Search Results (8)

According to many research groups, high glucose induces the overproduction of superoxide anions, with reactive oxygen species (ROS) generally being considered the link between high glucose levels and the toxicity seen at cellular levels. Respiratory complex anomalies can lead to the production of ROS. Calcium [Ca²⁺] at physiological levels serves as a second messenger in many physiological functions. Accordingly, mitochondrial calcium [Ca²⁺]_m overload leads to ROS production, which can be lethal to the mitochondria through various mechanisms. F1F0-ATPase (ATP synthase or complex V) is the enzyme responsible for catalyzing the final step of oxidative phosphorylation. This is achieved by F1F0-ATPase coupling the translocation of protons in the mitochondrial intermembrane space and shuttling them to the mitochondrial matrix for ATP synthesis to take place. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space, thereby blocking ATP synthesis and, in turn, leading to Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome. This study seeks to explore the possibility of [Ca²⁺]_m overload mediating the pathological roles of high glucose in defective respiratory chain-mediated mitochondrial stress. NARP cybrids are the in vitro experimental models of cells with F1FO-ATPase defects, with these cells harboring 98% of mtDNA T8993G mutations. Their counterparts, 143B osteosarcoma cell lines, are the parental cell lines used for comparison. We observed that NARP cells mediated and enhanced the death of cells (apoptosis) when incubated with hydrogen peroxide (H₂O₂) and high glucose, as depicted using the MTT assay of cell viability. Furthermore, using fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells were found to significantly enable mitochondrial reactive oxygen species (mROS) formation and enhance the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar-enhanced toxicity on the mitochondria may, in the future, help to alleviate the symptoms of patients with NARP syndromes and other neurodegenerative diseases. Full article

The integration of phosphorus chemistry with the mechanism of ATP synthesis/hydrolysis requires dynamical information during ATP turnover and catalysis. Oxygen exchange reactions occurring at β-catalytic sites of the F_OF₁-ATP synthase/F₁-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. They have been shown to provide valuable time-resolved information on enzyme catalysis during ATP synthesis and ATP hydrolysis. The present work conducts new experiments on oxygen exchange catalyzed by submitochondrial particles designed to (i) measure the relative rates of Pi–ATP, Pi–HOH, and ATP–HOH isotope exchanges; (ii) probe the effect of ADP removal on the extent of inhibition of the exchanges, and (iii) test their uncoupler sensitivity/resistance. The objectives have been realized based on new experiments on submitochondrial particles, which show that both the Pi–HOH and ATP–HOH exchanges occur at a considerably higher rate relative to the Pi–ATP exchange, an observation that cannot be explained by previous mechanisms. A unifying explanation of the kinetic data that rationalizes these observations is given. The experimental results in (ii) show that ADP removal does not inhibit the intermediate Pi–HOH exchange when ATP and submitochondrial particles are incubated, and that the nucleotide requirement of the intermediate Pi–HOH exchange is adequately met by ATP, but not by ADP. These results contradicts the central postulate in Boyer’s binding change mechanism of reversible catalysis at a F₁ catalytic site with $K_{eq}~1$ that predicts an absolute requirement of ADP for the occurrence of the Pi–HOH exchange. The prominent intermediate Pi–HOH exchange occurring under hydrolytic conditions is shown to be best explained by Nath’s torsional mechanism of energy transduction and ATP synthesis/hydrolysis, which postulates an essentially irreversible cleavage of ATP by mitochondria/particles, independent from a reversible formation of ATP from ADP and Pi. The explanation within the torsional mechanism is also shown to rationalize the relative insensitivity of the intermediate Pi–HOH exchange to uncouplers observed in the experiments in (iii) compared to the Pi–ATP and ATP–HOH exchanges. This is shown to lead to new concepts and perspectives based on ligand displacement/substitution and ligand permutation for the elucidation of the oxygen exchange reactions within the framework of fundamental phosphorus chemistry. Fast mechanisms that realize the rotation/twist, tilt, permutation and switch of ligands, as well as inversion at the γ-phosphorus synchronously and simultaneously and in a concerted manner, have been proposed, and their stereochemical consequences have been analyzed. These considerations take us beyond the binding change mechanism of ATP synthesis/hydrolysis in bioenergetics. Full article

