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• W2441488300 abstract "Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo. Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo. Shc proteins have three isoforms: p46, p52, and p66, and have major effects on metabolism (1Hagopian K. Tomilov A.A. Kim K. Cortopassi G.A. Ramsey J.J. Key glycolytic enzyme activities of skeletal muscle are decreased under Fed and Fasted states in mice with knocked down levels of Shc proteins.PloS one. 2015; 10e0124204Crossref PubMed Scopus (12) Google Scholar, 2Hagopian K. Tomilov A.A. Tomilova N. Kim K. Taylor S.L. Lam A.K. Cortopassi G.A. McDonald R.B. Ramsey J.J. Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis.Metabolism: Clinical and Experimental. 2012; 61: 1703-1713Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 3Tomilov A. Bettaieb A. Kim K. Sahdeo S. Tomilova N. Lam A. Hagopian K. Connell M. Fong J. Rowland D. Griffey S. Ramsey J. Haj F. Cortopassi G. Shc depletion stimulates brown fat activity in vivo and in vitro.Aging Cell. 2014; 2612267Google Scholar, 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar). p52Shc contacts the insulin receptor and regulates the signaling between the IRS1 and Ras pathway (5Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar). In 2004, it was demonstrated that the major mitochondrial Shc isoform is p46Shc (6Ventura A. Maccarana M. Raker V.A. Pelicci P.G. A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria.J. Biol. Chem. 2004; 279: 2299-2306Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), but since that time the mitochondrial partner and physiological function of p46Shc has been a mystery. Here we show the mitochondrial partner and function of p46Shc, and suggest how this could contribute to the obesity-resistance observed in ShcKO mice. There are three isoforms at the mammalian Shc locus, the highly expressed isoforms p46Shc and p52Shc, and the minor p66Shc. All three Shc isoforms are derived from a single DNA locus. First, two mRNA are produced: p66Shc and p52/46Shc by means of trans-splicing. The p66Shc mRNA has start codons for all three Shc isoforms. The p52/46Shc-mRNA does not have a start codon for p66Shc and only produces p52Shc and p46Shc (7Kisielow M. Kleiner S. Nagasawa M. Faisal A. Nagamine Y. Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.Biochem. J. 2002; 363: 1-5Crossref PubMed Scopus (75) Google Scholar). For this reason, knockdowns of either p66Shc, or all three Shc isoforms together have been achieved to date. There are two mouse models of Shc depletion produced to date: ShcL, also known as p66ShcKO developed by the Tom Prolla group (ShcProlla, Ref. 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar), and Shc mice developed by the Pelicci group (ShcP or ShcPelicci, also known as p66Shc(−/−, Refs. 3Tomilov A. Bettaieb A. Kim K. Sahdeo S. Tomilova N. Lam A. Hagopian K. Connell M. Fong J. Rowland D. Griffey S. Ramsey J. Haj F. Cortopassi G. Shc depletion stimulates brown fat activity in vivo and in vitro.Aging Cell. 2014; 2612267Google Scholar, 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar, 8Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. The p66shc adaptor protein controls oxidative stress response and life span in mammals.Nature. 1999; 402: 309-313Crossref PubMed Scopus (1475) Google Scholar, 9Ramsey J.J. Tran D. Giorgio M. Griffey S.M. Koehne A. Laing S.T. Taylor S.L. Kim K. Cortopassi G.A. Lloyd K.C. Hagopian K. Tomilov A.A. Migliaccio E. Pelicci P.G. McDonald R.B. The influence of shc proteins on life span in mice.J. Gerontol. 