Biochimica et Biophysica Acta 1864 (2017) 169–176 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Sirtuin 5 protects mitochondria from fragmentation and degradation during starvation Hala Guedouari a, Tanya Daigle a, Luca Scorrano b,c, Etienne Hebert-Chatelain a,⁎ a Department of Biology, University of Moncton, NB, Canada b Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, Padua, Italy c Department of Biology, University of Padua, Italy article info abstract Article history: During starvation, intra-mitochondrial sirtuins, NAD+ sensitive deacylating enzymes that modulate metabolic Received 17 May 2016 homeostasis and survival, directly adjust mitochondrial function to nutrient availability; concomitantly, mito- Received in revised form 13 September 2016 chondria elongate to escape autophagic degradation. However, whether sirtuins also impinge on mitochondrial Accepted 25 October 2016 dynamics is still uncharacterized. Here we show that the mitochondrial Sirtuin 5 (Sirt5) is essential for starvation Available online 15 November 2016 induced mitochondrial elongation. Deletion of Sirt5 in mouse embryonic fibroblasts increased levels of mito- chondrial dynamics of 51 kDa protein and mitochondrial fission protein 1, leading to mitochondrial accumulation Keywords: fi Sirtuin 5 of the pro- ssion dynamin related protein 1 and to mitochondrial fragmentation. During starvation, Sirt5 Mitochondrial fragmentation deletion blunted mitochondrial elongation, resulting in increased mitophagy. Our results indicate that starvation Mitochondrial degradation induced mitochondrial elongation and evasion from autophagic degradation requires the energy sensor Sirt5. © 2016 Elsevier B.V. All rights reserved. 1. Introduction lowered, and ROS production is increased [6–8]. The mitochondrial structure is also modified by nutrient availability. During starvation, mi- Mitochondrial oxidative phosphorylation (OXPHOS) converts most tochondria elongate in vitro and in vivo [9,10]. This elongation process of the energy found in nutrients into ATP, required for cellular ender- allows maintaining ATP production via lower mitochondrial degrada- gonic reactions. These key organelles also regulate several other impor- tion and increased dimerization and activity of ATP synthase [9].Nutri- tant physiological processes, such as calcium homeostasis, oxidative ent excess is associated with fragmented mitochondria in different stress and apoptosis [1]. Mitochondria are highly dynamic organelles: in vitro and in vivo models [11,12]. For instance, deletion of the protease they form a reticulum continuously remodeled by fission and fusion OMA1 impairs OPA1 processing and causes obesity [13]. Biopsies of events. In mammalian cells, mitochondrial fragmentation is mainly reg- skeletal muscle from subjects with type II diabetes and obesity reveal ulated by DRP1 [2]. During mitochondrial fission, DRP1 is recruited to mitochondria of smaller size [14,15]. Therefore, mitochondrial fusion the mitochondrial outer membrane allowing membranes constriction. and fission mechanisms represent a key step in the mitochondrial Fusion of mitochondria is primarily controlled by mitofusin (MFN) 1 adjustment to challenging nutrient conditions. and 2, located on the mitochondrial outer membrane, and OPA1, located Recently, sirtuins emerged as crucial molecular sensors of cellular in the inner membrane [3]. By controlling the shape, length and number energy balance [16,17]. Since sirtuin enzymatic activity requires NAD+ of mitochondria, fission and fusion events modulate mitochondrial as a cofactor, any energy stress that modifies NAD+ levels impacts on functions, including OXPHOS, reactive oxygen species (ROS) production their activity. For instance, several sirtuins target proteins are involved and apoptosis [4,5]. Because ATP production must be maintained, mito- in the increased lipid and amino acid oxidation to derive energy during chondrial structure and activity need to be finely regulated at different fasting [18,19]. There are seven sirtuin members in mammals, among levels to allow survival of cells in challenging metabolic conditions. which mitochondria-localized sirtuins (including Sirt3 and Sirt5) play Nutrient excess is associated with lower mitochondrial mass, lower a critical role in mitochondrial functions. During fasting, caloric restric- mitochondrial DNA (mtDNA) amount and impaired OXPHOS, whereas tion and exercise, the rise in NAD+ level increases Sirt3 expression and low nutrient availability promotes OXPHOS [6–9]. In obese and diabetic activity [20]. Activated Sirt3 deacetylates several proteins to impact on patients, mitochondrial respiration and ATP production are impaired, mitochondrial processes, such as