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The mitochondrial F1FO-ATPase, the key enzyme in cell bioenergetics, apparently works in the same way in mollusks and in mammals. We previously pointed out a raft-like arrangement in mussel gill mitochondrial membranes, which apparently distinguishes bivalve mollusks from mammals. To explore the relationship between the microenvironmental features and the enzyme activity, the physico-chemical features of mitochondrial membranes and the F1FO-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum). Similarly to the mussel, clam gill mitochondrial membrane lipids exhibit a high sterol content (42 mg/g protein), mainly due to phytosterols (cholesterol only attains 42% of total sterols), and abundant polyunsaturated fatty acids (PUFA) (70% of total fatty acids), especially of the n-3 family. However, the F1FO–ATPase activation energies above and below the break in the Arrhenius plot (22.1 °C) are lower than in mussel and mammalian mitochondria. Laurdan fluorescence spectroscopy analyses carried out at 10 °C, 20 °C and 30 °C on mitochondrial membranes and on lipid vesicles obtained from total lipid extracts of mitochondria, indicate a physical state without coexisting domains. This mitochondrial membrane constitution, allowed by lipid-lipid and lipidprotein interactions and involving PUFA-rich phospholipids, phytosterols (much more diversified in clams than in mussels) and proteins, enables the maintenance of a homogeneous physical state in the range 10–30 °C. Consistently, this molecular interaction network would somehow extend the temperature range of the F1FO–ATPase activity and may contribute to clam resilience to temperature changes.

The properties of the mitochondrial F1 FO -ATPase catalytic site, which can bind Mg2+ , Mn2+ , or Ca2+ and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca2+ -activated F1 FO -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg2+ activates the F1 FO -ATPase in competition with Mn2+ , Ca2+ is a noncompetitive inhibitor in the presence of Mg2+ . Selective F1 inhibitors (Is-F1 ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca2+ - or Mg2+ -activated F1 FO -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg2+ is replaced by Ca2+ . In a partially purified F1 domain preparation, Ca2+ -activated F1 -ATPase maintained Is-F1 sensitivity, and enzyme inhibition was accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the structural relationship between Ca2+ -activated F1 FO -ATPase and the PTP, and, in turn, on consequences, such as physiopathological cellular changes.

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F

Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F1FO-ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F1FO-ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.

open 6 no The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide. embargoed_20211117 Algieri C.; Nesci S.; Trombetti F.; Fabbri M.; Ventrella V.; Pagliarani A. Algieri C.; Nesci S.; Trombetti F.; Fabbri M.; Ventrella V.; Pagliarani A.

In mammalian cells enzymatic and non-enzymatic pathways produce H

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca

The eukaryotic F 1 F O -ATP synthase/hydrolase activity is coupled to H + translocation through the inner mitochondrial membrane. According to a recent model, two asymmetric H + half-channels in the a subunit translate a transmembrane vertical H + flux into the rotor rotation required for ATP synthesis/hydrolysis. Along the H + pathway, conserved aminoacid residues, mainly glutamate, address H + both in the downhill and uphill transmembrane movements to synthesize or hydrolyze ATP, respectively. Point mutations responsible for these aminoacid changes affect H + transfer through the membrane and, as a cascade, result in mitochondrial dysfunctions and related pathologies. The involvement of specific aminoacid residues in driving H + along their transmembrane pathway within a subunit, sustained by the literature and calculated data, leads to depict a model consistent with some mitochondrial disorders.

Based on recent advances on the Ca2+-activated F1FO-ATPase features, a novel multistep mechanism involving the mitochondrial F1FO complex in the formation and opening of the still enigmatic mitochondrial permeability transition pore (MPTP), is proposed. MPTP opening makes the inner mitochondrial membrane (IMM) permeable to ions and solutes and, through cascade events, addresses cell fate to death. Since MPTP forms when matrix Ca2+ concentration rises and ATP is hydrolyzed by the F1FO-ATPase, conformational changes, triggered by Ca2+ insertion in F1, may be transmitted to FO and locally modify the IMM curvature. These events would cause F1FO-ATPase dimer dissociation and MPTP opening.

