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.     

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.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

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.     

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.     

Deficit of complex I activity in human skin fibroblasts with chromosome 21 trisomy and overproduction of reactive oxygen species by mitochondria: involvement of the cAMP/PKA signalling pathway

DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.     

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles.

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.     

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.     

Pretreatment with Bacopa monnieri extract offsets 3-nitropropionic acid induced mitochondrial oxidative stress and dysfunctions in the striatum of prepubertal mouse brain

The present investigation was designed to determine the efficacy of Bacopa monnieri (Brahmi; BM) to offset 3-nitropropionic acid (3-NPA) induced oxidative stress and mitochondrial dysfunction in dopaminergic (N27) cells and prepubertal mouse brain. Pretreatment of N27 cells with BM ethanolic extract (BME) significantly attenuated 3-NPA-induced cytotoxicity. Further, we determined the degree of oxidative stress induction, redox status, enzymic antioxidants, and protein oxidation in the striatal mitochondria of mice given BME prophylaxis followed by 3-NPA challenge. While 3-NPA-induced marked oxidative stress in the mitochondria of the striatum, BME prophylaxis markedly prevented 3-NPA-induced oxidative dysfunctions and depletion of reduced glutathione and thiol levels. The activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione reductase, thioredoxin reductase), Na(+),K(+)-ATPase, and citric acid cycle enzymes in the striatum discernible among 3-NPA mice were significantly restored with BME prophylaxis. Interestingly, BME offered protection against 3-NPA-induced mitochondrial dysfunctions as evidenced by the restoration of the activities of ETC enzymes (NADH:ubiquinone oxidoreductase, NADH:cytochrome c reductase, succinate-ubiquinone oxidoreductase, and cytochrome c oxidase) and mitochondrial viability. We hypothesize that the neuroprotective effects of BME may be wholly or in part related to its propensity to scavenge free radicals, maintain redox status, and upregulate antioxidant machinery in striatal mitochondria.     

Phosphorylation and kinetics of mammalian cytochrome c oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.     

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.     

Essential role of A-kinase anchor protein 121 for cAMP signaling to mitochondria

A-Kinase anchor proteins (AKAPs) immobilize and concentrate protein kinase A (PKA) isoforms at specific subcellular compartments. Intracellular targeting of PKA holoenzyme elicits rapid and efficient phosphorylation of target proteins, thereby increasing sensitivity of downstream effectors to cAMP action. AKAP121 targets PKA to the cytoplasmic surface of mitochondria. Here we show that conditional expression of AKAP121 in PC12 cells selectively enhances cAMP.PKA signaling to mitochondria. AKAP121 induction stimulates PKA-dependent phosphorylation of the proapoptotic protein BAD at Ser(155), inhibits release of cytochrome c from mitochondria, and protects cells from apoptosis. An AKAP121 derivative mutant that localizes on mitochondria but does not bind PKA down-regulates PKA signaling to the mitochondria and promotes apoptosis. These findings indicate that PKA anchored by AKAP121 transduces cAMP signals to the mitochondria, and it may play an important role in mitochondrial physiology.     

Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential (P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production (P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by approximately 50% (P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20-30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls (P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.     

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.     

Mitochondrial targeting of intact CYP2B1 and CYP2E1 and N-terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae--role of protein kinase A in the mitochondrial targeting of CYP2E1

Previously we showed that intact rat cytochrome P450 2E1, cytochrome P450 2B1 and truncated cytochrome P450 1A1 are targeted to mitochondria in rat tissues and COS cells. However, some reports suggest that truncated cytochrome P450 2E1 is targeted to mitochondria. In this study, we used a heterologous yeast system to ascertain the conservation of targeting mechanisms and the nature of mitochondria-targeted proteins. Mitochondrial integrity and purity were established using electron microscopy, and treatment with digitonin and protease. Full-length cytochrome P450 2E1 and cytochrome P450 2B1 were targeted both to microsomes and mitochondria, whereas truncated cytochrome P450 1A1 (+ 5 and + 33/cytochrome P450 1A1) were targeted to mitochondria. Inability to target intact cytochrome P450 1A1 was probably due to lack of cytosolic endoprotease activity in yeast cells. Mitochondrial targeting of cytochrome P450 2E1 was severely impaired in protein kinase A-deficient cells. Similarly, a phosphorylation site mutant cytochrome P450 2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance of protein kinase A-mediated protein phosphorylation in mitochondrial targeting. Mitochondria-targeted proteins were localized in the matrix compartment peripherally associated with the inner membrane and their ethoxyresorufin O-dealkylation, erythromycin N-demethylase, benzoxyresorufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Cardiac H11 kinase/Hsp22 stimulates oxidative phosphorylation and modulates mitochondrial reactive oxygen species production: Involvement of a nitric oxide-dependent mechanism

H11 kinase/Hsp22 (Hsp22), a small heat shock protein upregulated by ischemia/reperfusion, provides cardioprotection equal to ischemic preconditioning (IPC) through a nitric oxide (NO)-dependent mechanism. A main target of NO-mediated preconditioning is the mitochondria, where NO reduces O₂ consumption and reactive oxygen species (ROS) production during ischemia. Therefore, we tested the hypothesis that Hsp22 overexpression modulates mitochondrial function through an NO-sensitive mechanism. In cardiac mitochondria isolated from transgenic (TG) mice with cardiac-specific overexpression of Hsp22, mitochondrial basal, ADP-dependent, and uncoupled O₂ consumption was increased in the presence of either glucidic or lipidic substrates. This was associated with a decrease in the maximal capabilities of complexes I and III to generate superoxide anion in combination with an inhibition of superoxide anion production by the reverse electron flow. NO synthase expression and NO production were increased in mitochondria from TG mice. Hsp22-induced increase in O₂ consumption was abolished either by pretreatment of TG mice with the NO synthase inhibitor l-N^G-nitroarginine methyl ester (l-NAME) or in isolated mitochondria by the NO scavenger phenyltetramethylimidazoline-1-oxyl-3-oxide. l-NAME pretreatment also restored the reverse electron flow. After anoxia, mitochondria from TG mice showed a reduction in both oxidative phosphorylation and H₂O₂ production, an effect partially reversed by l-NAME. Taken together, these results demonstrate that Hsp22 overexpression increases the capacity of mitochondria to produce NO, which stimulates oxidative phosphorylation in normoxia and decreases oxidative phosphorylation and reactive oxygen species production after anoxia. Such characteristics replicate those conferred by IPC, thereby placing Hsp22 as a potential tool for prophylactic protection of mitochondrial function during ischemia.     

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.     

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca^2 +, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R₂C₂) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).