The homozygous R504C mutation in MTO1 gene is responsible for ONCE syndrome.

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole-exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.

A study on the human mitochondrial RNA polymerase activity points to existence of a transcription factor B-like protein.

In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1+/-0.9 min(-1)). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.

[Human mitochondrial genetic system]. / Sistema genético mitocondrial humano.

The mitochondria are subcellular organelles devoted to energy production in form of ATP that contain their own genetic system. Mitochondrial DNA codify a small, but extremely important, number of polypeptides of the respiratory chain. The other mitochondrial proteins are encoded in the nucleus. Therefore, mitochondrial biogenesis require the coordinated expression of nuclear and mitochondrial genetic systems. The gene arrangement in mitochondrial DNA is extremely compact with the tRNA genes interspersed with the rRNA and protein-coding genes. This organization has its precise counterpart in the mode of expression and distinctive structural features of the RNAs. Both mitochondrial DNA strands are transcribed as a whole in the form of three polycistronic molecules that are later cut by specific enzymes that recognize the 5' and 3' end of the tRNA sequences, to produced the mature rRNA, mRNA and tRNA. The mitochondrial coded mRNAs are translated into proteins by a mitochondrial specific protein-synthesizing machinery. The genetics of the mitochondrial DNA differs from that of the nuclear DNA in several features. In particular, the mitochondrial genome is inherited from the mother that transmit their mitochondrial DNA to all her offsprings. Another characteristic of this genome is its tendency to mutate more frequently than the nuclear DNA. This provides a powerful tool for studying the evolution of man.

The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions.

The human mitochondrial transcription termination factor (mTERF) cDNA has been cloned and expressed in vitro, and two alternative precursors of the protein have been imported into isolated mitochondria and processed to the mature protein. The precursors contain a mitochondrial targeting sequence, and the mature mTERF (342 residues) exhibits three leucine zippers, of which one is bipartite, and two widely spaced basic domains. The in vitro synthesized mature protein has the expected specific binding capacity for a double-stranded oligonucleotide containing the tridecamer sequence required for directing termination, and produces a DNase I footprint very similar to that produced by the natural protein. However, in contrast to the latter, it lacks transcription termination-promoting activity in an in vitro system, pointing to another component(s) being required for making mTERF termination-competent. A detailed structure-function analysis of the recombinant protein and mutagenized versions of it by band shift assays has demonstrated that both basic domains and the three leucine zipper motifs are necessary for DNA binding. Furthermore, a variety of tests have shown that both the recombinant and the natural mTERF bind to DNA as a monomer, arguing against a dimerization role for the leucine zippers, and rather pointing, together with the results of mutagenesis experiments, to intramolecular leucine zipper interactions being required to bring the two basic domains in close register with the mTERF target DNA sequence.

Functional properties of a sarcoplasmic reticulum Ca(2+)-ATPase with an altered Ca(2+)-binding mechanism.

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

Limited carbodiimide derivatization modifies some functional properties of the sarcoplasmic reticulum Ca2+ release channel.

Sarcoplasmic reticulum membrane derived from the terminal cisternae region reacts with the carboxyl reagent N,N'-dicyclohexylcarbodiimide. The extension of this reaction is dependent on the reagent/protein ratio. By using a low ratio (10 microM reagent and 1 mg of protein/mL), we can selectively prevent the closure of the 450-kDa Ca2+ channel. Rapid filtration experiments indicate no alteration in the activating mechanism of Ca2+ release induced by Ca2+ or Sr2+ whereas the Ca2+ efflux inhibition by Ca2+, Mg2+, or ruthenium red disappears after the chemical treatment. The activating/inhibitory effect of ryanodine on the Ca2+ channel does not appear to be perturbed by N,N'-dicyclohexylcarbodiimide. The negligible incorporation of the 14C radioactive reagent to the 450-kDa band (the Ca2+ channel subunit) indicates the possibility of protein cross-linking in addition to simple derivatization. The functional alterations produced by this reagent suggest the presence of critical acidic residue(s) in a hydrophobic environment which are involved in the low-affinity cationic binding site. They can be tentatively associated with hydrophobic domains of the channel subunits contributing to the lining of the pore for Ca2+ release. The data also indicate that the channel activation by micromolar Ca2+ occurs in a different protein domain which is carbodiimide-insensitive under the experimental conditions tested.

Effect of diethylstilbestrol and related compounds on the Ca(2+)-transporting ATPase of sarcoplasmic reticulum.

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.