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 μM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE). Conversely, 300 μM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.

Background The mitochondrial F1FO-ATPase has the main role in synthesizing most of ATP, thus providing energy to living cells, but it also works in reverse and hydrolyzes ATP, depending on the transmembrane electrochemical gradient. Within the same complex the vital role of the enzyme of life coexists with that of molecular switch to trigger programmed cell death. The two-faced vital/lethal role makes the enzyme complex an intriguing biochemical target to fight pathogens resistant to traditional therapies and diseases linked to mitochondrial dysfunctions. A variety of post-translational modifications (PTMs) of selected F1FO-ATPase aminoacids have been reported to affect the enzyme function. Scope of review By reviewing the known PTMs of aminoacid side chains of both F1 and FO sectors according to the most recent advances, the main aim is to highlight how local chemical changes may constitute the molecular key leading to pathological or physiological events. Major conclusions PTMs represent the chemical tool to modulate the F1FO-ATPase activity in response to different stimuli. Some PTMs are required to ensure the enzyme catalysis or, conversely, to inactivate the enzyme function. Each covalent modification of the F1FO-ATPase, which occur in response to local changes, is the result of a selective molecular mechanism which, by translating a chemical modification into a biochemical effect, guarantees the enzyme tuning under changing conditions. General significance Once highlighted how the molecular mechanism works, some PTMs may be exploited to modulate the effect of drugs targeting the enzyme complex or constitute promising tools for F1FO-ATPase-targeted therapeutic strategies.

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg2+ or, with lower efficiency, by Ca2+. The latter may play a role when calcium concentration rises in mitochondria, a condition associated with cascade events leading to cell death. Common and distinctive features of these differently activated mitochondrial ATPases were pointed out in swine heart mitochondria. When Ca2+ replaces the natural cofactor Mg2+, the enzyme responsiveness to the transmembrane electrochemical gradient and to the classical F-ATPase inhibitors DCCD and oligomycin as well as the oligomycin sensitivity loss by thiol oxidation, are maintained. Consistently, the two mitochondrial ATPases apparently share the F1FO complex basic structure and mechanism. Peculiar cation-dependent properties, which may affect the F1 catalytic mechanism and/or the FO proton binding site features, may be linked to a different physiological role of the mitochondrial Ca-activated F-ATPase with respect to the Mg-activated F-ATPase.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F

The properties of the mitochondrial F

In mammalian cells enzymatic and non-enzymatic pathways produce H2S, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the H2S donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the F1FO-ATPase activated by the natural cofactor Mg2, but, when Mg2+ was replaced by Ca2+, a slight 15% enzyme inhibition at 100 µM NaHS was shown. Conversely, both the NADH-O2 and succinate-O2 oxidoreductase activities were totally inhibited by 200 μM NaHS with IC50 values of 61.6 ± 4.1 and 16.5 ± 4.6 μM NaHS, respectively. Since the mitochondrial respiration was equally inhibited by NaHS at both first or second respiratory substrates sites, the H2S generation may prevent the electron transfer from complexes I and II to downhill respiratory chain complexes, probably because H2S competes with O2 in complex IV, thus reducing membrane potential as a consequence of the cytochrome c oxidase activity inhibition. The Complex IV blockage by H2S was consistent with the linear concentration-dependent NADH-O2 oxidoreductase inhibition and exponential succinate-O2 oxidoreductase inhibition by NaHS, whereas the coupling between substrate oxidation and phosphorylation was unaffected by NaHS. Even if H2S is known to cause sulfhydration of cysteine residues, thiol oxidizing (GSSG) or reducing (DTE) agents, did not affect the F1FO-ATPase activities and mitochondrial respiration, thus ruling out any involvement of post-translational modifications of thiols. The permeability transition pore, the lethal channel which forms when the F1FO-ATPase is stimulated by Ca2+, did not open in the presence of NaHS, which showed a similar effect to ruthenium red, thus suggesting a putative Ca2+ transport cycle inhibition.

