Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency Maria Moran, Lorena Marín Buera, Maria Carmen Gil, Henry Rivera, Alberto Blazquez, Sara Seneca, Maria Vazquez Lopez, Joaquin Arenas, Miguel A Martin, Cristina Ugalde To cite this version: Maria Moran, Lorena Marín Buera, Maria Carmen Gil, Henry Rivera, Alberto Blazquez, et al.. Cel- lular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme de- ficiency. Human Mutation, Wiley, 2010, 31 (8), pp.930. 10.1002/humu.21294. hal-00552400 HAL Id: hal-00552400 https://hal.archives-ouvertes.fr/hal-00552400 Submitted on 6 Jan 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Human Mutation Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency For Peer Review Journal: Human Mutation Manuscript ID: humu-2010-0090.R1 Wiley - Manuscript type: Research Article Date Submitted by the 23-Apr-2010 Author: Complete List of Authors: Moran, Maria; Hospital Universitario 12 de Octubre, Centro de Investigacion Marín Buera, Lorena; Hospital Universitario 12 de Octubre, Centro de Investigacion Gil, Maria Carmen; Hospital Universitario 12 de Octubre, Centro de Investigacion Rivera, Henry; Hospital Universitario 12 de Octubre, Centro de Investigacion Blazquez, Alberto; Hospital Universitario 12 de Octubre, Centro de Investigacion Seneca, Sara; AZ-VUB, Center of Medical Genetics Vazquez Lopez, Maria; Hospital General Universitario Gregorio Marañón, Sección de Neuropediatría Arenas, Joaquin; Hospital Universitario 12 de Octubre, Centro de Investigación; Hospital Universitario 12 de Octubre, Centro de Investigacion Martin, Miguel; Hospital Universitario 12 de Octubre, Centro de Investigación; Hospital Universitario 12 de Octubre, Centro de Investigacion Ugalde, Cristina; Hospital Universitario 12 de Octubre, Centro de Investigacion Mitochondria, Respiratory chain , Complex III deficiency, BCS1L Key Words: mutations John Wiley & Sons, Inc. Page 1 of 44 Human Mutation Morán et al. 1 1 2 3 Cellular pathophysiological consequences of BCS1L mutations in mitochondrial 4 5 6 complex III enzyme deficiency 7 8 María Morán, 1,2,* Lorena Marín-Buera, 1,2,* M. Carmen Gil-Borlado, 1,2 Henry Rivera, 1,2 9 10 Alberto Blázquez, 1,2 Sara Seneca, 3 María Vázquez-López,4 Joaquín Arenas, 1,2 Miguel 11 12 1,2 1,2 13 A. Martín, and Cristina Ugalde 14 15 16 17 18 * These authors equally contributed to this work 19 20 For Peer Review 21 22 1Centro de Investigación, Hospital Universitario 12 de Octubre, Madrid, Spain; 2Centro 23 24 25 de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), U723, Spain; 26 27 3Center of Medical Genetics, AZ-VUB, Brussels, Belgium; and 4Sección de 28 29 Neuropediatría, Hospital General Universitario Gregorio Marañón, Madrid, Spain. 30 31 32 33 34 Corresponding author: Dr. Cristina Ugalde, Centro de Investigación, Hospital 35 36 Universitario 12 de Octubre. Avda. de Córdoba s/n. 28041 Madrid. Phone: +34 91 390 37 38 39 8763, FAX: +34 91 390 8544, e-mail: [email protected] 40 41 42 43 44 Abbreviated title: Role of mutated BCS1L in complex III deficiency 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 2 of 44 Morán et al. 2 1 2 3 ABSTRACT 4 5 6 Mutations in BCS1L, an assembly factor that facilitates the insertion of the 7 8 catalytic Rieske Iron-Sulphur subunit into respiratory chain complex III, result in a wide 9 10 variety of clinical phenotypes that range from the relatively mild Björnstad syndrome, to 11 12 13 the severe GRACILE syndrome. To better understand the pathophysiological 14 15 consequences of such mutations, we studied fibroblasts from six complex III-deficient 16 17 18 patients harbouring mutations in the BCS1L gene. Cells from patients with the most 19 20 severe clinical phenotypesFor exhibited Peer slow growth Review rates in glucose medium, variable 21 22 combined enzyme deficiencies and assembly defects of respiratory chain complexes I, 23 24 25 III and IV, increased H2O2 levels, unbalanced expression of the cellular antioxidant 26 27 defences, and apoptotic cell death induction. Besides, all patients showed cytosolic 28 29 accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, 30 31 32 assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial 33 34 networks, and decreased MFN2 protein levels. The observed structural alterations were 35 36 independent of the respiratory chain function and ROS production. Our results provide 37 38 39 new insights into the role of pathogenic BCS1L mutations in mitochondrial function and 40 41 dynamics. 42 43 44 45 46 KEYWORDS: Mitochondria; respiratory chain complex III deficiency; BCS1L 47 48 mutations. 