Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

Eupafolin: Effect on mitochondrial energetic metabolism

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.     

Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases

The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2′, 3′-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase γ, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level.     

The mitochondrial brain: From mitochondrial genome to neurodegeneration

Mitochondrial DNA mutations are an important cause of neurological disease. The clinical presentation is very varied in terms of age of onset and different neurological signs and symptoms. The clinical course varies considerably but in many patients there is a progressive decline, and in some evidence of marked neurodegeneration. Our understanding of the mechanisms involved is limited due in part to limited availability of animal models of disease. However, studies on human post-mortem brains, combined with clinical and radiological studies, are giving important insights into specific neuronal involvement.     

Apoptosis: a mitochondrial perspective on cell death

Mitochondria play an important role in both the life and death of cells. The past 7-8 years have seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Mitochondria have, next to their function in respiration, an important role in apoptotic signaling pathway. Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Apoptosis can be initiated by a wide array of stimuli, including multiple signaling pathways that, for the most part, converge at the mitochondria. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also "gatekeepers" that ultimately determine the fate of the cell. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death. In addition, several Bcl-2 family members and other upstream proteins also contribute to and regulate the apoptosis. In this review, we attempt to summarize our current view of the mechanism that leads to the influx and efflux of many proteins from/to mitochondria during apoptosis.     

Non-conventional mitochondrial permeability transition: Its regulation by mitochondrial dynamics

Mitochondrial permeability transition (MPT) is a phenomenon that the inner mitochondrial membrane (IMM) loses its selective permeability, leading to mitochondrial dysfunction and cell injury. Electrophysiological evidence indicates the presence of a mega-channel commonly called permeability transition pore (PTP) whose opening is responsible for MPT. However, the molecular identity of the PTP is still under intensive investigations and debates, although cyclophilin D that is inhibited by cyclosporine A (CsA) is the established regulatory component of the PTP. PTP can also open transiently and functions as a rapid mitochondrial Ca²⁺ releasing mechanism. Mitochondrial fission and fusion, the main components of mitochondrial dynamics, control the number and size of mitochondria, and have been shown to play a role in regulating MPT directly or indirectly. Studies by us and others have indicated the potential existence of a form of transient MPT that is insensitive to CsA. This “non-conventional” MPT is regulated by mitochondrial dynamics and may serve a protective role possibly by decreasing the susceptibility for a frequent or sustained PTP opening; hence, it may have a therapeutic value in many disease conditions involving MPT.     

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.     

Top down analysis ceramide-induced mitochondrial dysfunctions: role of mitochondrial swelling

Mitochondrial role in ceramide-induced apoptosis pathway remains unclear. Direct effects of ceramide on mitochondria (cytochrome c release, respiratory chain inhibition, oxygen radicals production...) have been reported [1, 2] and we previously showed that addition of ceramide to intact cells or isolated mitochondria triggers mitochondrial swelling which appeared to be insensitive to cyclosporin A (CsA) [3, 4]. The purpose of this work was to determine to which extent this CsA-insensitive mitochondrial swelling, therefore distinct from permeability transition, participates to ceramide-induced apoptosis. To achieve this, we applied Top-Down analysis of integrated mitochondrial function [5], in order to better understand ceramide-induced mitochondrial dysfunctions.     

Bird mitochondrial gene order: insight from 3 warbler mitochondrial genomes

Two main gene orders exist in birds: the ancestral gene order and the remnant control region (CR) 2 gene order. These gene orders differ by the presence of 1 or 2 copies of the CR, respectively. Among songbirds, Oscines were thought to follow the ancestral gene order, with the exception of the lyrebird and Phylloscopus warblers. Here, we determined the complete mitochondrial genome sequence of 3 non-Phylloscopus warblers species and found that the blackcap (Sylvia atricapilla) and the reed warbler (Acrocephalus scirpaceus) have 2 almost identical copies of the CR, whereas the eastern orphean warbler (Sylvia crassirostris) follows the remnant CR 2 gene order. Our results contradict previous studies suggesting that Acrocephalus and most sylvioid warblers exhibit the ancestral gene order. We were able to trace this contradiction to a misidentification of gene order from polymerase chain reaction length determination. We thus suggest that passerine gene order evolution needs to be revised.     

Trypanosomatids: mitochondrial RNA editing

RNA editing is a genetic regulatory process that was recently discovered in the mitochondrion of trypanosomatid parasites. It alters mRNA by the addition and deletion of uridines. Much remains to be learned about this process, including identification and characterization of the macromolecules that catalyze and regulate this process and the mechanism of editing.     

Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies

The ancient acquisition of the mitochondrion into the ancestor of modern‐day eukaryotes is thought to have been pivotal in facilitating the evolution of complex life. Mitochondria retain their own diminutive genome, with mitochondrial genes encoding core subunits involved in oxidative phosphorylation. Traditionally, it was assumed that there was little scope for genetic variation to accumulate and be maintained within the mitochondrial genome. However, in the past decade, mitochondrial genetic variation has been routinely tied to the expression of life‐history traits such as fertility, development and longevity. To examine whether these broad‐scale effects on life‐history trait expression might ultimately find their root in mitochondrially mediated effects on core bioenergetic function, we measured the effects of genetic variation across twelve different mitochondrial haplotypes on respiratory capacity and mitochondrial quantity in the fruit fly, Drosophila melanogaster. We used strains of flies that differed only in their mitochondrial haplotype, and tested each sex separately at two different adult ages. Mitochondrial haplotypes affected both respiratory capacity and mitochondrial quantity. However, these effects were highly context‐dependent, with the genetic effects contingent on both the sex and the age of the flies. These sex‐ and age‐specific genetic effects are likely to resonate across the entire organismal life‐history, providing insights into how mitochondrial genetic variation may contribute to sex‐specific trajectories of life‐history evolution.     

Hispidulin: antioxidant properties and effect on mitochondrial energy metabolism

Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3',4'-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05-0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H₂O₂ in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.     

Chicken domestication: an updated perspective based on mitochondrial genomes

Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A–I and W–Z. Common haplogroups A–G were shared by domestic chickens and red junglefowl. Rare haplogroups H–I and W–Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.     

Free fatty acid effects on mitochondrial permeability: an overview

A variety of experimental conditions elicit increases in mitochondrial permeability that can be differentiated from the classic cyclosporin A (CsA)-sensitive mitochondrial permeability transition (MPT). For example, butylated hydroxytoluene, signal peptides, and the hormone thyroxine have been shown to promote increases in mitochondrial permeability that are CsA-insensitive. Our laboratory has recently demonstrated that palmitic acid, a saturated 16-carbon free fatty acid (FFA), can also open a CsA-insensitive pore. This nonclassic permeability transition (NCPT) is further distinguished by a nonselective dependence on divalent cations and by spontaneous closure. To determine if induction of the NCPT is specific to palmitic acid and to resolve conflicting reports as to the mechanisms by which FFAs alter mitochondrial permeability, we examined in detail mitochondrial swelling induced by FFAs that differ in chain length and degree of saturation. The following results were obtained: (1) In the presence of modest Ca2+ concentrations (75 nmol/mg protein), medium-chain FFAs (C12-C18) were more effective in eliciting mitochondrial swelling than were shorter or longer FFAs; medium-chain alkanols and amines had no effect. (2) Under these conditions, saturated FFAs induced CsA-insensitive swelling with all the characteristics of the NCPT, while unsaturated FFAs triggered the MPT. (3) When matrix Ca2+ concentration was further elevated, unsaturated FFAs triggered the NCPT. (4) Mitochondrial swelling induced by saturated FFAs was inhibited by unsaturated FFAs but not by other saturated FFAs or medium-chain alkanols. These results suggest that ambient conditions can greatly influence the nature of the increase in mitochondrial permeability induced by FFAs. They are also consistent with our earlier proposal that Ca2+ (or Sr2+) binding to FFAs in the inner leaflet of the inner mitochondrial membrane underlies the NCPT.     

Trypanocidal CoQ analogues: their effect on other mitochondrial systems

1. A comparative study of compounds which inhibit the respiration of the infective form of the protozoan parasite Trypanosoma brucei brucei, such as salicylhydroxamic acid, other substituted benzhydroxamic acid, esters of 2,3- and 3,4-dihydroxybenzoic acid and structurally related compounds, showed that they have a remarkable degree of selectivity for the trypanosome as compared to rat liver mitochondria even though they are putative CoQ analogues and both respiratory systems are dependent on CoQ. 2. The minimal inhibition of mammalian mitochondrial function could not be assigned to inhibition of ubiquinone function in these mitochondria. 3. CoQ-reducing mitochondrial dehydrogenases from rat liver, trypanosomes and skunk cabbage (Symplocarpus foetidus) were insensitive to these inhibitors. 4. The alternative oxidase of skunk cabbage mitochondria was sensitive to a spectrum of trypanosome respiration inhibitors suggesting a similarity to the oxidase of the trypanosome although differing degrees of sensitivity and differing responses to alterations in the molecular structure of the inhibitors indicate that the milieu of the active sites are dissimilar.