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg(2+) effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F1FO-ATPase (F-ATPase) activity promoted by micromolar Hg(2+) concentrations were investigated. Hg(2+) was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg(2+) doses which inhibited the NADH-O2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg(2+) dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg(2+) interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria.

Polar lipids (PoL) represent a new promising dietary approach in the prevention and treatment of many human diseases, due to their potential nutritional value and unique biophysical properties. This study investigates the effects of catching season and oven baking on the fatty acid profiles (FAP) of PoL in four species of blue-back fish widely present in the North Adriatic Sea: anchovy (Engraulis encrasicholus), sardine (Sardina pilchardus), sprat (Sprattus sprattus), and horse mackerel (Trachurus trachurus). PoL levels (427-652 mg/100 g flesh) varied among the four species, with no significant seasonal variations within species. FAP of raw fillets were particularly high in polyunsaturated fatty acid (PUFA), especially docosahexaenoic acid (DHA) and EPA; total PUFA was constant in all species throughout the year, while long-chain n-3 polyunsaturated fatty acid (n-3 PUFA) rose in spring (except in sprat), especially due to the contribution of DHA. The FAP response for PoL to oven baking was species-specific and, among n-3 PUFA, DHA exhibited the greatest heat resistance; the influence of oven baking on FAP was found to be correlated with the catching season, especially for anchovy and sardine, while sprat PoL were not affected by cooking processes. The four species analyzed in this study presented very low n-6/n-3 fatty acid ratios and highly favorable nutritional indices, emphasizing their PoL qualities and promoting their role in increasing human n-3 PUFA intake. The four species can be considered as superior sources of n-3 PUFA and can be employed as supplements in functional food manufacturing and in pharmaceutical and cosmetic industries.

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 μg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 μg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82±0.07mM in presence of Mg2+ and 0.64±0.05mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.

The eukaryotic F

Based on recent advances on the Ca

The antibiotic oligomycin is known to inhibit mitochondrial F-type ATP synthases. The antibiotic inhibits both ATP synthesis and hydrolysis by blocking the proton translocation through FO which is coupled to the catalytic activity of F1. The amphiphilic organotin tri-n-butyltin (TBT), a known mitochondrial poison, can penetrate into biological membranes and covalently bind to electron-donor atoms of biomolecules such as sulfur. This study aims at exploring the mechanism(s) involved in the enzyme desensitization to oligomycin which occurs at concentrations >1 mM TBT. This poorly known effect of TBT, which only appeared at temperatures above the break in the Arrhenius plot of the enzyme activity, was found to be accompanied by the oxidation of isolated thiol groups of the mitochondrial complex. The oligomycin sensitivity was restored by the reducing agents glutathione and dithioerythritol and not influenced by antioxidants. The whole of data is consistent with the hypothesis that thiol oxidation is caused by TBT covalent binding to cysteine residues in a low-affinity site on FO and not by other possible oxidative events. According to this putative model, the onset of tin-sulfur bonds would trigger conformational changes and weaken the oligomycin interaction with FO.

Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells.

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H+ by coordinating the hydronium ion (H3O+) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

The mitochondrial F-ATPase can be activated either by the classical cofactor Mg

The mitochondrial FBy reviewing the known PTMs of aminoacid side chains of both FPTMs represent the chemical tool to modulate the FOnce highlighted how the molecular mechanism works, some PTMs may be exploited to modulate the effect of drugs targeting the enzyme complex or constitute promising tools for F

