They involved solely COX1 subunit and might be identical with subassemblies dCf from patient fibroblasts [14]

They involved solely COX1 subunit and might be identical with subassemblies dCf from patient fibroblasts [14]. association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the presence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis. oxidase, mitochondria, SCO2, SURF1 oxidase; CS, citrate synthase; DDM, n-dodecyl–D-maltoside; SDH, succinate: ubiquinone oxidoreductase; VDAC, voltage-dependent anion channel INTRODUCTION Eukaryotic COX (cytochrome oxidase), the terminal enzyme of the mitochondrial respiratory chain, is embedded in the inner mitochondrial membrane where it catalyses the transfer of electrons from reduced cytochrome to molecular oxygen and further couples this reaction with proton translocation across the inner membrane. Mammalian COX is usually a multisubunit complex of approx.?200?kDa composed of 13 subunits encoded by both the mitochondrial and nuclear genes. The mitochondrially encoded subunits COX1, COX2 and COX3 are evolutionarily conserved and form the catalytic and structural core of the enzyme [1]. The remaining ten evolutionarily younger subunits are encoded by the nuclear genome and are associated with the surface of the complex core. These small polypeptides are required for the stability and assembly of the holoenzyme and are also involved in modulation of its activity in response to various cellular stimuli [2]. Tissue-specific isoforms of subunits COX4, COX6A, COX6B and COX7A were identified in humans [3,4]. In addition to the constituent protein subunits, COX contains several redox-active prosthetic groups directly involved in electron transfer. These are two haem A moieties (and cause fatal infantile COX deficiency with the predominant symptoms being encephalopathy and hypertrophic cardiomyopathy. To date, all patients identified were either compound heterozygotes for 1541G A (where 1541G A denotes the guanine adenine nucleotide transition at the position 1541 of the DNA) mutation, with the other allele carrying either a nonsense or missense mutation, or homozygotes for this common 1541G A transition, predicting a E140K amino acid substitution near the highly conserved CXXXC putative copper-binding motif [9C11]. The most severe cases (early onset) are compound heterozygotes, while patients homozygous for E140K substitution have a comparatively milder phenotype (delayed onset, less progressive). Human SURF1 is usually a 30?kDa transmembrane protein localized in the inner mitochondrial membrane [12,13]. The precise function of this protein is still unknown, but recently it was suggested that human SURF1 promotes the association of COX2 with the COX assembly 4-Hydroxytamoxifen intermediate composed of COX1, COX4 and COX5A [14], which was originally described as COX1- and COX4-made up of assembly intermediate S2 [15]. Mutations in human cause Leigh syndrome, a fatal neurological disorder associated with severe isolated COX deficiency and characterized mainly by bilaterally symmetrical necrotic lesions in the basal ganglia and brainstem [16,17]. Nearly all reported patients carried loss-of-function mutations that predict either truncated and unstable protein product or unstable mRNA [18]. Histochemical studies and enzyme activity measurements show that mutations result in a tissue-specific decrease of COX activity, with heart and 4-Hydroxytamoxifen skeletal muscle being most severely affected. In 4-Hydroxytamoxifen contrast, cultured fibroblasts and liver were shown to retain high residual activity [9C11], indicating tissue-specific differences in COX biogenesis or maintenance. The COX activity was reduced to approx.?10% of control values in patient fibroblasts [14], and the skeletal muscle of patients was repeatedly shown to retain approx.?20% of residual COX activity [19]. In fibroblasts, Rabbit Polyclonal to MAP9 the reduction of COX activity was shown to be accompanied by a similar decrease in holoenzyme levels and also by a marked accumulation of COX subcomplexes, suggesting that the residual enzyme is fully active and that the enzyme deficiency stems from impaired assembly or maintenance of the protein complex [14]. The aim of the present study was to examine and compare the consequences of and mutations in various human tissues. We have investigated the steady-state levels of COX holoenzyme and the presence and composition of COX subcomplexes in tissues and primary fibroblast cultures from three patients harbouring mutations and from three patients carrying mutations in mutations result in severely decreased levels of mutant SCO2 protein in heart, brain, liver and fibroblasts. The subunit composition of COX subcomplexes identified demonstrates the participation of human being SCO2 in biogenesis or maintenance of COX2 and suggests an addition to the present style of the human being COX set up pathway..