The mitochondrial FF-ATPase exploits the dithiol redox state to modulate the permeability transition pore.

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the FF-ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg and at 150 μM when it is activated by Ca. The PAO-driven FF-ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE).

The inhibition of gadolinium ion (Gd) on the mitochondrial FF-ATPase is linked to the modulation of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (PTP), which drives regulated cell death when Ca concentration suddenly increases in mitochondria, was related to changes in the Ca-activated FF-ATPase. The effects of the gadolinium cation (Gd), widely used for diagnosis and therapy, and reported as PTP blocker, were evaluated on the FF-ATPase activated by Mg or Ca and on the PTP. Gd more effectively inhibits the Ca-activated FF-ATPase than the Mg-activated FF-ATPase by a mixed-type inhibition on the former and by uncompetitive mechanism on the latter.

Vitamin K Vitamers Differently Affect Energy Metabolism in IPEC-J2 Cells.

The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to study the effect of compounds at the intestinal level.

Molecular and Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System: Implications for Pathology.

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 FF-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.

Relationship between serum concentration, functional parameters and cell bioenergetics in IPEC-J2 cell line.

The foetal bovine serum (FBS) concentration could influence functional parameters of IPEC-J2 cells. IPEC-J2 is a non-transformed continuous epithelial cell line that represents an established in vitro model to study porcine gut inflammation and alterations of intestinal integrity. This cell line also represents a good translational model thanks to the high similitudes between pig and human gastrointestinal tract.

Biological characteristics and metabolic profile of canine mesenchymal stem cells isolated from adipose tissue and umbilical cord matrix.

Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production.

Sulfide affects the mitochondrial respiration, the Ca-activated FF-ATPase activity and the permeability transition pore but does not change the Mg-activated FF-ATPase activity in swine heart mitochondria.

In mammalian cells enzymatic and non-enzymatic pathways produce HS, a gaseous transmitter which recently emerged as promising therapeutic agent and modulator of mitochondrial bioenergetics. To explore this topic, the HS donor NaHS, at micromolar concentrations, was tested on swine heart mitochondria. NaHS did not affect the FF-ATPase activated by the natural cofactor Mg, but, when Mg was replaced by Ca, a slight 15% enzyme inhibition at 100 µM NaHS was shown.

Ca as cofactor of the mitochondrial H -translocating F F -ATP(hydrol)ase.

The mitochondrial F F -ATPase in the presence of the natural cofactor Mg acts as the enzyme of life by synthesizing ATP, but it can also hydrolyze ATP to pump H . Interestingly, Mg can be replaced by Ca , but only to sustain ATP hydrolysis and not ATP synthesis. When Ca inserts in F , the torque generation built by the chemomechanical coupling between F and the rotating central stalk was reported as unable to drive the transmembrane H flux within F .

Incoming news on the F-type ATPase structure and functions in mammalian mitochondria.

•Recent findings of cryo-EM structures of mammalian FF-ATPase.•The membrane-embedded domain of the FF-ATPase and the permeability transition pore.•The Ca-activated F-ATPase role in the mPTP is consistent with recent cryo-EM findings.

Mitochondrial FF-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis.

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 increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial FF-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg by Ca in the FF-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg- or Ca-dependent FF-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 Mg-dependent FF-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery.

1,5-Disubstituted-1,2,3-triazoles as inhibitors of the mitochondrial Ca -activated F F -ATP(hydrol)ase and the permeability transition pore.

The mitochondrial permeability transition pore (mPTP), a high-conductance channel triggered by a sudden Ca concentration increase, is composed of the F F -ATPase. Since mPTP opening leads to mitochondrial dysfunction, which is a feature of many diseases, a great pharmacological challenge is to find mPTP modulators. In our study, the effects of two 1,5-disubstituted 1,2,3-triazole derivatives, five-membered heterocycles with three nitrogen atoms in the ring and capable of forming secondary interactions with proteins, were investigated.

Effects of Hydrogen Sulfide Donor NaHS on Porcine Vascular Wall-Mesenchymal Stem Cells.

Hydrogen sulfide (HS) is now considered not only for its toxicity, but also as an endogenously produced gas transmitter with multiple physiological roles, also in maintaining and regulating stem cell physiology. In the present work, we evaluated the effect of a common HS donor, NaHS, on porcine vascular wall-mesenchymal stem cells (pVW-MSCs). pVW-MSCs were treated for 24 h with increasing doses of NaHS, and the cell viability, cell cycle, and reactive oxygen species (ROS) production were evaluated.

Mitochondrial F-type ATP synthase: multiple enzyme functions revealed by the membrane-embedded F structure.