The main and best known role of the mitochondrial ATP synthase is to synthesize ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. However, under different conditions, the same enzyme can also switch to the opposite function of ATP hydrolysis and exploits its energy to pump protons against their gradient and energize the membrane. The change in functionality is linked to the change of direction of rotation of the two matched sectors of this unique complex, namely the hydrophilic F1, which performs the catalysis, and the hydrophobic membrane-embedded FO, which channels protons. Accordingly, viewed from the matrix side, ATP synthesis is driven by counterclockwise rotation and ATP hydrolysis by clockwise rotation of the FO rotor which is transmitted to F1. ATP dissipation through this mechanism features some diseases such as myocardial ischemia. Increasing evidence shoulders the hypothesis that the asymmetry of the a subunit of FO and particularly the steric arrangement of the two inner semi-channels for protons, play a key role in conferring to the coupled bi-functional complex the ability to reverse rotation by switching from ATP synthesis to ATP hydrolysis and vice versa. Accordingly, the conserved steric arrangement of the chiral a subunit of FO yields the same direction of rotation for all the ATP synthases. According to this hypothesis, the a subunit chirality imposes the direction of rotation of the rotor according to the proton gradient across the membrane. It seems likely that the direction of rotation of the membrane-embedded c-ring, which is adjacent to the a-subunit and acts as a rotor, may be under multiple control, being rotation essential to make the whole enzyme machinery work. However, the asymmetric features of the a subunit would make it the master regulator, thus directly determining which of the two functions, ATP production or ATP dissipation, will be performed. The handedness of a subunit should be considered in drug design to counteract tissue damage under all pathological conditions linked to functional impairment of ATP synthase.

Among butyltin compounds, tributyltin (TBT), widely exploited in the past in antifouling paints for its biocidal properties, is long known as one of the most harmful sea contaminants. Among the ascertained and universal toxicity mechanisms, TBT targeting F1FO-ATPase and thus impairing cell bioenergetics, is here reviewed. While TBT effects on F1FO-ATPase have been investigated for decades, the possible impact of the derivatives dibutyltin (DBT) and monobutyltin (MBT), produced by abiotic and/or biotic dealkylation of TBT and usually considered far less toxic, have been poorly explored up until now. Butyltin effects on F1FO-ATPase and their underlying action mechanism seem to be tightly structure dependent. Butyltins are membrane-active toxicants. Owing to its more pronounced lipophilicity TBT targets the transmembrane FO sector, blocks ionic translocation and causes a dose-dependent loss of sensitivity to FO inhibitors such as oligomycin and N,N′-dicyclohexylcarbodiimide. DBT strongly inhibits F1FO-ATPase activity by competing with the Mg+2 cofactor in the F1 catalytic site but is ineffective on the enzyme sensitivity to FO inhibitors. MBT is apparently ineffective. The possible contribution of DBT to the overall butyltin toxicity on membrane systems may not be neglectable since usually TBT coexists with its derivatives in organotin-exposed animal tissues. Copyright © 2013 John Wiley & Sons, Ltd.

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg

In mussel digestive gland mitochondria the environmental pollutant tri-n-butyltin (TBT), other than strongly inhibiting ATPase activity at

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss.

Tributyltin (TBT), widely employed in the past in antifouling paints, is one of the most toxic organic pollutants. Although recently banned, it still threatens coastal water ecosystems and accumulates in filterfeeding molluscs. TBT is known to act as amembrane-active toxicant; however data on mussels are scanty and exposure effects on mitochondrial ATPase activities remain hitherto unexplored. TBT effects on the mitochondrial Mg-ATPase activities in the digestive gland of Mytilus galloprovincialis were investigated both in vitro and in TBT-exposed mussels. Both an oligomycin-sensitive Mg-ATPase (OS Mg-ATPase) (70% of total Mg-ATPase activity) and an oligomycin-insensitive ATPase (OI Mg-ATPase) (30%) were found. The OS-Mg-ATPase was as much as 70% in vitro inhibited by 0.7 μM(203 μg/L) TBT, while higher concentrations promoted a partial inhibition release up to 5.0 μM TBT; higher than 10.0 μMTBT concentrations yielded nearly complete enzyme inhibition. Concentrations higher than 1 μM TBT enhanced the OI Mg-ATPase. Mussels exposed to 0.5 and 1.0 μg/L TBT in aquaria showed a 30% depressed OS Mg-ATPase activity, irrespective of TBT dose and exposure time (24 and 120 h). The OI Mg-ATPase activity was apparently refractory to TBT exposure and halved both in control and TBT-exposed mussels after 120 h exposure.

