title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier.
abstract: GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.
authors.affiliation: Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Martínez-Zamora
authors.affiliation: Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Meseguer
authors.affiliation: Laboratory of Intracellular Protein Degradation and Rare Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Esteve
authors.affiliation: Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.
authors.forename: Magda
authors.initials: M
authors.lastname: Villarroya
authors.affiliation: Laboratory of Intracellular Protein Degradation and Rare Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.|Centro de Investigación Biomédica En Red de Enfermedades Raras (CIBERER), node U721, Valencia, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Aguado
authors.affiliation: Departamento de Desarrollo y Reparación Cardiovascular, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.|Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: J Antonio
authors.initials: JA
authors.lastname: Enríquez
authors.affiliation: Laboratory of Intracellular Protein Degradation and Rare Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.|Centro de Investigación Biomédica En Red de Enfermedades Raras (CIBERER), node U721, Valencia, Spain.
authors.forename: Erwin
authors.initials: E
authors.lastname: Knecht
authors.affiliation: Laboratory of RNA Modification and Mitochondrial Diseases, Centro de Investigación Príncipe Felipe, Valencia, Spain.|Centro de Investigación Biomédica En Red de Enfermedades Raras (CIBERER), node U721, Valencia, Spain.
authors.forename: M-Eugenia
authors.initials: ME
authors.lastname: Armengod
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006019:Glycolysis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D027501:Monocarboxylic Acid Transporters; D010085:Oxidative Phosphorylation; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D000071246:Uncoupling Protein 2
chemical_list: D027321:Anion Transport Proteins; D002148:Calmodulin-Binding Proteins; D005227:Fatty Acids; D007473:Ion Channels; C573718:MPC1 protein, human; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D027501:Monocarboxylic Acid Transporters; C541710:NDUFAF3 protein, human; C519296:NDUFAF4 protein, human; D012357:RNA, Transfer, Lys; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2; D000255:Adenosine Triphosphate; D055372:AMP-Activated Protein Kinases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D019204:GTP-Binding Proteins; C498989:GTPBP3 protein, human; D042967:Electron Transport Complex I