2014; 69: 1177-1185Crossref Scopus (35) Google Scholar). Names in literature for these two models can be described by these definitions: ShcL, ShcProlla; ShcKO, ShcP = ShcPelicci = p66Shc(−/−). Briefly, ShcL or ShcProlla mice have a deletion of only the minor p66Shc isoform and have no health benefits: ShcL mice are not lean, do not resist weight gain on high fat diets (HFD), 2The abbreviations used are: HFDhigh fat dietBLIbio-layer interferometryOCRoxygen consumption rateBNblue nativeACAA23-ketoacyCoA thiolase. and are not longer-lived on HFD, and do not have improved insulin sensitivity (4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar). Thus p66Shc deletion by itself does not result in health benefits. high fat diet bio-layer interferometry oxygen consumption rate blue native 3-ketoacyCoA thiolase. By contrast, ShcKO have in addition to the deletion of minor p66Shc isoform, a major reduction of p46Shc and p52Shc expression in main energy-expending tissues: muscles (cardiac and skeletal), brown fat, and liver (3Tomilov A. Bettaieb A. Kim K. Sahdeo S. Tomilova N. Lam A. Hagopian K. Connell M. Fong J. Rowland D. Griffey S. Ramsey J. Haj F. Cortopassi G. Shc depletion stimulates brown fat activity in vivo and in vitro.Aging Cell. 2014; 2612267Google Scholar, 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar). ShcKO have several health benefits, including a lean phenotype (10Berniakovich I. Trinei M. Stendardo M. Migliaccio E. Minucci S. Bernardi P. Pelicci P.G. Giorgio M. p66Shc-generated oxidative signal promotes fat accumulation.J. Biol. Chem. 2008; 283: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), resistance to genetic obesity (11Ranieri S.C. Fusco S. Panieri E. Labate V. Mele M. Tesori V. Ferrara A.M. Maulucci G. De Spirito M. Martorana G.E. Galeotti T. Pani G. Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13420-13425Crossref PubMed Scopus (84) Google Scholar), resistance to high fat diets (HFD) (4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar) and higher capacity for mitochondria to oxidize lipid iv vitro and iv vivo (2Hagopian K. Tomilov A.A. Tomilova N. Kim K. Taylor S.L. Lam A.K. Cortopassi G.A. McDonald R.B. Ramsey J.J. Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis.Metabolism: Clinical and Experimental. 2012; 61: 1703-1713Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 3Tomilov A. Bettaieb A. Kim K. Sahdeo S. Tomilova N. Lam A. Hagopian K. Connell M. Fong J. Rowland D. Griffey S. Ramsey J. Haj F. Cortopassi G. Shc depletion stimulates brown fat activity in vivo and in vitro.Aging Cell. 2014; 2612267Google Scholar, 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar), and a metabolic shift toward increasing mitochondrial lipid oxidation (1Hagopian K. Tomilov A.A. Kim K. Cortopassi G.A. Ramsey J.J. Key glycolytic enzyme activities of skeletal muscle are decreased under Fed and Fasted states in mice with knocked down levels of Shc proteins.PloS one. 2015; 10e0124204Crossref PubMed Scopus (12) Google Scholar, 2Hagopian K. Tomilov A.A. Tomilova N. Kim K. Taylor S.L. Lam A.K. Cortopassi G.A. McDonald R.B. Ramsey J.J. Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis.Metabolism: Clinical and Experimental. 2012; 61: 1703-1713Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Enzymatic activity of mitochondrial thiolase is significantly increased in these ShcKO tissues, which have reduced expression of mitochondrial p46Shc isoform (2Hagopian K. Tomilov A.A. Tomilova N. Kim K. Taylor S.L. Lam A.K. Cortopassi G.A. McDonald R.B. Ramsey J.J. Shc proteins influence the activities of enzymes involved in fatty acid oxidation and ketogenesis.Metabolism: Clinical and Experimental. 2012; 61: 1703-1713Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We carried out mass spectrometry, two-dimensional gel electrophoresis, and bio-layer interferometry (BLI) (12Abdiche Y.N. Malashock D.S. Pinkerton A. Pons J. Exploring blocking assays using Octet, ProteOn, and Biacore biosensors.Anal. Biochem. 