Expression and processing of the TMEM70 protein.

TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29kDa precursor protein that is processed to a 21kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy.

We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in individuals with isolated mitochondrial ATP synthase deficiency and identified disease-causing mutations in TMEM70. Complementation of the cell lines of these individuals with wild-type TMEM70 restored biogenesis and metabolic function of the enzyme complex. Our results show that TMEM70 is involved in mitochondrial ATP synthase biogenesis in higher eukaryotes.

HIF and reactive oxygen species regulate oxidative phosphorylation in cancer.

A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency.

BACKGROUND: To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205deltaTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. RESULTS: Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines--M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects. CONCLUSION: Our analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.