Due to the increasing evidences on dinoflagellate contribution to mucilage phenomena in the Mediterranean Sea, this study was aimed at identifying biochemical and/or genetic markers that could help in relating Gonyaulax fragilis growth with mucilage formation when microscope identification was difficult. The biochemical characterization considered the fatty acid and sterol composition of cultured strains and mucilage samples. The cultured algae displayed the typical fatty acid pattern of dinoflagellates as the 18:0 was the prevalent saturated fatty acid (SFA), 18:1n-7 and 18:1n-9 were the major monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were dominated by 18:5n-3 and 22:6n-3. Two groups of sterols were identified in algal cells: desmethylsterols, dominated by cholesterol and β-sitosterol, and 4α-methylsterols, having dinosterol as the prevailing sterol. The mucilage lipid pattern was characterized by low amounts of PUFAs and appeared deeply influenced by environment dynamics; however, the 20:5n-3/22:6n-3 ratio

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents "the enzyme of life" of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the common drug binding site of the enzyme is somehow destabilized, thus weakening the enzyme-drug interactions and making the ATP synthase insensitive to drug inhibition. On these bases we hypothesize that the selective oxidation of these cysteine thiols can be exploited to desensitize the mitochondrial ATP synthase to drugs which target FO and maintain their inhibitory potency on bacterial ATP synthases. According to our hypothesis, this strategy could represent an intriguing tool to prevent adverse effects of antimicrobial drugs in mammals, thus enhancing the number of natural and synthetic compounds which can be used in therapy. To this aim studies should be addressed to the identification and formulation of compounds and/or treatments able to selectively oxidize the crucial cysteine thiols of c-subunits without affecting the overall functionality of the mitochondrial ATP synthase and other thiol containing proteins.

The possibility of increasing n-3 and n-6 long-chain polyunsaturated fatty acids (PUFA) content in microalgal mixtures used to feed Tapes philippinarum larvae was explored by lowering culture temperature from 26 to 14 °C. Although fatty acid composition of different microalgal species has a genetic basis, the algal cultures grown at 14 °C significantly increased the content of long-chain n-3 PUFA in Isocrysis galbana and in Thalassiosira pseudonana, while in Tetraselmis tetrathelo, the PUFA increase only involved shorter chain PUFA, namely 16:4n-3 and 18:4n-3. However, larvae fed on the PUFA enriched microalgal mixture did not show improvements in growth and survival performances with respect to the control group fed the microalgal mixture grown at 26 °C. From a biochemical perspective, two key aspects emerged from the results: (i) clam larvae have adequate biotransformation and selection skills to adjust fatty acid profile to their requirements as they can even modulate the incorporation of essential long-chain PUFA as 20:5n-3 and 22:6n-3 when the dietary supply exceeds the physiological requirements; (ii) bivalve can biosynthesize non-methylene-interrupted dienoic (NMID) fatty acids as confirmed by the constancy of relative proportion with larvae growth in spite of the NMID fatty acid absence in the diet.

The mitochondrial F1FO-ATP synthase has not only the known life function in building most cellular ATP, but also, as recently hinted, an amazing involvement in cell death. Accordingly, the two-faced enzyme complex, which catalyzes both ATP synthesis and ATP hydrolysis, has been involved in the mitochondrial permeability transition, the master player in apoptosis and necrosis. Nitrite, a cellular nitric oxide reservoir, has a recognized role in cardiovascular protection, through still unclear mechanisms.In swine heart mitochondria the effect of nitrite on the F1FO-ATPase activity activated by Ca(2+), henceforth defined as Ca-ATPase(s), or by the natural cofactor Mg(2+), was investigated by evaluating ATP hydrolysis under different assay conditions.Ca(2+) is far less efficient than the natural cofactor Mg(2+) in the ATPase activation. However, when activated by Ca(2+) the ATPase activity is especially responsive to nitrite, which acts as uncompetitive inhibitor and up to 2 mM inhibits the Ca2+-activated-ATPase(s), probably by promoting dytirosine formation on the enzyme proteins, leaving the Mg-ATPase(s) unaffected. Most likely these ATPases refer to the same F1FO complex, even if coexistent ATPases may overlap.The preferential inhibition by nitrite of the Ca-ATPase(s), due to post-translational tyrosine modifications, may prevent the calcium-dependent functionality of the mitochondrial F1FO complex and related events.In mitochondria the preferential inhibition of the Ca-ATPase activity/ies by nitrite concentrations which do not affect the coexistent Mg-ATPase(s) may quench the negative events linked to the calcium-dependent functioning mode of the F1FO complex under pathological conditions.

