Anna Signorile

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function.

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Oxidative protein damage in human diabetic eye: evidence of a retinal participation.

Considerable evidence indicates that the maintenance of protein redox status is of fundamental importance for cell function, whereas structural changes in proteins are considered to be among the molecular mechanisms leading to diabetic complications. In this study, protein redox status and antioxidant activity were investigated in the lens and vitreous of diabetic and non‐diabetic subjects. A significantly lower content of sulphydryl proteins was found in lens and vitreous of diabetic patients than in those of non‐diabetic and control subjects. Moreover, an increased formation of protein‐bound free sulphydryls and carbonyl proteins, indices of oxidative damage to proteins, was noted in diabetic patients. All these parameters were shown to be altered particularly when diabetes was complicated with retinal alterations. In addition, glutathione peroxidase activity and ascorbic acid levels, known to exert important antioxidant functions in the eye compartment, were found to be significantly decreased in the lens of diabetic patients, especially in the presence of retinal damage. This study indicates an alteration of protein redox status in subjects affected by diabetes mellitus; lens and vitreous proteins were found to be oxidized to a greater extent in the presence of retinal disease, together with a marked decrease of eye antioxidant systems. These results suggest that oxidative events are involved in the onset of diabetic eye complications, in which the decrease in free radical scavengers was shown to be associated with the oxidation of vitreous and lens proteins. Protein oxidation may, therefore, represent an important mechanism in the onset of eye complications in diabetic patients.

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.

Occurrence of A-kinase anchor protein and associated cAMP-dependent protein kinase in the inner compartment of mammalian mitochondria.

Evidence showing the existence in the inner compartment of rat‐heart mitochondria of AKAP121 and associated PKA is presented. Immunoblotting analysis and trypsin digestion pattern show that 90% or more of mitochondrial C‐PKA, R‐PKA and AKAP121 is localized in the inner mitochondrial compartment, when prepared both from isolated mitochondria or cardiomyocyte cultures. This localization is verified by measurement of the specific catalytic activity of PKA, radiolabelling of R‐PKA by 32P‐phosphorylated C‐PKA and of AKAP by 32P‐phosphorylated R‐PKA and electron microscopy of mitochondria exposed to gold‐conjugated AKAP121 antibody.

The Oxidative Phosphorylation System in Mammalian Mitochondria

The chapter provides a review of the state of art of the oxidative phosphorylation system in mammalian mitochondria. The sections of the paper deal with: (i) the respiratory chain as a whole: redox centers of the chain and protonic coupling in oxidative phosphorylation (ii) atomic structure and functional mechanism of protonmotive complexes I, III, IV and V of the oxidative phosphorylation system (iii) biogenesis of oxidative phosphorylation complexes: mitochondrial import of nuclear encoded subunits, assembly of oxidative phosphorylation complexes, transcriptional factors controlling biogenesis of the complexes. This advanced knowledge of the structure, functional mechanism and biogenesis of the oxidative phosphorylation system provides a background to understand the pathological impact of genetic and acquired dysfunctions of mitochondrial oxidative phosphorylation.

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 patients 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.

cAMP response element‐binding protein (CREB) is imported into mitochondria and promotes protein synthesis

The cAMP response element‐binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that, once phosphorylated, promotes transcription of cAMP response element‐regulated genes. We have studied the mitochondrial import of CREB and its effect on the expression of mtDNA‐encoded proteins. [35S]Methionine‐labelled CREB, synthesized in vitro in the Rabbit Reticulocyte Lysate system using a construct of the human cDNA, was imported into the matrix of isolated rat liver mitochondria by a membrane potential and TOM complex‐dependent process. The imported CREB caused cAMP‐dependent promotion of the synthesis of mitochondrially encoded subunits of oxidative phosphorylation enzyme complexes. Thus, CREB moves from the cytosol to mitochondria, in addition to the nucleus, and, when phosphorylated by cAMP‐dependent protein kinase, promotes the expression of mitochondrial genes.

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 Downs 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.

Respiratory chain complex I, a main regulatory target of the cAMP/PKA pathway is defective in different human diseases

In mammals, complex I (NADH‐ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace‐maker of mitochondrial respiration. The work reviewed here shows that the cAMP/PKA pathway regulates the biogenesis, assembly and catalytic activity of complex I and mitochondrial oxygen superoxide production. The structural, functional and regulatory complexity of complex I, renders it particularly vulnerable to genetic and sporadic pathological factors. Complex I dysfunction has, indeed, been found, to be associated with several human diseases. Knowledge of the pathogenetic mechanisms of these diseases can help to develop new therapeutic strategies.

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.