cAMP regulates the functional activity, coupling efficiency and structural organization of mammalian FOF1 ATP synthase.

The present study shows that in isolated mitochondria and myoblast cultures depletion of cAMP, induced by sAC inhibition, depresses both ATP synthesis and hydrolysis by the FOF1 ATP synthase (complex V) of the oxidative phosphorylation system (OXPHOS). These effects are accompanied by the decrease of the respiratory membrane potential, decreased level of FOF1 connecting subunits and depressed oligomerization of the complex. All these effects of sAC inhibition are prevented by the addition of the membrane-permeant 8-Br-cAMP. These results show, for the first time, that cAMP promotes ATP production by complex V and prevents, at the same time, its detour to a mitochondrial membrane leak conductance, which is involved in cell death.

Regulation of the biogenesis of OXPHOS complexes in cell transition from replicating to quiescent state: involvement of PKA and effect of hydroxytyrosol.

A study is presented on the expression of mitochondrial oxidative phosphorylation complexes in exponentially growing and serum-starved, quiescent human fibroblast cultures. The functional levels of respiratory complexes I and III and complex V (adenosine triphosphate (ATP) synthase) were found to be severely depressed in serum-starved fibroblasts. The depression of oxidative phosphorylation system (OXPHOS) complexes was associated with reduced levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and the down-stream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). In serum-starved fibroblasts decrease of the catalytic activity of AMP cyclic dependent protein kinase (PKA) and phosphorylation of cAMP response element-binding protein (CREB), the transcription coactivator of the PGC-1α gene, was found. Hydroxytyrosol prevented the decline in the expression of the PGC-1α transcription cascade of OXPHOS complexes in serum-starved fibroblast cultures. The positive effect of HT was associated with activation of PKA and CREB phosphorylation. These results show involvement of PKA, CREB and PGC-1α in the regulation of OXPHOS in cell transition from the replicating to the quiescent state.

Respiratory chain complex I, a main regulatory target of the cAMP/PKA pathway is defective in different human diseases.

In mammals, complex I (NADH-ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace-maker of mitochondrial respiration. The work reviewed here shows that the cAMP/PKA pathway regulates the biogenesis, assembly and catalytic activity of complex I and mitochondrial oxygen superoxide production. The structural, functional and regulatory complexity of complex I, renders it particularly vulnerable to genetic and sporadic pathological factors. Complex I dysfunction has, indeed, been found, to be associated with several human diseases. Knowledge of the pathogenetic mechanisms of these diseases can help to develop new therapeutic strategies.

The ß-adrenoceptor agonist isoproterenol promotes the activity of respiratory chain complex I and lowers cellular reactive oxygen species in fibroblasts and heart myoblasts.

A study is presented on the effect of the ß-adrenoceptor agonist isoproterenol on mitochondrial oxygen metabolism in fibroblast and heart myoblast cultures. Isoproterenol treatment of serum-limited fibroblasts and proliferating myoblasts results in the promotion of mitochondrial complex I activity and decrease of the cellular level of reactive oxygen species. These effects of isoproterenol are associated with cAMP-dependent phosphorylation of complex I subunit(s). Addition of okadaic acid, inhibitor of protein phosphatase(s), reverses the decline of complex I activity in serum-limited fibroblast cultures and activates the complex in proliferating myoblast cultures. The effects of isoproterenol on complex I activity and reactive oxygen species balance can contribute to the therapeutic effect of the drug.

Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain.

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS-PAGE electrophoresis, (32)P labelling and immunodetection show that "in vitro" PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. (32)P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.

Structure and expression of the atp operon coding for F1F0-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727.

Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp operon coding for F1F0-ATP synthase of Nonomuraea sp. ATCC 39727 was determined. It consisted of ten open reading frames arranged in the order atpI (encoding the i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH (delta subunit), atpA (alpha subunit), atpG (gamma subunit), atpD (beta subunit) and atpC (epsilon subunit). The orfX coded for a putative small hydrophobic 71 amino acid peptide of unknown function related to several bacterial permeases. Its presence appeared to be a distinctive feature of the atp operon of phylogenetically distant actinobacteria. Transcription of the atp operon was evaluated. The results of northern blot and RT-PCR experiments demonstrated that the atp genes were co-transcribed into a single polycistronic mRNA. Real-time RT-PCR data provided evidence showing that transcription of the atp operon was biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at the end of the exponential growth phase, and then moderately increased during the early stationary phase when, in contrast, the levels of ctaC, encoding the cytochrome c oxidase subunit II, progressively decreased. Western blot analysis confirmed that ATP synthase was also present in the membrane during the stationary phase. These results together with previous data demonstrate that oligomycin-sensitive ATP-driven proton pumping activity remained constant in the stationary phase; in contrast, the activity and cytochrome content of the respiratory enzymes became negligible.