The vascular functional and structural integrity is essential for the maintenance of the whole organism and it has been demonstrated that different types of vascular progenitor cells resident in the vessel wall play an important role in this process. The purpose of the present research was to observe the effect of tributyltin (TBT), a risk factor for vascular disorders, on porcine Aortic Vascular Precursor Cells (pAVPCs) in term of cytotoxicity, gene expression profile, functionality and differentiation potential. We have demonstrated that pAVPCs morphology deeply changed following TBT treatment. After 48h a cytotoxic effect has been detected and Annexin binding assay demonstrated that TBT induced apoptosis. The transcriptional profile of characteristic pericyte markers has been altered: TBT 10nM substantially induced alpha-SMA, while, TBT 500nM determined a significant reduction of all pericyte markers. IL-6 protein was detected in the medium of pAVPCs treated with TBT at both doses studied and with a dose response. TBT has interfered with normal pAVPCs functionality preventing their ability to support a capillary-like network. In addition TBT has determined an increase of pAVPCs adipogenic differentiation. In conclusion in the present paper we have demonstrated that TBT alters the vascular stem cells in term of structure, functionality and differentiating capability, therefore effects of TBT in blood should be deeply explored to understand the potential vascular risk associated with the alteration of vascular stem cells physiology.

The ATP synthase or F1FO-ATPase is the key enzyme in cell bioenergetics, due to its main role to build ATP, but it can also work “in reverse” to hydrolyze ATP. The enzyme complex, increasingly involved as key molecular switch between life and death, is finely tuned by multiple and only partially known mechanisms, widely operative in health and disease. Among them, the enzyme regulation through the chemical modification of protein thiols, namely cysteine side chains, is thought to play a prominent role. Thiols are known to have high biological impact, to be easily oxidized, susceptive to multiple post translational oxidative modifications and to occur both in the catalytic sector F1 and in the membrane-embedded rotor FO which form the F1FO complex. Even if thiol properties mirror the chemical attitudes of sulfur, not all cysteine thiols are equally prone to chemical modifications, being strongly influenced by their molecular environment. Cysteine thiol modifications, which, according to the ambient, may be reversible, interchangeable and even irreversible, not only modulate the enzyme catalytic and proton channeling activities, but also its response to co-occurring effectors. Additionally, they mirror the ambient redox state and the availability of reactive species involved in cell signaling. In short, within the F1FO complex, thiols act as chemical interface between the environment and the enzyme function. In this chapter the knowledge on the thiol-related F1FO-ATPase modulation is reviewed, with special focus on mammalian mitochondria, aiming at contributing to shed light on a key molecular mechanism under physiological and pathological conditions.

The mitochondrial F₁F₀ complex is highly sensitive to macrolide antibiotics and especially targeted by oligomycins. These compounds bind to the membrane-embedded sector F₀ and block proton conductance through the inner membrane, thus inhibiting both ATP synthesis and hydrolysis. Oligomycin sensitivity is universally recognized as a clue of the functional integrity and matching between F₀ and F₁. Since oligomycin binding implies multiple interactions with amino acid residues of F₀, amino acid substitutions often affect the inhibition efficiency. Moreover, variegated factors spanning from membrane properties to xenobiotic incorporation and detachment of the oligomycin-insensitive F₁ sector can alter the oligomycin sensitivity of the enzyme complex. The overview on the multiple factors involved strengthens the link between altered oligomycin sensitivity and physiopathological conditions associated with defective ATPases. An improved understanding of the mechanisms involved may also favor drug design to counteract oxidative damage, which stems from most mitochondrial dysfunctions.

