Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death

RESEARCH ARTICLE Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death Emanuele Bernardinelli1☯, Roberta Costa1☯, Giada Scantamburlo1, Janet To2, Rossana Morabito3, Charity Nofziger1, Carolina Doerrier4, Gerhard Krumschnabel4, Markus Paulmichl5, Silvia Dossena1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute of Pharmacology and Toxicology, Paracelsus Medical University, Salzburg, Austria, 2 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 3 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy, 4 Oroboros Instruments, Innsbruck, Austria, 5 Center for Health and Bioresources, Austrian Institute of Technology, Vienna, Austria ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Bernardinelli E, Costa R, Scantamburlo G, To J, Morabito R, Nofziger C, et al. (2017) Mistargeting of the mitochondrial protein LIPT2 leads to apoptotic cell death. PLoS ONE 12(6): e0179591. https://doi.org/10.1371/journal. pone.0179591 Editor: Ferenc Gallyas, Jr., University of PECS Medical School, HUNGARY Received: February 15, 2017 Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis. Accepted: June 1, 2017 Published: June 19, 2017 Copyright: © 2017 Bernardinelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: GK and CD are employees of the commercial company Oroboros Instruments. Oroboros Instruments provided support in the form of salaries for authors GK and CD and research instrumentation (Oxygraph-2k), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of Introduction Lipoylation is a post-translational modification involving five lipoate-dependent enzymes which catalyze essential redox reactions in humans. Two of these enzymes (α-ketoglutarate dehydrogenase and pyruvate dehydrogenase, PDH) play an essential role in mitochondrial energy metabolism, and three (branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the glycine cleavage system, GCS) are involved in amino acid metabolism. The first four enzymes are collectively denoted as 2-oxoacid dehydrogenases. The lipoate-dependent enzymes are multicomplex proteins, and lipoylation involves the E2 subunit/E3 binding protein and the H-protein of 2-oxoacid dehydrogenases and GCS, respectively [1]. Lipoic acid (6,8 dithiooctanoic acid) is a small hydrophobic molecule consisting of eight carbons and two sulfhydryl groups, first identified in association with PDH [2]. While being well characterized in E. coli, the biosynthesis of lipoic acid in eukaryotes is not completely understood and relies on recent studies in yeast. In eukaryotic cells, the activity of lipoatedependent enzymes appears to rely exclusively on de novo intramitochondrial lipoic acid PLOS ONE | https://doi.org/10.1371/journal.pone.0179591 June 19, 2017 1 / 26 LIPT2 and apoptosis these authors are articulated in the author contributions’ section. Competing interests: GK and CD are employees of Oroboros Instruments, a company that designed and manufacturers the high resolution respirometry instruments (Oxygraph-2k) used in this study. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. synthesis [3]. In the mitochondrial fatty acid synthesis (mtFAS) pathway, octanoic acid—the precursor of lipoic acid—is synthesized from malonate and conjugated to an acyl carrier protein (ACP). Then, octanoic acid is transferred to the H protein of GCS system via action of lipoyl(octanoyl) transferase 2 (LIP2 in yeast; LIPT2, putative, in humans) (Fig 1). In the reaction, the free carboxyl group of octanoic acid is attached via an amide linkage to the epsilonamino group of a conserved lysine residue within a conserved lipoyl domain. Octanoylated H protein is the substrate for insertion of two sulfur atoms at C-6 and C-8 positions to obtain lipoylated H protein in a reaction catalyzed by the iron-sulphur (Fe-S) cluster protein lipoic acid synthetase (LIP5 in yeast; LIAS in humans). An additional enzyme (LIP3 in yeast; LIPT1 in humans) catalyzes the transfer of octanoic/lipoic acid to the E2 subunits of the 2-oxoacid dehydrogenase complexes [4,5] (Fig 1). The relevance of lipoic acid biosynthesis in mammalians was elucidated by the use of cell lines [6] or knockout mice [7] in which the expression of key components of this pathway was compromised. These studies evidenced that mtFAS and lipoylation of mitochondrial proteins are tightly related and essential for mitochondrial function, cell survival and energy utilization. Lipoic acid biosynthesis defects were reported to be associated with human disease for the first time in 2011, when mutations in genes encoding for Fe-S cluster proteins (LIAS, MIM 60703) [8] or proteins involved in their biogenesis (NFU1, MIM 608100; and BOLA3, MIM 613183) [9,10] were identified as responsible for the patients’ phenotype. Later, mutations affecting lipoic acid transfer pathway (LIPT1, MIM 610284) have also been described [5,11]. Patients with lipoic acid deficiency present common, early-onset clinical features such as psychomotor retardation, leukoencephalopathy and hypotonia; pulmonary hypertension and cardiomyopathy may also be present. Abnormalities of biochemical parameters include altered levels of organic acids (lactate, 2-ketoglutarate) and glycine, and PDH deficiency. The gravity of symptoms often results in fatalities [5]. Very recently, mutations involving the LIPT2 gene (c.89T>C; c.377T>G) were identified by exome sequencing in a 8-year-old boy with Fig 1. Lipoic acid biosynthesis. mtFAS generates octanoyl-ACP, that enters the lipoic acid biosynthesis pathway. The octanoyl moiety is then transferred fr (...truncated)