2009; 386: 172-180Crossref PubMed Scopus (65) Google Scholar, 13Abdiche Y.N. Miles A. Eckman J. Foletti D. Van Blarcom T.J. Yeung Y.A. Pons J. Rajpal A. High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.PloS One. 2014; 9e92451Crossref PubMed Scopus (78) Google Scholar, 14Wilson J.L. Scott I.M. McMurry J.L. Optical biosensing: Kinetics of protein A-IGG binding using biolayer interferometry.Biochem. Mol. Biol. Educ. 2010; 38: 400-407Crossref PubMed Scopus (21) Google Scholar) assays to identify the major mitochondrial partner of p46Shc. These assays identified mitochondrial 3-ketoacylCoA thiolase (ACAA2) as the p46Shc major partner. Furthermore, p46Shc modulation iv vitro and iv vivo modulates ACAA2 activity. Mitochondrial lipid oxidation is a stepwise pathway resulting in the production of acetoacetylCoA and acetylCoA, which enters the TCA cycle to form citrate. ACAA2 thiolase catalyzes the last step of the spiral, to form acetylCoA. Specific small molecule regulators of ACAA2 activity have been shown to regulate flux through the pathway (15Brunengraber H. Boutry M. Lowenstein J.M. Fatty acid and 3- -hydroxysterol synthesis in the perfused rat liver. Including measurements on the production of lactate, pyruvate, -hydroxy-butyrate, and acetoacetate by the fed liver.J. Biol. Chem. 1973; 248: 2656-2669Abstract Full Text PDF PubMed Google Scholar, 16Raaka B.M. Lowenstein J.M. Inhibition of fatty acid oxidation by 2-bromooctanoate. Evidence for the enzymatic formation of 2-bromo-3-ketooctanoyl coenzyme A and the inhibition of 3-ketothiolase.J. Biol. Chem. 1979; 254: 6755-6762Abstract Full Text PDF PubMed Google Scholar, 17Raaka B.M. Lowenstein J.M. Inhibition of fatty acid oxidation by 2-bromooctanoate. Including effects of bromooctanoate on ketogenesis and gluconeogenesis.J. Biol. Chem. 1979; 254: 3303-3310Abstract Full Text PDF PubMed Google Scholar). Data suggest p46Shc to be an inhibitor of mitochondrial thiolase activity, and that p46Shc depletion activates mitochondrial thiolase and increased lipid oxidation. ShcKO mice were originally characterized by Refs. 8Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. The p66shc adaptor protein controls oxidative stress response and life span in mammals.Nature. 1999; 402: 309-313Crossref PubMed Scopus (1475) Google Scholar, 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar; these mice were denoted “ShcP” in Ref. 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar. ShcL mice were previously described in Ref. 4Tomilov A.A. Ramsey J.J. Hagopian K. Giorgio M. Kim K.M. Lam A. Migliaccio E. Lloyd K.C. Berniakovich I. Prolla T.A. Pelicci P. Cortopassi G.A. The Shc locus regulates insulin signaling and adiposity in mammals.Aging Cell. 2011; 10: 55-65Crossref PubMed Scopus (51) Google Scholar. Both strains, ShcKO and ShcL were re-derived using IVF into C57BL6 background. Mice were kept pathogen-free throughout the study in a barrier facility at UC Davis. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were performed in compliance with local, state, and federal regulations. Diet was LM-485 (Teklad 7012) with nutrient composition: protein, 19.9%; carbohydrates, 53.7%; fat, 5.7%. The high fat diet was: protein, 17.7%; carbohydrates, 7.3%; fat, 60.0%. The source of lipid was soybean oil, protein (soybean meal), energy source (ground oats). Antibodies were from Cell Signaling Inc. (Danvers, MA) except for rabbit anti-Shc, mouse anti-Shc, and anti-cytochrome c; these antibodies were from BD Biosciences (San Diego, CA). Rabbit anti-PCX and mouse anti-ACADVL antibody were from Novus Biologicals (Littletown, CO), mouse anti-tubulin, and mouse anti-ACAA2 antibody were from Sigma; mouse anti-ACAA1 was from Mito Science Inc. (Eugene, OR). Rabbit anti-CPT2 was from Epitomics Inc. (Burlingame, CA), goat anti-ETFA antibodies were from Everest Biotech (San Diego, CA). The infrared dye 700 and 800 labeled streptavidin and secondary antibody were from Li-Cor Biosciences (Lincoln, NE). The transfection reagent TransIT-LT1 was purchased from Mirus Bio LLC (Madison, WI). Plasmids were purchased from GeneCopoeia (Rockville, MD): EX-H9090-M62 for expression of human C-terminally biotinylated p46Shc, EX-C0702-M48 and EX-T3030-M48 for expression of mouse and human N-terminally biotinylated ACAA2, respectively, EX-EGFP-M48 for expression of N-terminally biotinylated GFP, and EX-T3030-M48 for expression of N-terminally biotinylated p52Shc. ShcδPTB and ShcδSH2 plasmids were constructed based on EX-T3030-M48 and EX-H9090-M62, respectively, using the following primers: 5′-gagttgcgcttcaaacaatacctc-3′, 5′-gtcgttgggatgcagccagcc-3′ to create