Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) – a natural polyphenol component of green tea – to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content.In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.     

Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role.

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.     

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 NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade

Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cgamma-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg(2+)-dependent and inhibited by Ca(2+). In human and murine fibroblast and myoblast cultures "in vivo", elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration.Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.     

cAMP controls oxygen metabolism in mammalian cells

The impact of cAMP on ROS-balance in human and mammalian cell cultures was studied. cAMP reduced accumulation of ROS induced by serum-limitation, under conditions in which there was no significant change in the activity of scavenger systems. This effect was associated with cAMP-dependent activation of the NADH-ubiquinone oxidoreductase activity of complex I. In fibroblasts from a patient a genetic defect in the 75 kDa FeS-protein subunit of complex I resulted in inhibition of the activity of the complex and enhanced ROS production, which were reversed by cAMP. A missense genetic defect in the NDUFS4 subunit, putative substrate of PKA, suppressed, on the other hand, the activity of the complex and prevented ROS production.     

cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction

Evidence has been obtained for the occurrence of a cAMP-dependent serine protein kinase associated with the inner membrane/matrix of mammalian mitochondria. The catalytic site of this kinase is localized at the inner side of the inner membrane, where it phosphorylates a number of mitochondrial proteins. One of these has been identified as the AQDQ subunit of complex I. cAMP-dependent phosphorylation of this protein promotes the activity of complex I and mitochondrial respiration. A 5 bp duplication in the nuclear gene encoding this protein has been found in a human patient, which eliminates the phosphorylation site. PKA anchoring proteins have recently been identified in the outer membrane of mammalian mitochondria, which could direct phosphorylation of proteins at contact sites with other cell structures.     

Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.     

Minor participation of cAMP on the protein kinase phosphorylation of mitochondrial and cytosolic fractions from Ascaris suum: a comparative study with porcine heart muscle

In contrast to porcine heart muscle in which cAMP effectively activated the phosphorylation of cytosolic proteins, cAMP exerted a minor effect on the phosphorylation of proteins from the soluble fraction of Ascaris suum muscle. Similarly, cAMP did not enhance the kinase activity in the mitochondrial membranes from porcine heart and A. suum, although major differences in protein phosphorylation were observed between both fractions. However, cAMP-dependent protein kinases (PKA) were evidenced in the parasitic soluble mitochondrial fraction, since the phosphorylation of histone IIA and kemptide was augmented in this fraction, in the presence of cAMP. An increase in the phosphorylation of exogenously added A. suum phosphofructokinase was also obtained when cAMP was added to the parasite soluble mitochondrial fraction. The phosphorylation of phosphofructokinase by this fraction was inhibited when kemptide and cAMP were included in the reaction mixture, suggesting substrate competition for the same PKA. Although PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent PKAs, did not affect the endogenous phosphorylation of proteins in the various A. suum fractions, an inhibition on the phosphorylation of exogenously added kemptide and phosphofructokinase was observed when PKI (6-22) was incubated with the parasite mitochondrial soluble fraction.     

The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

Regulation of NADH/CoQ Oxidoreductase: Do Phosphorylation Events Affect Activity?

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.     

Progesterone synthesis by human placental mitochondria is sensitive to PKA inhibition by H89

The transfer of cholesterol to mitochondria, which might involve the phosphorylation of proteins, is the rate-limiting step in human placental steroidogenesis. Protein kinase A (PKA) activity and its role in progesterone synthesis by human placental mitochondria were assessed in this study. The results showed that PKA and phosphotyrosine phosphatase D1 are associated with syncytiotrophoblast mitochondrial membrane by an anchoring kinase cAMP protein-121. The 32P-labeled of four major proteins was analyzed. The specific inhibitor of PKA, H89, decreased progesterone synthesis in mitochondria while in mitochondrial steroidogenic contact sites protein-phosphorylation was diminished, suggesting that PKA plays a role in placental hormone synthesis. In isolated mitochondria, PKA activity was unaffected by the addition of cAMP suggesting a constant activity of this kinase in the syncytiotrophoblast. The presence of PKA and phosphotyrosine phosphatase D1 anchored to mitochondria by an anchoring kinase cAMP protein-121 indicated that syncytiotrophoblast mitochondria contain a full phosphorylation/dephosphorylation system.     

Proteolytic regulation of the mitochondrial cAMP-dependent protein kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.     

Human Tau may Modify Glucocorticoids-Mediated Regulation of cAMP-dependent Kinase and Phosphorylated cAMP Response Element Binding Protein

Phosphorylation of the cAMP response element binding protein (CREB) by cAMP-dependent kinase (PKA) is critical to memory formation. However, activation of PKA can also increase tau phosphorylation, which may contribute to memory impairment. Therefore, the regulation of PKA may be part of the mechanism by which glucocorticoids (GCs) influence memory. Additionally, the cellular response to GCs may be affected by the presence of human tau. The goal of this paper was to study GCs-mediated regulation of PKA as well as CREB and tau phosphorylation in wild-type HEK293 cells and HEK293 cells stably expressing human tau441 (HEK293/tau441 cells). By using dexamethasone (DEX) as GCs, we found that DEX induced a tau-dependent selective decrease in the level of PKA RIIβ subunit protein. The observed decrease in RIIβ expression was not due to alterations of mRNA levels and was reversed by inhibiting the proteasome with lactacystin. Moreover, the decrease in RIIβ did not diminish the co-localization of the catalytic subunit of PKA with tau and might contribute to the DEX-induced increase in tau phosphorylation at Ser-214. DEX also induced a tau-dependent decrease in CREB phosphorylation that could not be reversed by activating PKA with forskolin. Taken together, these results show that human tau protein may alter the GCs-mediated regulation of PKA activity and CREB phosphorylation.     

Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.     

Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabolism

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src- and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism.