cAMP/PKA Signaling Modulates Mitochondrial Supercomplex Organization.

The oxidative phosphorylation (OXPHOS) system couples the transfer of electrons to oxygen with pumping of protons across the inner mitochondrial membrane, ensuring the ATP production. Evidence suggests that respiratory chain complexes may also assemble into supramolecular structures, called supercomplexes (SCs). The SCs appear to increase the efficiency/capacity of OXPHOS and reduce the reactive oxygen species (ROS) production, especially that which is produced by complex I. Studies suggest a mutual regulation between complex I and SCs, while SCs organization is important for complex I assembly/stability, complex I is involved in the supercomplex formation. Complex I is a pacemaker of the OXPHOS system, and it has been shown that the PKA-dependent phosphorylation of some of its subunits increases the activity of the complex, reducing the ROS production. In this work, using in ex vivo and in vitro models, we show that the activation of cAMP/PKA cascade resulted in an increase in SCs formation associated with an enhanced capacity of electron flux and ATP production rate. This is also associated with the phosphorylation of the NDUFS4 subunit of complex I. This aspect highlights the key role of complex I in cellular energy production.

Mitochondrial cAMP prevents apoptosis modulating Sirt3 protein level and OPA1 processing in cardiac myoblast cells.

Mitochondria, responding to a wide variety of signals, including oxidative stress, are critical in regulating apoptosis that plays a key role in the pathogenesis of a variety of cardiovascular diseases. A number of mitochondrial proteins and pathways have been found to be involved in the mitochondrial dependent apoptosis mechanism, such as optic atrophy 1 (OPA1), sirtuin 3 (Sirt3), deacetylase enzyme and cAMP signal. In the present work we report a network among OPA1, Sirt3 and cAMP in ROS-dependent apoptosis. Rat myoblastic H9c2 cell lines, were treated with tert-butyl hydroperoxide (t-BHP) to induce oxidative stress-dependent apoptosis. FRET analysis revealed a selective decrease of mitochondrial cAMP in response to t-BHP treatment. This was associated with a decrease of Sirt3 protein level and proteolytic processing of OPA1. Pretreatment of cells with permeant analogous of cAMP (8-Br-cAMP) protected the cell from apoptosis preventing all these events. Using H89, inhibitor of the protein kinase A (PKA), and protease inhibitors, evidences have been obtained that ROS-dependent apoptosis is associated with an alteration of mitochondrial cAMP/PKA signal that causes degradation/proteolysis of Sirt3 that, in turn, promotes acetylation and proteolytic processing of OPA1.

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.

Intramitochondrial adenylyl cyclase controls the turnover of nuclear-encoded subunits and activity of mammalian complex I of the respiratory chain.

In mammalian cells the nuclear-encoded subunits of complex I are imported into mitochondria, where they are assembled with mt-DNA encoded subunits in the complex, or exchanged with pre-existing copies in the complex. The present work shows that in fibroblast cultures inhibition by KH7 of cAMP production in the mitochondrial matrix by soluble adenylyl cyclase (sAC) results in decreased amounts of free non-incorporated nuclear-encoded NDUFS4, NDUFV2 and NDUFA9 subunits of the catalytic moiety and inhibition of the activity of complex I. Addition of permeant 8-Br-cAMP prevents this effect of KH7. KH7 inhibits accumulation in isolated rat-liver mitochondria and incorporation in complex I of "in vitro" produced, radiolabeled NDUFS4 and NDUFV2 subunits. 8-Br-cAMP prevents also this effect of KH7. Use of protease inhibitors shows that intramitochondrial cAMP exerts this positive effect on complex I by preventing digestion of nuclear-encoded subunits by mitochondrial protease(s), whose activity is promoted by KH7 and H89, an inhibitor of PKA.

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.

A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level.

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

cAMP-dependent protein kinase regulates post-translational processing and expression of complex I subunits in mammalian cells.

Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.

Mammalian complex I: a regulable and vulnerable pacemaker in mitochondrial respiratory function.

In this paper the regulatory features of complex I of mammalian and human mitochondria are reviewed. In a variety of mitotic cell-line cultures, activation in vivo of the cAMP cascade, or direct addition of cAMP, promotes the NADH-ubiquinone oxidoreductase activity of complex I and lower the cellular level of ROS. These effects of cAMP are found to be associated with PKA-mediated serine phosphorylation in the conserved C-terminus of the subunit of complex I encoded by the nuclear gene NDUFS4. PKA mediated phosphorylation of this Ser in the C-terminus of the protein promotes its mitochondrial import and maturation. Mass-spectrometry analysis of the phosphorylation pattern of complex I subunits is also reviewed.

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.