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.     

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.     

The human complex I NDUFS4 subunit: from gene structure to function and pathology

Complex I is the first and largest enzyme of the oxidative phosphorylation system. It consists of at least 43 subunits. Recent studies have shown that the NDUFS4 subunit of complex I contributes to the activation of the complex through cAMP dependent phosphorylation of a conserved site (RVS) located at the C-terminal region of this protein. This report focuses on the NDUFS4 subunit. Summarized is the current knowledge of this subunit, from gene structure to function and pathology.     

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.     

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.     

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 first nuclear-encoded complex I mutation in a patient with Leigh syndrome.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.     

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.     

cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.     

A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome

Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population.     

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.     

Minireview: The NADH: Ubiquinone Oxidoreductase (Complex I) of the Mammalian Respiratory Chain and the cAMP Cascade

Recent work has revealed cAMP-dependent phosphorylation of the 18-kDa IP subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene (chromosome 5). Phosphorylation of this protein has been shown to take place in fibroblast cultures in vivo, as well as in isolated mitochondria, which in addition to the cytosol also contain, in the inner-membrane matrix fraction, a cAMP-dependent protein kinase. Mitochondria appear to have a Ca²⁺-inhibited phosphatase, which dephosphorylates the 18-kDa phosphoprotein. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18-kDa protein is associated with potent 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. In one case consisting of a 5 bp duplication, which destroyed the phosphorylation site, cAMP-dependent activation of complex I was abolished in the patient's fibroblast cultures. In another case consisting of a nonsense mutation, leading to termination of the protein after only 14 residues of the putative mitochondria targeting peptide, a defect in the assembly of complex I was found in fibroblast cultures.     

Mutations in structural genes of complex I associated with neurological diseases

This paper summarizes observations on the genetic and biochemical basis of hereditary defects of complex I (NADH-ubiquinone oxidoreductase) of the respiratory chain in human neurological patients. Two different types of functional defects of the complex are described. In one type mutations in the NDUFS1 and NDUFS4 nuclear structural genes of the complex were identified in two unrelated families. Both NDUFS1 and NDUFS4 neurological disorders were transmitted by autosomic recessive inheritance. The two mutations resulted in different impact on cellular metabolism. The NDUFS4 mutation, giving a more severe, fatal pathological pattern, resulted in a defective assembly of the complex and complete suppression of the enzymatic activity. The NDUFS1 mutation, with less severe progressive pathology, caused only partial inhibition of the complex but enhanced production of oxygen free radicals. In the second type of deficiencies extensive mutational analysis did not reveal pathogenic mutations in complex I genes but a decline in the level and activity of complex I, III, and IV were found, apparently associated with alteration in the cardiolipin membrane distribution.     

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals.     

Clinical and molecular findings in children with complex I deficiency

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.