title: An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters.
abstract: Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ(0) cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK(-) A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ(0) state. Pathway and protein-protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ(0) cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ(0) cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ(0) cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ(0) cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ(0) cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.
authors: From the ‡Max Planck Institute for Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany; §Freie Universität Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustraße 3, 14195 Berlin, Germany;
authors: Ina
authors: I
authors: Aretz
authors: From the ‡Max Planck Institute for Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany;
authors: Christopher
authors: C
authors: Hardt
authors: ¶Functional Proteomics, Faculty of Medicine, Goethe-University, Theodor Stern Kai 7, Haus 26, D-60590 Frankfurt am Main, Germany.
authors: Ilka
authors: I
authors: Wittig
authors: From the ‡Max Planck Institute for Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany; meierhof@molgen.mpg.de.
authors: David
authors: 0000-0002-0170-868X
authors: D
authors: Meierhofer
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D045744:Cell Line, Tumor; D002952:Citric Acid Cycle; D004272:DNA, Mitochondrial / Q000235:genetics*; D015536:Down-Regulation; D004734:Energy Metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019289:Pyruvic Acid / Q000378:metabolism*; D017384:Sequence Deletion; D054875:Ubiquitination
chemical_list: D000596:Amino Acids; D004272:DNA, Mitochondrial; D020543:Proteome; D019289:Pyruvic Acid