Triphosphate Synthase Research Articles

Monitoring oocyte/embryo respiration using electrochemical-based oxygen sensors

This paper presents a disposable three-electrode, Clark-type biosensor suitable for mitochondrial respirometry in single oocytes and embryos. The biosensor was embedded in a PMMA (polymethyl methacrylate) micro-chamber to allow investigation of single oocytes/embryos immersed in up to 100 μl of respiration buffer. The micro-chamber was sealed to avoid oxygen exchange between the inside of the chamber and the atmosphere, while being maintained at a temperature of 38.5 °C to preserve cell viability. Using amperometry, the oxygen consumption of cells inside the micro-chamber was measured. The measurements from the sensor system revealed basal cell respiration supported by endogenous substrates, respiration associated with proton leak induced by inhibition of the adenosine triphosphate (ATP) synthase (complex V) with oligomycin, and the maximal non-coupled respiratory capacity revealed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) titration. Potential applications of this oxygen sensor system include evaluating effects of metabolic therapies on oocyte bioenergetics and monitoring mitochondrial function throughout oocyte maturation and blastocyst development to predict embryo viability to compliment assisted reproductive technologies.

Active transport of cytoophidia in Schizosaccharomyces pombe.

The metabolic enzyme cytidine triphosphate synthase has recently been found to form micrometer-sized filamentous structures termed cytoophidia, which are evolutionarily conserved across prokaryotes and eukaryotes. The cytoophidium represents a novel type of membraneless organelle and behaves dynamically inside the cell. The question of how cytoophidia transport is mediated, however, remains unanswered. For the first time, we detected in this study the active transport of cytoophidia, taking advantage of the fission yeast Schizosaccharomyces pombe as an excellent model for studying membraneless organelles. We demonstrated that actin filaments, not microtubules, are responsible for this transport. Furthermore, we determined that Myo52, a type of myosin V, is required for the active transport of cytoophidia. These results reveal the major players critical to the dynamics of cytoophidia and extend our understanding of intracellular transport of membraneless organelles.—Li, H., Ye, F., Ren, J.-Y., Wang, P.-Y., Du, L.-L., Liu, J.-L. Active transport of cytoophidia in Schizosaccharomyces pombe.

Structure, mechanism, and regulation of the chloroplast ATP synthase.

The chloroplast adenosine triphosphate (ATP) synthase uses the electrochemical proton gradient generated by photosynthesis to produce ATP, the energy currency of all cells. Protons conducted through the membrane-embedded Fo motor drive ATP synthesis in the F1 head by rotary catalysis. We determined the high-resolution structure of the complete cF1Fo complex by cryo-electron microscopy, resolving side chains of all 26 protein subunits, the five nucleotides in the F1 head, and the proton pathway to and from the rotor ring. The flexible peripheral stalk redistributes differences in torsional energy across three unequal steps in the rotation cycle. Plant ATP synthase is autoinhibited by a β-hairpin redox switch in subunit γ that blocks rotation in the dark.

Protons find a path

Molecular Motors Adenosine triphosphate (ATP) synthases are dynamos that interconvert rotational and chemical energy. Capturing the complete structure of these multisubunit membrane-bound complexes has been hindered by their inherent ability to adopt multiple conformations. Srivastava et al. used

Region of Interest analysis using mass spectrometry imaging of mitochondrial and sarcomeric proteins in acute cardiac infarction tissue

Matrix-assisted laser desorption ionization image mass spectrometry (MALDI-IMS) has been developed for the identification of peptides in various tissues. The MALDI-IMS signal distribution patterns and quantification of the signal intensities of the regions of interest (ROI) with healthy regions were compared for identification of the disease specific biomarkers. We performed a new ROI analysis using the conventional t-test and data number independent Cohen’s d-value analysis. Using these techniques, we analysed heart tissues after acute myocardial infarction (AMI). As a result, IMS signals of mitochondrial adenosine triphosphate synthase alpha subunit (ATP5A), myosin-6/7(MYH6/7), aortic actin, and the myosin light chain 3 (MYL3) were identified in the infarcted region. In particular, the signals of MYH7 are significantly greater in the infarcted region using ROI analysis. ROI analysis using MALDI-IMS may be a promising technique for the identification of biomarkers for pathological studies that involve the comparison of diseased and control areas.

