Mitochondrial disorders due to mutations in SDHAF1and TMEM70 : assembly factors of human mitochondrial Respiratory Chain Complexes II and V

Abstract

Mitochondrial disorders are clinical syndromes associated with abnormalities of the oxidative phosphorylation (OXPHOS) system, the main responsible for the production of energy in the cell. The subunits constituting these multimeric complexes have a dual genetic origin, mitochondrial or nuclear. Hence, mitochondrial syndromes can be due to mutations of mitochondrial DNA or to abnormalities in nuclear genes. The biogenesis of the mitochondrial respiratory chain complexes (MRC) is an intricate and finely tuned process. The majority of the inherited mitochondrial disorders are due to nuclear genes, and many of them encode ancillary factors: proteins necessary for the proper assembly/stability of the MRC even if they are not incorporated in the final complexes. The detailed mechanisms of these processes are not fully understood and the exact function of many such factors remains obscure. During the period of my PhD I focused my attention on the role of two new nuclear genes SDHAF1 and TMEM70 necessary for the correct assembly of Complex II and Complex V, respectively. Succinate dehydrogenase or Complex II participates in the electron transfer, in the respiratory chain and in succinate catabolism in the Krebs cycle where it functions as a succinate dehydrogenase (SDH), catalyzing the oxidation and dehydration of succinate to fumarate. CII takes part in the MRC by coupling this reaction to the reduction of ubiquinone to ubiquinol, that in turn funnels electrons to CIII (Ernster and Dallner 1995). Succinate dehydrogenase consists of four subunits, all encoded by the nuclear genome (Bugiani M. et al., 2006). The two larger subunits, SDHA and SDHB, are catalytic. and are linked to tSDHC and SDHD, two small hydrophobic polypeptides that contain a heme b moiety and anchor the complex to the inner mitochondrial membrane (Sun F. et al. 2005). Isolated complex II deficiency is a relatively rare cause of mitochondrial disease encompassing 2–8% of OXPHOS defective cases (Munnich and Rustin, 2001; Ghezzi D.et al., 2009). Fenotipically two main clinical presentations are known: mitochondrial encephalomyopathy and familial paragangliomas (tumors of chromaffin cells). Several proteins assisting assembly of CII have been described (Rutter et al., 2010), but only the two recently discovered factors SDHAF1 (Ghezzi D. et al., 2009) and SDHAF2 ( Hao HX et al., 2009) directly and specifically promote CII assembly. In the last year of my PhD I studied the biochemical and molecular features of a 7 years old girl born from an inbred family originating from Bangladeshi who manifested clinical aspects of leukencephalopathy similar to the ones described for patients with mutations in SDHAF1. This girl was born at term without complications. Developmental milestones were normal until 13-months of age, when she was referred to medical evaluation for a 10 days history of acute psychomotor regression, clumsy crawling, partial loss of voluntary sitting, and hypotonia. Histochemical studies of a muscle biopsy performed at age 13 months showed a markedly-diffuse reduction of SDH in all muscle fibers. At the age of 7 years, the patient showed a severe spastic-dystonic tetraparesis, moderate mental retardation, mild dysphagia, poor growth and showed clonic, myoclonic and tonic-clonic seizures several times a day, scarcely responsive to antiepileptic drugs. Therapy with Riboflavine led to improved vigilance and alertness, lower frequency of epileptic seizures and some developmental progess. We performed a spectrophotometric determination of respiratory chain enzymes activity and we observe a profound, isolated defect of complex II (residual activity was 30% of the lowest control value after normalization with citrate synthase), and a severe impairment (<70% of normal values) of complex V activity in the direction of ATP synsthesis when succinate was used as substrate; whereas, normal values in the presence of malate. After Western blotting analysis we noticed that the reduced complex II activity in muscle and fibroblasts mitochondria was associated with a decreased amount of complex II subunits SDH70 and SDH30; whereas, complex I, IV, and V subunits were normally expressed. Moreover, BN-PAGE in the first dimension and Tricine/SDS-PAGE in the second dimension followed by Western blotting revealed a drastic decrease of the holocomplex II with normal amount of holocomplexes I and IV. So after these results we decided to perform a genetically screening of CII structural and assemblatory factors and we identified a novel, homozygous c.103G>T mutation in SDHAF1, predicting a premature protein truncation at amino acid residue 35 (p.E35X). The mutation was heterozygous in the parents and it was not found in 200 control chromosomes. Several lines of evidence support the pathogenic role of the new c.103G>T variant. First, the mutation segregates with disease in the family being heterozygous in the healthy father and mother and homozygous in the affected child. Second, the mutation was not detected in 200 control alleles and affects a highly conserved residue