OXPHOS, fatty acid oxidation, ketone activities of the respiratory chain and tricarboxylic cycle enzymes are body production and oxidative stress management [20]. Disruption of Sirt3 either by genetic ablation or during high-fat diet results in hyperacetylation of mitochondrial proteins, mitochondrial dysfunctions ⁎ Corresponding author at: Department of Biology, University of Moncton, 18 Av. Antonine-Maillet, Moncton, NB E1A 3E9, Canada. and accelerated development of metabolic abnormalities [21,22]. Sirt5 E-mail address: [email protected] (E. Hebert-Chatelain). was recently shown to possess a strong desuccinylase activity [23–25]. http://dx.doi.org/10.1016/j.bbamcr.2016.10.015 0167-4889/© 2016 Elsevier B.V. All rights reserved. 170 H. Guedouari et al. / Biochimica et Biophysica Acta 1864 (2017) 169–176 Analyses of the lysine succinylation proteome in wild-type (Sirt5+/+) Proteins were immunodetected using antibodies against Sirt5 (Cell and Sirt5 knock out cells (Sirt5−/−) revealed several potential targets Signaling; 8782), α-tubulin (Cell Signaling; 3873 RRID:AB_1904178), of this sirtuin within mitochondria, including enzymes involved in SDHA (Abcam; ab14715), succinyllysine (PTM Biolabs; PTM-401), OXPHOS, tricarboxylic cycle, amino acid degradation and fatty acid OPA1 (BD Biosciences; 612607), DRP1 (BD Biosciences; 611113), metabolism [25,26]. Therefore, sirtuins represent key modulators of pDRP1-S616 (Cell Signaling; 3455), pDRP1-S637 (Cell Signaling; mitochondria, allowing this organelle to adjust according to nutrient 4867), MFN2 (Sigma; M6444), MFN1 (Abnova; H00055669-M04 availability, to maintain metabolic homeostasis and eventually, survival. RRID:AB_581724), Fis1 (ProteinTech; 10956-1-AP RRID:AB_2102532), Despite the apparently convergent effect on the metabolic adapta- MiD49 (ProteinTech; 16413-1-AP), MiD51 (ProteinTech; 20164-1-AP), tion to challenging metabolic conditions, the interaction between MFF (Sigma; HPA010968), β-actin (Sigma; A1978 RRID:AB_476692), sirtuins and mitochondrial dynamics is still uncharacterized. We set FLAG (Sigma; F1804), Hsp60 (Santa Cruz; sc-13966 RRID:AB_2121457), out to genetically investigate how Sirt5 impacts on mitochondrial activ- NDUFA9 (Abcam; ab14713 RRID:AB_301431), COX1 (Abcam; ab14705 ity and structure. We show that deletion of Sirt5 leads to mitochondrial RRID:AB_2084810) and MnSOD (Santa Cruz; sc-30080). Primary anti- DRP1 accumulation, mitochondrial fragmentation and accordingly to bodies were revealed with a horseradish peroxidase-conjugated F(ab mitochondrial degradation during autophagy. These results suggest ′)2 fragment of anti-mouse or anti-rabbit fragment-specific (Jackson that Sirt5 modulate mitochondrial dynamics in order to maintain ImmunoResearch Laboratories, West Grove, PA). The blots were visual- metabolic homeostasis during challenging metabolic conditions. ized using enhanced chemiluminescence (ECL) Plus from Amersham. Labels were quantified by densitometric analysis using the Image J (NIH) software. 2. Materials and methods 2.3. Oxygraphic measurements 2.1. Cell culture and transfection Mitochondrial oxygen consumption assays were performed in a Immortalized wild-type (Sirt5+/+) and Sirt5 knock-out (Sirt5−/−) glass chamber equipped with a Clark oxygen electrode (Hansatech). mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. − Cell respiration was measured using 2 × 106 cells ml 1 at 37 °C in a Yingming Zhao (University of Chicago) and Dr. David Lombard (Univer- 2 ml chambers at a stirring rate of 750 rpm. Three different states of sity of Michigan). MEFs were maintained in culture with high glucose endogenous respiration with intact cells were measured: (i) basal respi- (4.5 g l−1) DMEM (Dulbecco's modified Eagle's medium) supplemented ration representing the endogenous physiological coupled state, (ii) res- with 2 mM glutamine, 1 mM pyruvate and 10% (v/v) fetal bovine serum. − piration with oligomycin (2 μgml 1) representing the non-coupled All of the cells were kept in a 5% CO atmosphere at 37 °C and the 2 resting respiration, and (iii) maximal uncoupled respiration induced medium was renewed every 2 to 3 days. Analysis and cell harvesting by FCCP (0.5 μMstepswith2μM final concentration) providing a mea- were always performed when confluency was about 80–90%. sure of the maximal capacity of ETS under conditions of physiological Mitochondrially targeted dsRED (mtRFP) was a gift from M. Zaccolo substrate supply in the intact cell. Respiratory control ration (RCR) (Venetian Institute of Molecular Medicine, Padua, Italy). Sirt5-FLAG was calculated from the ratio between uncoupled and non-coupled was obtained from Addgene (plasmid 13816). Plasmids were respiration. transfected using polyethylenimine