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 3 of 44 Human Mutation Morán et al. 3 1 2 3 INTRODUCTION 4 5 6 Mitochondrial respiratory chain complex III (CIII, ubiquinol-cytochrome c 7 8 oxidoreductase or cytochrome bc1 complex, E.C.1.10.2.2) catalyzes the transfer of 9 10 electrons from reduced coenzyme Q to cytochrome c with a concomitant translocation 11 12 13 of protons across the inner membrane (Baum et al., 1967). The purified bovine complex 14 15 is a ~450 kDa symmetric homodimer (Iwata 1998; Xia et al., 1997). Each monomer is 16 17 18 composed of eight structural subunits, and three catalytic subunits: cytochrome b, the 19 20 only subunit encodedFor in the mitochondrial Peer genome, Review cytochrome c1, and the Rieske Iron- 21 22 Sulphur Protein (RISP). 23 24 25 Mitochondrial complex III enzyme deficiency (MIM #124000 ) is a relatively 26 27 infrequent diagnosed defect of the oxidative phosphorylation (OXPHOS) system (Benit 28 29 30 et al., 2009). The vast majority of nuclear mutations leading to complex III deficiency 31 32 have been reported in the BCS1L gene (MIM #603647), which encodes a mitochondrial 33 34 inner membrane protein necessary for the correct biogenesis of respiratory chain 35 36 37 complex III (Cruciat et al., 1999; Fernandez-Vizarra et al., 2007; Fernandez-Vizarra et 38 39 al., 2009; Nobrega et al., 1992). BCS1L mutations lead to three main clinical 40 41 ) 42 phenotypes (Ramos-Arroyo et al., 2009): i) Björnstad syndrome (MIM #262000 , an 43 44 autosomal recessive disorder characterized by sensorineural hearing loss and pili torti 45 46 ( 47 (Hinson et al., 2007); ii) GRACILE syndrome MIM #603358), a Finnish-heritage 48 49 disease caused by the homozygous p.S78G mutation (Fellman 2002; Visapaa et al., 50 51 2002), which is characterized by fetal growth retardation, aminoaciduria, cholestasis, 52 53 54 iron overload, lactic acidosis, and early death; and iii) complex III deficiency in 55 56 neonates or infants presenting with encephalopathy, alone or in combination with 57 58 visceral involvement (Blazquez et al., 2009; de Lonlay et al., 2001; De Meirleir et al., 59 60 2003; Fernandez-Vizarra et al., 2007; Gil-Borlado et al., 2009). John Wiley & Sons, Inc. Human Mutation Page 4 of 44 Morán et al. 4 1 2 3 Human BCS1L protein consists of 419 aminoacids, and shares functional 4 5 6 homology and 50% identity with yeast Bcs1p (Nobrega et al., 1992; Petruzzella et al., 7 8 1998) . In yeast, Bcs1p acts as an ATP-dependent chaperone, maintaining pre-complex 9 10 III in a competent state for the subsequent assembly of the Rieske and Qcr10 subunits 11 12 13 (Cruciat et al., 1999). The absence of Bcs1p prevents the assembly of Rieske within 14 15 complex III and leads to a complete respiratory deficiency. Bcs1p spans three different 16 17 18 domains: i) the N-terminal domain comprises aminoacid residues 1 to 126, which 19 20 contain all the requiredFor information Peer for the mitochondrialReview targeting and sorting of the 21 22 protein (Folsch et al., 1996; Petruzzella et al., 1998); ii) a Bcs1p-specific domain 23 24 25 contains aminoacid residues that are important for Bcs1p activity and stability (Nouet et 26 27 al., 2009); and iii) the C-terminal region contains a ~200 aminoacids domain that 28 29 encompasses two motifs corresponding to nucleotide binding sites, a characteristic 30 31 32 feature of the AAA (for ATPases associated with various cellular activities) protein 33 34 superfamily (Frickey and Lupas, 2004). 35 36 Pathogenic mutations have been described in the three different domains of 37 38 39 human BCS1L, although the pathophysiological mechanisms that contribute to the 40 41 clinical manifestations of the disease remain poorly understood. In some BCS1L - 42 43 44 mutated patients presenting with Björnstad syndrome or complex III deficiency, an 45 46 assembly defect of the RISP subunit led to a mitochondrial complex III enzyme defect 47 48 (Blazquez et al., 2009; Fernandez-Vizarra et al., 2007; Gil-Borlado et al., 2009; Hinson 49 50 51 et al., 2007). However, in patients presenting with the characteristic BCS1L mutation 52 53 c.232A>G (p.S78G) leading to GRACILE syndrome, complex III activity was 54 55 essentially normal despite low BCS1L levels (Fellman et al., 2008). Iron overload has 56 57 58 been reported in some patients with BCS1L mutations leading to GRACILE syndrome 59 60 or complex III deficiency, but not Björnstad Syndrome, suggesting a second hypothetic role for BCS1L in iron metabolism (De Meirleir et al., 2003; Visapaa et al., 2002).