of the protein (p.E35) transformed in a premature stop codon, that generate a truncated polypeptide of only 35 amminoacids. Finally, the reduced complex II activity in muscle and fibroblasts mitochondria and the drastic decrease of the holocomplex II amount support the idea that SDHAF1 could be necessary for the correct assembly of SDH complex. In fact SDHAF1, stands for SDH Assembly Factor 1, is a small protein containing a tripeptide sequence LYR, a proposed signature for proteins involved in Fe–S metabolism. Hence, SDHAF1 could play a role in the insertion or retention of the Fe–S clusters within the protein backbone of CII. So overall our studies contribute to expand the array of mutational spectrum of SDHAF1and also corroborate the idea that although SDHAF1 resides in the mitochondrial matrix while CII is membrane bound, mutation in this protein cause a drastic decrease of CII activity and the corrispond amount in human muscles and fibroblast. Furthermore, we underline how mutations in this assembly factor could bear, together with SDHA genetic defects, to a clinical presentation of mitochondrial encephalomyopathy. In fact, to date no mutation in SDHAF1 has been reported in patients with paragaglioma. Among the huge number of cases I’ve analyzed during my Phd work , I’ve also collected two patients displaing peculiar clinical features characterized by hypertrophyc cardiomiopathy, 3-methyl glutaconic aciduria, ipotonia and isolated complex V defect. Both patients resulted mutated in TMEM70 gene, with patient Pt1 being homozygote for the widely described c.317-2A>G splice site mutation and patient Pt2 compound heterozygote for c.317-2A>G and a new c.628A>C missense mutation. Several lines of evidence support the pathogenic role of the new p.209T>R variant. First, the mutation segregates with disease in the family being heterozygous in the healthy father and in combination with the c.317-2A>G in the two affected siblings. Second, the mutation was not detected in 200 control alleles and affects a highly conserved residue of the protein (p.209Thr>Arg). Third, the amount of TMEM70 was undetectable in patient Pt1 and decreased up to 90% in patient Pt2. Finally, in patient Pt2 the level of several complex V subunits was decreased as well as the total amount of complex V. My studies further support the essential role that TMEM70 has in the assembly/stability of ATPsynthase. Both patients, in fact, show a reduction in the amount of fully assembled complex V and accumulation of F1 subcomplexes, as shown by BNGE analysis. In order to confirm these results I decided to analyze the effect of oligomycin or conditions altering the sensitivity to oligomycin, like sonication, towards complex V activity. In fact, it is well known that oligomycin inhibits ATP synthesis and hydrolysis by different mechanisms. Oligomycin binds to the ATPase 6 subunit by blocking proton translocation. Isolated F1 does not make ATP, it is not sensitive to oligomycin, and shows a higher ATP hydrolysis than the F1F0 complex. The hydrolytic activity of complex V without or with oligomycin [namely ATPase and oligomycin sensitive-ATPase (OS-ATPase), respectively] was 30% reduced in patient 1 in both conditions; whereas, in patient 2 was similar to the controls mean values. When mitochondria of both patients were submitted to sonication, a rapid and significant loss of the sensitivity to oligomycin was evident at 20 seconds of sonications. Contrariwise, cells of controls displayed no significant changes. Therefore, the reduction in the amount of fully assembled complex V and the accumulation of F1 subcomplexes shown by BNGE analysis is further supported by hydrolysis experiments in which both patients lose earlier the sensitivity to oligomycin than controls when subjected to mechanical stress, underling a weak bound of the F1 sector to the membrane. In previous studies TMEM70 has been described as a chaperon directing the assembly of complex V, but any attempt to show a direct interaction of this protein with the complex has failed (Houstek J., 2010). We show, for the first time, the presence of TMEM70 in high molecular complexes of about 470 and 550 kDa when Hela cells, expressing a HA tagged form of the protein, were when subjected to BNGE. The immunoprecipitation of complex V clearly demonstrates that the interaction of the protein with ATP synthase is specific, although any attempt to identify the specific interacting protein was unsuccessful. Previous studies have shown that mutations in the mitochondrially encoded subunits (a, A6L) cause reduction of ATP synthase holoenzyme and accumulation of subcomplexes (Jonckheere A.I, 2011). Our patients display a pattern of subassemblies overlapping with the one observed in rho zero cells. In this light is tempting to speculate that TMEM70 may stabilize F1 sector by binding it and assisting the insertion of the mitochondrially encoded subunits. Nevertheless many questions remain to be answered for example why this protein is present only in the higher Eukaryotes, how it can affect complex V activity/assembly, whether it can affect the lipid milieu since it is hydrophobic protein. Further studies are needed to clarify the functional role of this protein.