EX-T3030-M48-p562Shcδ(44–198)PTB, and 5′-gagcggaaactgtacctcgagt-3′, 5′-gagctgctcagccatggacac-3′ to create EX-H9090-M62-p46Shcδ(329–425)SH2, using Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, Inc., Rockford, IL). Livers were extracted from 3 m/o C57Bl6 male mice and homogenized in ice-cold fractionation buffer: 250 mm sucrose, 10 mm Tris-HCl, 1 mm EGTA, pH 7.4. Cell debris (P1) was separated from homogenates (Tot) for 5 min at 400 × g, and the supernatant (S1) centrifuged for 10 min at 700 × g for the separation of nuclei or intact unbroken cells (P2) and the supernatant (S2) centrifuged at 7000 × g for 15 min for the isolation of mitochondria (P3) and cytosolic fraction (S3). The pellet containing the mitochondria was washed twice and finally resuspended in 0.2 m mannitol, 50 mm sucrose, 1 mm EDTA, 10 mm HEPES-NaOH, pH 7.4 for the further separation of the heavy mitochondrial fraction (He), the light mitochondrial (Li), and the microsomal (MS) fractions by sequential centrifugation 10 min each at 3000 × g, 15,000 × g, and 100,000 × g, respectively. Polarographic analysis were performed by using Clark type Hansatech Oxygraph in 125 mm KCl, 10 mm MOPS-Tris, 1 mm inorganic phosphate, 100 μm EGTA, pH 7.4 with the addition of 5 mm glutamate/malate, 400 μm ADP or 1–10 mm dinitrophenol to induce, respectively, state 3 or the uncoupled state. For stable transfection of FL83B cells we used lentiviral vectors pLKO.1 empty, PLKO.1 scramble, pLKO.1 RMM 3981-97976309 (shShc-1), pLKO.1 RMM 3981-9622377 (shShc-2), pLKO.1 RMM 3981-9622378 (shShc-3), pLKO.1 RMM 3981-9622379 (shShc-4), pLKO.1 RMM 3981-9622381 (shShc-5), and pLKO.1 RMM 3981-97076308 (shShc-6) from Open Biosystems, GE Healthcare Dharmacon Inc. (Piscataway, NJ). Mitochondria were isolated according to Ref. 18Frezza C. Cipolat S. Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.Nat. Protocols. 2007; 2: 287-295Crossref PubMed Scopus (859) Google Scholar. Briefly, livers from fasted mice were placed into ice-cold isolation medium (220 mm mannitol, 70 mm sucrose, 1 mm EDTA, 20 mm Tris, 0.1% FA-free BSA, pH 7.4), chopped, rinsed from blood, placed in 10% (w/v) isolation medium, and homogenized with six strokes at 1000 rpm. The homogenate was centrifuged at 500 × g for 10 min at 4 °;C, and the resulting supernatant was decanted into clean centrifuge tubes and centrifuged at 10,000 × g for 10 min (4 °;C). The supernatant was discarded, and the pellet was resuspended and washed in isolation medium with and without BSA, followed by centrifugation at 10,000 × g. The mitochondrial pellet was suspended in isolation medium without BSA (220 mm mannitol, 70 mm sucrose, 1 mm EGTA, 20 mm Tris, pH 7.4). Mitochondria were plated (10 μg/50 μl protein per well), spun at 4 °;C for 20 min at 3000 × g, and covered with 450 μm MAS-3 Seahorse assay medium (115 mm KCl, 10 mm KH2PO4, 2 mm MgCl2, 3 mm HEPES, 1 mm EGTA, 0.2% FA-free BSA), supplemented with either 10 mm succinate+2 μm rotenone, 10 mm pyruvate+5 mm malate, or 20 μm palmitoyl-l-carnitine+10 mm malonate to measure fluxes through respective oxidative processes. Oxygen consumption rates (OCR) were measured starting from basal respiration followed by sequential regimens of 2 mm ADP, 1 μm oligomycin, 50 μm FCCP, and 4 μm antimycin A/2 μm rotenone. Basal respiration was assayed in 2 cycles of mix (50 s) and measuring (2 min), ADP in one cycle of mix (50 s) and measuring (8 min), oligomycin in 1 cycle of mix (50 s) and measuring (4 min), FCCP in 1 cycle of mix (50 s) and measuring (4 min), antimycin A/rotenone in 1 cycle of mix (50 s), and measuring (4 min). Unbuffered DMEM was used for glucose-driven OCR measurements, and KHB (111 mm NaCl, 4.7 mm KCl, 2 mm MgSO4, 1.2 mm Na2HPO4, 0.5 mm carnitine, 0.6% fat-free BSA, pH 7.4) was used to measure palmitate-driven OCR of cultured cells. FL83B cells (200 μl) of DMEM, 25 mm glucose, 10% FBS (50.000/well) onto Seahorse plates and let attach overnight; medium was changed to 2.5 mm glucose DMEM for 24 h and then changed to KHB. Regimens: BSA/sodium palmitate 0.25%/800 μm complex (palmitate), oligomycin, 1 μm; FCCP, 5 μm; rotenone, 0.1 μm; antimycin A, 1 μm. Total protein in each well was used for normalization. HeLa cells were transfected with p66Shc specific or TotalShc-specific si-RNA as described in Ref. 7Kisielow M. Kleiner S. Nagasawa M. Faisal A. Nagamine Y. Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.Biochem. J. 2002; 363: 1-5Crossref PubMed Scopus (75) Google Scholar, and 150,000 cells were seeded in 200 μl of DMEM, 10% FBS, 25 mm glucose medium, and left to attach overnight. Medium was changed to KHB; cells were equilibrated for 1 h, and OCR was assayed with following regiments concentrations: BSA/sodium palmitate 0.6%/200 μm complex, oligomycin, 1 μm; FCCP, 5 μm; rotenone, 0.1 μm; antimycin A, 1 μm. Total protein in each well was used for normalization. Mouse liver mitochondria were tested for quality as described, lysed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) lysis buffer (pH 7.5) containing 20 mm HEPES, 150 mm KCl, 0.5% CHAPS, 10% (v/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, and EDTA-free complete protease inhibitor mixture (Roche). 5 mg of lysates were incubated in 600 μl of lysis buffer with 10 μg of rabbit IgG or rabbit polyclonal anti-Shc antibody and 50 μl of Dynabeads Protein G (Invitrogen Inc., Grand Island, NY) at 4 °;C for 2 h. Precipitates were analyzed by Western blot with the indicated antibody. Precipitates were separated on 4–12% Bis-Tris PAGE, stained with colloid Coomassie Blue; bands were excised, and sent for mass spectrometry analysis. Proteins were isolated with cell lysis buffer (Cell Signaling Technologies), and Westerns were analyzed with the indicated antibody. BN gel electrophoresis and second dimension SDS-PAGE followed by Western blotting were performed according to the protocol described by Wittig and Schagger (19Wittig I. Schägger H. Electrophoretic methods to isolate protein complexes from mitochondria.Methods Cell Biol. 2007; 80: 723-741Crossref PubMed Scopus (32) Google Scholar). Mitochondria were solubilized with digitonin for 15 min on ice; buffer: 2 mm 6-aminohexanoic acid, 50 mm Bis-Tris, and 0.5 mm EDTA, pH 7.0. A digitonin/protein ratio was 6:1, as empirically determined. Samples were centrifuged 30 min at 20,000 × g at 4 °;C, Coomassie Blue G-250 was added to the supernatant with ratio 8:1 (w/w). 100 μg of proteins per line were separated on Native-PAGE Novus 4–16% Bis-Tris gradient gel (Invitrogen). To verify intact protein complexes, in-gel activity of OXPHOS complexes and supercomplexes was performed as described (19Wittig I. Schägger H. Electrophoretic methods to isolate protein complexes from mitochondria.Methods Cell Biol. 2007; 80: 723-741Crossref PubMed Scopus (32) Google Scholar, 20Smet J. De Paepe B. Seneca S. Lissens W. Kotarsky H. De Meirleir L. Fellman V. Van Coster R. Complex III staining in blue native polyacrylamide gels.J. Inherit. Metab. Dis. 2011; 34: 741-747Crossref PubMed Scopus (19) Google Scholar, 21Schägger H. von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1906) Google Scholar, 22Acín-Pérez R. Fernández-Silva P. Peleato M.L. Pérez-Martos A. Enriquez J.A. Respiratory active mitochondrial supercomplexes.Mol. Cell. 2008; 32: 529-539Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). For the second dimension, strips of BN gels were denatured in 50 mm TrisHCl (pH 7.0), 10% glycerol, 200 mm DTT, 2% SDS, 0.1% bromphenol blue; Western blots were performed. Proteins we expressed in HEK T293 cells: terminally biotinylated p46Shc protein, p46Shc version lacking the PTB domain (p46ShcδPTB), p46Shc lacking the SH2 domain (p46ShcδSH2), GFP, and ACAA2 were produced using the described plasmids. Expression, purity, and biotinylation levels of proteins were verified by Western blotting. Proteins were purified with avidin agarose, and p46Shc or p46ShcδSH2 were loaded onto sets of Octet RED 384 SA biosensors (Pall ForteBio LLC., Menlo Park, CA) to a density of 7 nm followed by blocking of non-occupied streptavidin residues of biosensors with 200 μm biotin. Sensors were tested against titration series of concentrations of purified ACAA2 or GFP as follows: 3.5 μm, 1.7 μm, 0.9 μm, 433.7 nm, 216.9 nm, 108.4 nm, 54.2 nm, and 27.1 nm for both ACAA2 and GFP. Series dilutions were prepared in BLI Kinetic Buffer (Pall ForteBio LLC., Menlo Park, CA) containing 1% BSA. We tested the full size p46Shc in parallel with p46ShcδPTB, p46ShcδSH2, and GFP against indicated concentrations of ACAA2. GFP sensors were used to compensate for possible signal drifting due to buffer changes or nonspecific bindings of ACAA2 to the sensors. A typical binding sensorgram is presented in Fig. 4a. After a stable baseline was achieved, biosensors were moved into solution that contain ACAA2 or GFP (the association step) for 900 s, then biosensors were moved into a buffer without ACAA2 or GFP (the dissociation step); log plots of responses [nm] were created against log C [nm] of thiolase, and EC50 and Hill slopes were measured. AcylCoA dehydrogenase activity, using palmitoyl-CoA as substrate, was determined at 600 nm (∈ = 21 mm−1 cm−1) by measuring the decrease in absorbance due to acylCoA-dependent reduction of 2,6-dichlorophenolindophenol (DCPIP) in the presence of phenazine methosulfate (PMS). The activity of 3-hydroxyacylCoA dehydrogenase, using acetoacetylCoA as substrate, was determined at 340 nm (∈ = 6.22 mm−1 cm−1) by measuring the oxidation of NADH. The activity of 3-oxoacylCoA thiolase, using acetoacetylCoA as substrate, was determined at 303 nm (∈ = 16.9 mm−1 cm−1) by measuring the breakdown of the thioester bond. Statistical analysis was performed with SAS 9.4 and Excel Statistical Data Analysis Tool package 2007. Unless indicated otherwise, p values were determined with one-way ANOVA followed by Scheffe’s post hoc planed comparison test; *, p < 0.1; **, p < 0.05. Survival curves were analyzed with Logrank Test using Prizm 4 for Windows Version 4.02 Software. p46Shc was previously reported to have cryptic N-terminal mitochondrial signal sequence and to be targeted to the mitochondria (6Ventura A. Maccarana M. Raker V.A. Pelicci P.G. A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria.J. Biol. Chem. 2004; 279: 2299-2306Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). p46Shc was fused with RFP and cotransfected with mito-GFP into NIH3T3 cells (Fig. 1a); Mito-GFP and p46Shc::RFP fluorescence overlapped. This is consistent with mitochondrial localization of p46Shc. Moreover, p46Shc::GFP fusion was transfected into NIH3T3 cells. As previously observed (6Ventura A. Maccarana M. Raker V.A. Pelicci P.G. A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria.J. Biol. Chem. 2004; 279: 2299-2306Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) p46Shc showed a clear mitochondrial pattern of distribution iv vitro (Fig. 1a). Livers from C57BL6 mice were fractionated: the highest-respiring fractions had more of the p46Shc isoform (Fig. 1b), and only p46Shc, but not p52Shc was very abundant in the purified mitochondrial fraction (He). These results are consistent with mitochondrial localization of p46Shc. This is also consistent with data published earlier by others where p46Shc was noted to be mitochondrially localized (6Ventura A. Maccarana M. Raker V.A. Pelicci P.G. A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria.J. Biol. Chem. 2004; 279: 2299-2306Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Ljubicic V. Menzies K.J. Hood D.A. Mitochondrial dysfunction is associated with a pro-apoptotic cellular environment in senescent cardiac muscle.Mech. Ageing Dev. 2010; 131: 79-88Crossref PubMed Scopus (39) Google Scholar). We isolated mitochondria from wild type C57Bl6 mouse liver, and tested samples for purity in an effort to isolate only physiologically active mitochondria. Samples were analyzed to be free from cytosolic contamination; Western blot analysis for mitochondrial markers MnSOD, CytC, VDAC, and cytosolic markers such as tubulin is" @default.
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• W2441488300 title "p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria" @default.
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