title: Effects of Lon protease down-regulation on the mitochondrial function and proteome.
abstract: The Lon protease is an ATP-dependent protease of the mitochondrial matrix that contributes to the degradation of abnormal and oxidized proteins in this compartment. It is also involved in the stability and regulation of the mitochondrial genome. The effects of a depletion of this protease on the mitochondrial function and the identification of oxidized target proteins of Lon have been performed using as cellular model HeLa cells in which Lon level expression can be down-regulated. The expression level of proteins playing a role in the stress response was first determined. The amount of ClpP, another protease in charge of protein degradation of the mitochondrial matrix, and the amount of several chaperones have been evaluated. The expression level of respiratory chain subunits was also measured with or without Lon depletion. The mitochondrial compartment morphology was monitored in different stress conditions, and measured using a parameter devoted to the evaluation of the mitochondrial dynamics. None of these investigations showed a significant phenotype resulting from Lon down-regulation A possible impact of Lon depletion on oxidized mitochondrial proteins level was then sought. 1D gel electrophoresis after the derivatization of protein carbonyl groups with 2,4-dinitrophenyl hydrazine (DNPH) revealed an increase in carbonylated proteins more important in mitochondrial extracts than in total cellular extracts. 2D difference gel electrophoresis (DIGE) experiments provide results consistent with these observations with some enlightenments. Performed with fluorescent dyes labelling either proteins or their carbonyl groups, these experiments indicated proteome modifications in cells with Lon down-regulation both at the level of protein expression and at the level of protein oxidation. These variations are noted in proteins acting in different cellular activities, i.e. metabolism, protein quality control and cytoskeleton organization.
authors.affiliation: UMR CNRS UPMC 8256, INSERM U1164 "Biological Adaptation and Aging" (Université Pierre et Marie Curie), Paris, France. Electronic address: mariepaulehamon@gmail.com.
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Hamon
authors.affiliation: UMR CNRS UPMC 8256, INSERM U1164 "Biological Adaptation and Aging" (Université Pierre et Marie Curie), Paris, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bayot
authors.affiliation: UMR CNRS UPMC 8256, INSERM U1164 "Biological Adaptation and Aging" (Université Pierre et Marie Curie), Paris, France.
authors.forename: Monique
authors.initials: M
authors.lastname: Gareil
authors.affiliation: UMR5254 CNRS UPPA, Laboratory of Bio-inorganic Analytical Chemistry (Université de Pau et des Pays de l'Adour), Pau, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chavatte
authors.affiliation: Inserm U1016, CNRS UMR 8104, Université Paris Descartes UMR-S1016 (Institut Cochin), Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Lombès
authors.affiliation: UMR CNRS UPMC 8256, INSERM U1164 "Biological Adaptation and Aging" (Université Pierre et Marie Curie), Paris, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Friguet
authors.affiliation: UMR5254 CNRS UPPA, Laboratory of Bio-inorganic Analytical Chemistry (Université de Pau et des Pays de l'Adour), Pau, France.
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Bulteau