In the last fifteen years the presence and distribution of large aggregates in the Mediterranean Sea have begun to be studied suggesting that these particles could play a relevant role in the carbon biogeochemistry of the basin. However the massive formation is a sporadic event and it is often difficult to detect. During the autumn of 2000 high densities of mucilage aggregates were observed in the major part of the coastal zone of the Tyrrhenian Sea (western Mediterranean Sea). The chemical composition and origin of these aggregates were evaluated. The carbohydrates, proteins, lipids and the humic and fulvic acid fractions constitute 45–68% of the organic carbon (OC). Marine origin was suggested by UV spectra and C/N ratios of humic substances extracted from mucilage samples. The polysaccharide component was isolated and purified by extraction of four fractions of different solubility and the saccharide carbon percentage ranged from 13 to 20% of the OC. By comparison the qualitative and quantitative monosaccharide, fatty acid and sterol compositions were very similar to those obtained from Adriatic pelagic mucilages, while on the other hand the results showed remarkable differences in comparison to benthic aggregates. Scanning electron microscopy (SEM) on native samples showed a complex morphology with the presence of fibrillar structures forming a highly branched tri-dimensional network similar to those found in Adriatic pelagic samples.

The ATP synthase can be imagined as a reversible H(+)-translocating channel embedded in the membrane, FO portion, coupled to a protruding catalytic portion, F1. Under physiological conditions the F1FO complex synthesizes ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. Alternatively, under other patho-physiological conditions it exploits ATP hydrolysis to energize the membrane by uphill pumping protons. The reversibility of the mechanism is guaranteed by the structural coupling between the hydrophilic F1 and the hydrophobic FO. Which of the two opposite processes wins in the energy-transducing membrane complex depends on the thermodynamic balance between the protonmotive force (Δp) and the phosphorylation potential of ATP (ΔG P). Accordingly, while Δp prevalence drives ATP synthesis by translocating protons from the membrane P-side to the N-side and generating anticlockwise torque rotation (viewed from the matrix), ΔG P drives ATP hydrolysis by chemomechanical coupling of FO to F1 with clockwise torque. The direction of rotation is the same in all the ATP synthases, due to the conserved steric arrangement of the chiral a subunit of FO. The ability of this coupled bi-functional complex to produce opposite rotations in ATP synthesis and hydrolysis is explained on the basis of the a subunit asymmetry.

Background The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases. Methods The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction. Results When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE. Conclusions The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT. General significance The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.

1. 1. In Mytilus galloprovincialis Lam. both the Na + + K + )- and the Na + -ATPases show a tissue-and dose-dependent response to mercury (0.1 and 0.25 mg · L −1 Hg 2+ ), zinc (0.5 and 1.0 mg · L −1 Zn 2+ ) and ammonia (0.4 and 1.0 mg · L −1 ammonia-N). 2. 2. The two ATPases were generally more susceptive to the toxicants in the gills than in the mantle; in the case of heavy metals this finding complies with the higher metal incorporation in the gills with respect to the mantle. 3. 3. In the gills the two ATPase inhibition by mercury was enhanced by the co-presence of environmental 0.4 mg · L −1 ammonia-N; zinc removed the ( Na + + K + )-ATPase inhibition by 0.1 mg · L −1 Hg 2+ as well as the enzyme activation promoted by 0.4 mg · L −1 ammonia-N; both ATPases were inhibited by the 1.0 mg · L −1 ammonia-N treatment. 4. 4. Mantle ATPases were apparently refractory to mercury but not to zinc: the ( Na + + K + )-ATPase was similarly stimulated by the exposure to 1.0 mg · L −1 Zn 2+ and to the mixture Zn 2+ + Hg 2+ and unaffected by 0.5 mg · L −1 Zn 2+ alone; the Na + -ATPase was depressed by the exposures to 1.0 mg · L −1 ammonia-N and to the mixture Zn 2+ + ammonia-N and conversely stimulated by Hg 2+ + ammonia-N, whereas all other treatments were uneffective. 5. 5. In vitro approaches pointed out a susceptivity to the toxicants only for the (Na + + K + )-ATPase. The enzyme response in vitro often differed from that in vivo .