Research paper Effect of Lon protease knockdown on mitochondrial function in HeLa cells Aurélien Bayot a, d , Monique Gareil a , Laurent Chavatte b,1 , Marie-Paule Hamon a , Caroline L’Hermitte-Stead e , Florian Beaumatin c , Muriel Priault c , Pierre Rustin d , Anne Lombès e , Bertrand Friguet a, * , Anne-Laure Bulteau a,1 a UR4 e Vieillissement, Stress, Inflammation, Sorbonne Universités, UPMC Univ Paris 06, Université Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France b Centre de recherche de Gif-sur-Yvette, FRC 3115, Centre de Génétique Moléculaire, CNRS, UPR3404, 91198 Gif-sur-Yvette Cedex, France c Institut de Biochimie et Génétique Cellulaires, UMR 5095, CNRS, Université Bordeaux 2, France d Inserm, Hopital Robert Debré, 75019 Paris, France e Inserm, Institut Cochin, 75014 Paris, France article info Article history: Received 23 July 2013 Accepted 4 December 2013 Available online 17 December 2013 Keywords: Lon protease Mitochondria HeLa cells Protein oxidation Cellular redox status abstract ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI- 38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Mitochondria are a major source of intracellular reactive oxygen species (ROS), the production of which increases with ageing and cancer. The deleterious effects of ROS may be responsible for the impairment of mitochondrial function observed during various pathophysiological states associated with oxidative stress, ageing and cancer. These organelles are also targets of oxidative damage (oxidation on mtDNA, lipids, proteins) [1]. An important factor for protein maintenance in the presence of oxidative stress is the enzymatic removal of oxidative modifications and/or protein degradation. Two soluble ATP-dependent proteases, Lon and ClpXP, are found in the matrix of mammalian mitochondria. Lon is a homo-oligomeric complex whereas ClpXP is a hetero-oligomeric complex. This hetero-oligomer of ClpXP is comprised of two sub- units: ClpP, the proteolytic component, and ClpX, the ATPase component [2]. Currently, information regarding the role and the identity of specific protein substrates for each of the ATP- dependent proteases is limited [3e7]. The Lon protease is espe- cially efficient in the recognition and proteolysis of misfolded and damaged protein structures [8]. Reduced Lon activity has been associated with insufficient degradation and accumulation of oxidized protein, impaired mitochondrial function and cell death in WI-38 fibroblasts [9]. The Lon protease has been reported to exhibit an age-related impairment in skeletal muscle, liver and heart that was associated with a buildup of oxidatively modified protein [10,11]. Age-dependent increases in the levels of oxidatively modified protein may therefore be due, in part, to decreased clearance of damaged protein by proteases such as Lon. Lon Abbreviations: ROS, reactive oxygen species; MTT, 3,4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazoliumbromide; COX, cytochrome c oxidase; VO 2 , oxygen con- sumption rate; FCCP, carbonyl cyanide-trifluoromethoxy phenylhydrazine; CCCP, carbonyl cyanide-chloro phenylhydrazine; HBSS, Hank’s buffered salt solution. * Corresponding author. Laboratoire de Biologie Cellulaire du Vieillissement e UR4 e Vieillissement, Stress, Inflammation, Université Pierre et Marie Curie e Paris 6, case courrier 256, 4 Place Jussieu, 75252 Paris Cedex 05, France. Tel.: þ33 1 44 27 32 05; fax: þ33 1 44 27 51 40. E-mail address: bertrand.friguet@snv.jussieu.fr (B. Friguet). 1 Present address: Laboratoire de Chimie Analytique Bio-Inorganique et Envi- ronnement, CNRS/UPPA, UMR5254, 64053 Pau Cedex, France. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.12.005 Biochimie 100 (2014) 38e47