Complex I and the cAMP cascade in human physiopathology

A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca²⁺-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.     

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.     

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.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

Mutation in the NDUFS4 gene of complex I abolishes cAMP-dependent activation of the complex in a child with fatal neurological syndrome

Evidence is presented showing that in a patient with fatal neurological syndrome, the homozygous 5 bp duplication in the cDNA of the NDUFS4 18 kDa subunit of complex I abolishes cAMP-dependent phosphorylation of this protein and activation of the complex. These findings show for the first time that human complex I is regulated via phosphorylation of the subunit encoded by the NDUFS4 gene.     

Activation of the cAMP cascade in human fibroblast cultures rescues the activity of oxidatively damaged complex I

A study of the relationship between cAMP/PKA-dependent phosphorylation and oxidative damage of subunits of complex I of the mitochondrial respiratory chain is presented. It is shown that, in fibroblast cultures, PKA-mediated phosphorylation of the NDUFS4 subunit of complex I rescues the activity of the oxidatively damaged complex. Evidence is presented showing that this effect is mediated by phosphorylation-dependent exchange of carbonylated NDUFS4 subunit in the assembled complex with the de novo synthesized subunit. These results indicate a potential use for β-adrenoceptor agonists in preventing/reversing the detrimental effects of oxidative stress in the mitochondrial respiratory system.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

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, ³²P labelling and immunodetection show that “in vitro” PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. ³²P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.     

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.     

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.     

Activity,tissue distribution and site-directed mutagenesis of a human peptide methionine sulfoxide reductase of type B: hCBS1

Immunochemical and functional evidence showing the existence in the inner membrane and matrix fraction of mammalian mitochondria of serine/threonine phosphatases acting on cAMP-dependent phosphoproteins is presented. Mg(2+)-dependent Ca(2+)-inhibitable PP2C phosphatase, associated to the inner membrane, dephosphorylates the 18 kDa (NDUFS4 gene) of complex I.     

Dictyostelium discoideum mitochondrial DNA encodes a NADH:Ubiquinone oxidoreductase subunit which is nuclear encoded in other eukaryotes

Complex I, a key component of the mitochondrial electron transport system, is thought to have evolved from at least two separate enzyme systems prior to the evolution of mitochondria from a bacterial endosymbiont, but the genes for one of the enzyme systems are thought to have subsequently been transferred to the nuclear DNA. We demonstrated that the cellular slime mold Dictyostelium discoideum retains the ancestral characteristic of having mitochondria encoding at least one gene (80-kDa subunit) that is nuclear encoded in other eukaryotes. This is consistent with the cellular slime molds of the family Dictyosteliaceae having diverged from other eukaryotes at an early stage prior to the loss of the mitochondrial gene in the lineage giving rise to plants and animals. The D. discoideum mitochondrially encoded 80-kDa subunit of complex I exhibits a twofold-higher mutation rate compared with the homologous chromosomal gene in other eukaryotes, making it the most divergent eukaryotic form of this protein.Correspondence to: K.L. Williams     

Mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase and succinate dehydrogenase complexes contain plant specific subunits

Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial samples and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their individual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9–10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis. Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.     

Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.     

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.     

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.     

Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB protein of Escherichia coli

A stable DNA/protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.