Function of Mitochondrial Stat3 in Cellular Respiration
Naresh B. V. Sepuri4,
Darren P. Baker10,
Medhat O. Hassan12,
Jennifer I. Drake14,
Edward J. Lesnefsky9,15, and
Andrew C. Larner1
1 Department of Biochemistry and Molecular Biology and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
2 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland.
3 Department of Biology, Cleveland State University, Cleveland, OH 44114, USA.
4 Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
5 Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
6 Department of Immunology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland.
7 Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
8 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
9 Division of Cardiology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
10 BiogenIdec, 14 Cambridge Center, Cambridge, MA 02142, USA.
11 Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
12 Pathology and Laboratory Medicine Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA.
13 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
14 Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
15 Medical Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA.
Fig. 1.. Stat3 in the mitochondria from mouse heart and liver. (A) Whole cell (WCE), cytoplasmic (Cyto), and mitochondrial (Mito) extracts were separated by SDS-PAGE. The blots were probed with antisera against Stat3, -tubulin, calreticulin, and GRIM-19. (B) Increasing amounts of heart and liver mitochondria probed for Stat3, tubulin, and cytochrome c. (C) mStat3 is proteinase K–resistant. Mitochondria were incubated with (lanes 2 and 3) or without (lane 1) proteinase K (PK). To disrupt mitochondrial integrity, triton X-100 was added in the digestion buffer (lane 3). Samples were probed for Stat3, porin, Bcl-2, and GRIM-19. (D) mStat3 is serine phosphorylated. Purified mitochondria, as well as cytosolic and whole cell extracts were prepared from WT mice hearts. The immunoblots were probed for either total Stat3 or serine phosphorylated Stat3, as well as voltage-dependent anion channel (VDAC) and -tubulin as controls for mitochondrial purity. A fluorescent conjugated secondary antibody was used to develop the blots allowing the relative amount of total and serine phosphorylated Stat3 to be measured. The ratio of total Stat3 to serine phosphorylated Stat3 in whole cell extracts was 2.5, in cytosolic extracts was 2.3, and in mitochondria extracts was 0.3.
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Fig. 2.. Stat3 in complex I immunoprecipitates (IP). Antibody to complex I (CxI) or a nonspecific isotype-matched IgG were incubated with liver mitochondrial extracts. Immunoprecipitates were resolved on SDS-PAGE and probed for Stat3, GRIM-19, NDUFA9, Cx II Fp subunit, Cx III core protein 2, Cx IV subunit I, and Cx V subunit.
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Fig. 3.. Depressed mitochondrial respiration in Stat3–/– pro-B cells. (A) Mitochondrial oxidase activity in WT and Stat3–/– cells: NADH oxidase (left) and DHQ oxidase (right). Oxygen consumption is expressed as nanogram atom of oxygen per minute per milligram of mitochondrial protein (nAtom O/min/mg) and is presented as mean ± SD. (B) ETC activities of complexes I, II, III, and IV. Activities are expressed as milliunits (nanomoles per minute) per milligram of mitochondrial protein and were normalized to citrate synthase activity. Results are presented as mean ± SD. Error bars indicate SD; asterisks indicate P < 0.05.
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Fig. 4.. Decreased rates of O2 consumption in Stat3–/– heart mitochondria. Mitochondria isolated from littermates of WT (left) and Stat3–/– (right) hearts were incubated in an oxygen sensor chamber, and O2 consumption (y axis) as a function of incubation time (x axis) was recorded. In the upper panels, the mitochondria were incubated with pyruvate/malate (complex I substrates), and they were incubated with succinate (complex II substrate) in the lower panels. Different concentrations of ADP were added to the mitochondria to measure state 3 respiration (0.2 mM ADP) or state 3 Vmax rates of respiration (2 mM ADP). State 4 rates of respiration were calculated when 0.2 mM ADP was depleted from the reaction.
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