Of the two main sectors of the F-type ATP synthase, the membrane-intrinsic F domain is the one which, during evolution, has undergone the highest structural variations and changes in subunit composition. The F complexity in mitochondria is apparently related to additional enzyme functions that lack in bacterial and thylakoid complexes. Indeed, the F-type ATP synthase has the main bioenergetic role to synthesize ATP by exploiting the electrochemical gradient built by respiratory complexes.

Sperm function and mitochondrial activity: An insight on boar sperm metabolism.

In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to link them to sperm function. Boar spermatozoa were collected, diluted at 30 × 10 spz/mL and incubated for 1 h with: Rotenone (ROT), complex I inhibitor, Dimethyl-malonate (DMM), complex II inhibitor, antimycin A (ANTI), complex III inhibitor, oligomycin (OLIGO), ATP synthase inhibitor, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), uncoupling agent, 2-deoxy-glucose (2DG), glucose agonist, and Dimethyl sulphoxide (DMSO) as control vehicle. Viability and mitochondrial membrane potential (Sybr14/PI/JC1 staining) and sperm motility (using CASA system) were assayed after incubation.

Phenylglyoxal inhibition of the mitochondrial FF-ATPase activated by Mg or by Ca provides clues on the mitochondrial permeability transition pore.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the FF-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the FF-ATPase either sustained by the natural cofactor Mg or by Ca 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.

Nicotinamide Nucleotide Transhydrogenase as a Sensor of Mitochondrial Biology.

The enzyme nicotinamide nucleotide transhydrogenase (NNT) transfers hydride from NADH to NADP coupled to H translocation across the inner mitochondrial membrane. In a recent study, Kampjut and Sazanov reveal that the bifunctional NNT mechanism rules the NAD(P)/NAD(P)H interconversion ratio, which in turn regulates antioxidant defense and sirtuin actions.

Mitochondrial Ca -activated F F -ATPase hydrolyzes ATP and promotes the permeability transition pore.

The properties of the mitochondrial F F -ATPase catalytic site, which can bind Mg , Mn , or Ca and hydrolyze ATP, were explored by inhibition kinetic analyses to cast light on the Ca -activated F F -ATPase connection with the permeability transition pore (PTP) that initiates cascade events leading to cell death. While the natural cofactor Mg activates the F F -ATPase in competition with Mn , Ca is a noncompetitive inhibitor in the presence of Mg . Selective F inhibitors (Is-F ), namely NBD-Cl, piceatannol, resveratrol, and quercetin, exerted different mechanisms (mixed and uncompetitive inhibition) on either Ca - or Mg -activated F F -ATPase, consistent with the conclusion that the catalytic mechanism changes when Mg is replaced by Ca .

Emerging Roles for the Mitochondrial ATP Synthase Supercomplexes.

As pointed out by Gu et al. (Science 2019) in mammalian mitochondria, the H-shaped tetrameric structure of the ATP synthase, the cell powerhouse, consists of two V-shaped dimers linked by two IF1 in antiparallel arrangement. This supramolecular structure reveals new functional/structural roles of the enzyme complex in mitochondria.

A Therapeutic Role for the FF-ATP Synthase.

Recently, the FF-ATP synthase, due to its dual role of life enzyme as main adenosine triphosphate (ATP) maker and of death enzyme, as ATP dissipator and putative structural component of the mitochondrial permeability transition pore (mPTP), which triggers cell death, has been increasingly considered as a drug target. Accordingly, the enzyme offers new strategies to counteract the increased antibiotic resistance. The challenge is to find or synthesize compounds able to discriminate between prokaryotic and mitochondrial FF-ATP synthase, exploiting subtle structural differences to kill pathogens without affecting the host.

Characterization of metabolic profiles and lipopolysaccharide effects on porcine vascular wall mesenchymal stem cells.

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.

Crucial aminoacids in the F sector of the FF-ATP synthase address H across the inner mitochondrial membrane: molecular implications in mitochondrial dysfunctions.

The eukaryotic FF-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.

Lipid-protein interactions in mitochondrial membranes from bivalve mollusks: molecular strategies in different species.

The mitochondrial FF-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 FF-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum).

From the Ca-activated FF-ATPase to the mitochondrial permeability transition pore: an overview.

Based on recent advances on the Ca-activated FF-ATPase features, a novel multistep mechanism involving the mitochondrial FF 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 Ca concentration rises and ATP is hydrolyzed by the FF-ATPase, conformational changes, triggered by Ca insertion in F, may be transmitted to F and locally modify the IMM curvature.

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

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.

Post-translational modifications of the mitochondrial FF-ATPase.

Background: The mitochondrial FF-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.