) for 48 h in order to mimic conditions in high-risk individuals. In some experiments, cells were also treated with 1 μmol/l MG132 (474790; Calbiochem, San Diego, CA) for 24 h to inhibit proteasome function in hepatocytes (27Ishii M. Maeda A. Tani S. Akagawa M. Palmitate induces insulin resistance in human HepG2 hepatocytes by enhancing ubiquitination and proteasomal degradation of key insulin signaling molecules.Arch. Biochem. Biophys. 2015; 566: 26-35Crossref PubMed Scopus (74) Google Scholar, 28Joshi-Barve S. Barve S.S. Butt W. Klein J. McClain C.J. Inhibition of proteasome function leads to NF-kappaB-independent IL-8 expression in human hepatocytes.Hepatology. 2003; 38: 1178-1187Crossref PubMed Scopus (68) Google Scholar). Cells were processed for immunofluorescence with anti-PEX5, anti-catalase, anti-HNE, or anti-KDEL antibodies (see supplemental Table S1 for antibody information); the labeled area was quantified in six randomly selected microscopic fields (×20) per well using the ImageJ software (1.47v; National Institutes of Health). For dihydroethidium (DHE) staining, cells were incubated with 5 μmol/l DHE (Invitrogen) in PBS containing 1% (weight/vol) BSA at 37°C for 5 min; the stained area was quantified" @default.
• W2994893002 created "2019-12-26" @default.
• W2994893002 creator A5000042801 @default.
• W2994893002 creator A5001825767 @default.
• W2994893002 creator A5006953866 @default.
• W2994893002 creator A5011043460 @default.
• W2994893002 creator A5012857972 @default.
• W2994893002 creator A5017202435 @default.
• W2994893002 creator A5034464186 @default.
• W2994893002 creator A5046743882 @default.
• W2994893002 creator A5053328406 @default.
• W2994893002 creator A5062571519 @default.
• W2994893002 creator A5066912379 @default.
• W2994893002 creator A5068490300 @default.
• W2994893002 creator A5078832883 @default.
• W2994893002 creator A5083514157 @default.
• W2994893002 creator A5086564739 @default.
• W2994893002 date "2020-02-01" @default.
• W2994893002 modified "2023-10-17" @default.
• W2994893002 title "Lipid droplet-associated kinase STK25 regulates peroxisomal activity and metabolic stress response in steatotic liver" @default.
• W2994893002 cites W1664030242 @default.
• W2994893002 cites W1964697500 @default.
• W2994893002 cites W1965580938 @default.
• W2994893002 cites W1967618979 @default.
• W2994893002 cites W1968628675 @default.
• W2994893002 cites W1974744093 @default.
• W2994893002 cites W1975684232 @default.
• W2994893002 cites W1981390999 @default.
• W2994893002 cites W1987264857 @default.
• W2994893002 cites W1991161256 @default.
• W2994893002 cites W2004934188 @default.
• W2994893002 cites W2006765846 @default.
• W2994893002 cites W2008441088 @default.
• W2994893002 cites W2011597748 @default.
• W2994893002 cites W2014443494 @default.
• W2994893002 cites W2022983144 @default.
• W2994893002 cites W2030123555 @default.
• W2994893002 cites W2032261812 @default.
• W2994893002 cites W2034853241 @default.
• W2994893002 cites W2037199116 @default.
• W2994893002 cites W2042904851 @default.
• W2994893002 cites W2043274355 @default.
• W2994893002 cites W2046320949 @default.
• W2994893002 cites W2050410337 @default.
• W2994893002 cites W2057095795 @default.
• W2994893002 cites W2059613020 @default.
• W2994893002 cites W2061013234 @default.
• W2994893002 cites W2064178372 @default.
• W2994893002 cites W2073543481 @default.
• W2994893002 cites W2073751076 @default.
• W2994893002 cites W2082514180 @default.
• W2994893002 cites W2086500674 @default.
• W2994893002 cites W2086782515 @default.
• W2994893002 cites W2090365424 @default.
• W2994893002 cites W2094783619 @default.
• W2994893002 cites W2096173332 @default.
• W2994893002 cites W2098940158 @default.
• W2994893002 cites W2099343588 @default.
• W2994893002 cites W2106272768 @default.
• W2994893002 cites W2117738749 @default.
• W2994893002 cites W2118013260 @default.
• W2994893002 cites W2123830703 @default.
• W2994893002 cites W2123831432 @default.
• W2994893002 cites W2125219081 @default.
• W2994893002 cites W2125372126 @default.
• W2994893002 cites W2127212889 @default.
• W2994893002 cites W2128057304 @default.
• W2994893002 cites W2144535719 @default.
• W2994893002 cites W2146528837 @default.
• W2994893002 cites W2155142785 @default.
• W2994893002 cites W2157240187 @default.
• W2994893002 cites W2158138282 @default.
• W2994893002 cites W2161629955 @default.
• W2994893002 cites W2163857095 @default.
• W2994893002 cites W2165825213 @default.
• W2994893002 cites W2169061341 @default.
• W2994893002 cites W2177861841 @default.
• W2994893002 cites W2179972854 @default.
• W2994893002 cites W2188093405 @default.
• W2994893002 cites W2260924587 @default.
• W2994893002 cites W2262954976 @default.
• W2994893002 cites W2274781935 @default.
• W2994893002 cites W2463195069 @default.
• W2994893002 cites W2469284364 @default.
• W2994893002 cites W2520119373 @default.
• W2994893002 cites W2570688801 @default.
• W2994893002 cites W2591604204 @default.
• W2994893002 cites W2605342494 @default.
• W2994893002 cites W2759997926 @default.
• W2994893002 cites W2771009053 @default.
• W2994893002 cites W2801929531 @default.
• W2994893002 cites W2808368206 @default.
• W2994893002 cites W2810629608 @default.
• W2994893002 cites W2818331228 @default.
• W2994893002 cites W2898850054 @default.
• W2994893002 cites W2899760200 @default.
• W2994893002 cites W2904854417 @default.
• W2994893002 cites W2906678048 @default.

• next