title: Analysis of the mitochondrial proteome of cybrid cells harbouring a truncative mitochondrial DNA mutation in respiratory complex I.
abstract: Transmitochondrial cytoplasmic hybrids (cybrids) are well established model systems to reveal the effects of mitochondrial DNA (mtDNA) mutations on cell metabolism excluding the interferences of a different nuclear background. The m.3571insC mutation in the MTND1 gene of respiratory complex I (CI) is commonly detected in oncocytic tumors, in which it causes a severe CI dysfunction leading to an energetic impairment when present above 83% mutant load. To assess whether the energetic deficit may alter the mitochondrial proteome, OS-78 and OS-93 cybrid cell lines bearing two different degrees of the m.3571insC mutation (78% and 92.8%, respectively) and control cybrids bearing wild-type mtDNA (CC) were analyzed. Two-dimensional electrophoresis and mass spectrometry revealed significant alterations only in cybrids above the threshold (OS-93). All differentially expressed proteins are decreased. In particular, the levels of the pyruvate dehydrogenase E1 chain B subunit (E1β), of lipoamide dehydrogenase (E3), the enzyme component of pyruvate and 2-oxoglutarate dehydrogenase complexes, and of lactate dehydrogenase B (LDHB) were reduced. Moreover, a significant decrease of the pyruvate dehydrogenase complex activity was found when OS-93 cybrid cells were grown in galactose medium, a metabolic condition that forces cells to use respiration. These results demonstrate that the energetic impairment caused by the almost homoplasmic m.3571insC mutation perturbs cellular metabolism leading to a decreased steady state level of components of very important mitochondrial NAD-dependent dehydrogenases.
authors.affiliation: CNR - Institute of Biomembranes and Bioenergetics, Bari, Italy. c.musicco@ibbe.cnr.it.
authors.forename: Clara
authors.initials: C
authors.lastname: Musicco
authors.forename: Antonella
authors.initials: A
authors.lastname: Cormio
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Calvaruso
authors.forename: Luisa
authors.initials: L
authors.lastname: Iommarini
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gasparre
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Porcelli
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Timperio
authors.forename: Lello
authors.initials: L
authors.lastname: Zolla
authors.forename: Maria Nicola
authors.initials: MN
authors.lastname: Gadaleta
mesh_terms: D045744:Cell Line, Tumor; D019069:Cell Respiration; D003470:Culture Media; D004272:DNA, Mitochondrial / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D006822:Hybrid Cells; D045022:Lactate Dehydrogenases / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics*; D020543:Proteome / Q000378:metabolism*; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000378:metabolism
chemical_list: D003470:Culture Media; D004272:DNA, Mitochondrial; D008058:Dihydrolipoamide Dehydrogenase; D005690:Galactose; D045022:Lactate Dehydrogenases; D009245:NADH Dehydrogenase; D020543:Proteome; D030481:Pyruvate Dehydrogenase (Lipoamide); C071195:D-lactate dehydrogenase; C111305:pyruvate dehydrogenase E1 beta subunit; C515075:NADH dehydrogenase subunit 1, human