Oxygen exchange reactions occurring at β-catalytic sites of the F_OF₁-ATP synthase/F₁-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F₁-ATPase (MF₁). The apparent rate constant of oxygen exchange governing the intermediate Pi–HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1–5000 μM) and a corresponding ~200-fold range of reaction velocity (3.5–650 [moles of Pi/{moles of F₁-ATPase}⁻¹ s⁻¹]). Isotopomer distributions of [¹⁸O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F₁-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F₁-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F₁-ATPase have been discussed. Current models of ATP hydrolysis by F₁-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath’s torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F₁-ATPase. Full article

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase. Full article

F₁·F_o-ATP synthases/ATPases (F₁·F_o) are molecular machines that couple either ATP synthesis from ADP and phosphate or ATP hydrolysis to the consumption or production of a transmembrane electrochemical gradient of protons. Currently, in view of the spread of drug-resistant disease-causing strains, there is an increasing interest in F₁·F_o as new targets for antimicrobial drugs, in particular, anti-tuberculosis drugs, and inhibitors of these membrane proteins are being considered in this capacity. However, the specific drug search is hampered by the complex mechanism of regulation of F₁·F_o in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis. In this review, we consider the current state of the problem of “unidirectional” F₁·F_o catalysis found in a wide range of bacterial F₁·F_o and enzymes from other organisms, the understanding of which will be useful for developing a strategy for the search for new drugs that selectively disrupt the energy production of bacterial cells. Full article

The F₁F_O-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F₁F_O-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF₁), and chloroplasts (ε and γ disulfide) emerged to block the F₁F_O-ATPase activity selectively. In this study, we analyze how these F₁F_O-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F₁F_O-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF₁ likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria. Full article

Cucumber (Cucumis sativus L.) is a warm-season crop that is sensitive to chilling temperatures and a maternally inherited cold tolerance exists in the heirloom cultivar ‘Chipper’ (CH). Because the organelles of cucumber show differential transmission (maternal for chloroplast and paternal for mitochondrion), this cold tolerance is hypothesized to be chloroplast-associated. The goal of this research was to characterize the cold tolerant phenotype from CH and determine its genetic basis. Doubled haploid (DH) lines were produced from CH and cold susceptible cucumbers, reciprocal hybrids with identical nuclear genotypes were produced, and plants were subjected to cold treatments under lights at 4 °C for 5.5 h. Hybrid plants with CH as the maternal parent had significantly higher fresh and dry weights 14 days after cold treatment compared to the reciprocal hybrid, revealing an enhanced cold recovery phenotype maternally conferred by CH. Results from analyses of the nuclear transcriptome and reactive oxygen species (ROS) between reciprocal hybrids were consistent with the cold recovery phenotype. Sequencing of the chloroplast genome and transcriptome of the DH parents and reciprocal hybrids, respectively, revealed one maternally transmitted non-synonymous single nucleotide polymorphism (SNP) in the chloroplast F₁F_O-ATP synthase (CF₁F_O-ATPase) beta-subunit gene (atpB) of CH which confers an amino acid change from threonine to arginine. Protein modeling revealed that this change is located at the interface of the alpha- and beta-subunits in the CF₁F_O-ATPase complex. Polymorphisms in the CF₁F_O-ATPase complex have been associated with stress tolerances in other plants, and selection for or creation of polymorphic beta-subunit proteins by chloroplast transformation or gene editing could condition improved recovery from cold stress in plants. Full article

The vacuolar ATPase (V-ATPase) complex of yeast (Saccharomyces cerevisiae) is comprised of two sectors, V₁ (catalytic) and V_O (proton transfer). The hexameric (A₃B₃) cylinder of V₁ has a central cavity that must accommodate at least part of the rotary stalk of V-ATPase, a key component of which is subunit D (Vma8p). Recent electron microscopy (EM) data for the prokaryote V-ATPase complex (Thermus thermophilus) suggest that subunit D penetrates deeply into the central cavity. The functional counterpart of subunit D in mitochondrial F₁F_O-ATP synthase, subunit γ, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit g, we probed the location of the C-terminus of Vma8p by attachment of a large protein adduct, green fluorescent protein (GFP). We found that truncated Vma8p proteins lacking up to 40 C-terminal residues fused to GFP can be incorporated into functional V-ATPase complexes, and are able to support cell growth under alkaline conditions. We conclude that large protein adducts can be accommodated at the top of the central cavity of V₁ without compromising V-ATPase function, arguing for structural flexibility of the V₁ sector. Full article