Stress and damage mechanisms in Dendrobium nobile Lindl. protocorm-like bodies during pre- and post-liquid nitrogen exposure in cryopreservation revealed by iTRAQ proteomic analysis

Liquid nitrogen (LN) exposure, a crucial step in cryopreservation, is mainly responsible for the reduced viability of protocorm-like bodies (PLBs) in Dendrobium nobile Lindl. To characterize the stress and damage mechanisms during pre- and post-LN exposure, the isobaric tags for relative and absolute quantification (iTRAQ)-labeling method was used to evaluate proteome profiles of PLBs in D. nobile ‘Hamana Lake Dream’ undergoing the cooling and rewarming process. In total, 1747 proteins were detected; a cluster of orthologous group (COG) analysis of the identified proteins revealed that posttranslational modification, protein turnover, and chaperone-related proteins were the predominant protein classes. Of these, 108 proteins were differentially changed and grouped into different functional categories, which included protein turnover, stress and defense, carbohydrate and energy metabolism, signaling transduction, metabolism, membrane, and transport. Furthermore, a quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that some proteins, such as annexin, l-ascorbate peroxidase 1, copper/zinc superoxide dismutase, glutathione S-transferase dehydroascorbate reductase 2-like, and an adenosine triphosphate (ATP) synthase, were regulated at their transcriptional levels. Based on functional analysis of these proteins, it was found that protein synthesis, processing, and degradation might be the main strategies used to reestablish cell balance after the cooling and rewarming process. Moreover, the production of reactive oxygen species (ROS) and the decline in energy production, signaling transduction, and membrane transport during pre- and post-LN exposure might be responsible for the viability loss of the PLBs. This work provides potential protein candidates for exploring the stress and cryo-injury mechanism in cryopreservation.

High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.

Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.

Mitochondrial ATP synthase dimers spontaneously associate due to a long-range membrane-induced force

Adenosine triphosphate (ATP) synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. From yeast to vertebrates, cryoelectron tomograms of these membranes have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. The rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. In this study, we present a quantitative molecular-simulation analysis that strongly suggests that the dimers of ATP synthases organize into rows spontaneously, driven by a long-range attractive force that arises from the relief of the overall elastic strain of the membrane. The strain is caused by the V-like shape of the dimers, unique among membrane protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of direct protein-protein interactions or a specific lipid composition of the membrane. We further hypothesize that, once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations by causing macroscopic membrane ridges that ultimately become the edges of cristae. In this way, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline key experiments that would be required to verify or refute this hypothesis.

Loss of hypoxia inducible factor-1α aggravates γδ T-cell-mediated inflammation during acetaminophen-induced liver injury.

Acetaminophen (APAP)‐induced liver injury is closely associated with acute hepatic inflammation. Hypoxia‐inducible factor‐1 (HIF‐1) is activated during immunological processes and regulates gene expressions in various types of immune cells. Although HIF‐1 controls the differentiation and functions of conventional T cells in chronic inflammation, the pathological importance of HIF‐1 in innate‐like T cells during acute inflammation remains unknown. Here, we investigated the role of HIF‐1 in innate‐like γδ T cells during APAP‐induced acute liver injury. In response to APAP administration, T‐cell‐specific Hif‐1α gene knockout mice sustained severe liver damage compared to wild‐type control mice but without any impacts on the initial hepatic insult. This severe liver damage was accompanied by excessive neutrophil infiltration into the liver, increased serum interleukin (IL)‐17A levels, and increased hepatic expressions of C‐X‐C chemokine ligand (Cxcl) 1 and Cxcl2. Neutrophil depletion and IL‐17A neutralization completely abolished the aggravated phenotypes in T‐cell‐specific Hif‐1α gene knockout mice. Loss of the Hif‐1α gene enhanced the aberrant accumulation of IL‐17A‐producing innate‐like γδ T cells in the affected liver with no apparent effects on their IL‐17A‐producing ability. Adoptive transfer of Hif‐1α‐deficient splenic γδ T cells into recombination activating gene 2 (Rag2)‐deficient mice aggravated APAP‐induced liver injury with increased neutrophil accumulation in the liver compared to that of wild‐type γδ T cells. Furthermore, Hif‐1α‐deficient γδ T cells selectively showed aberrantly enhanced migratory ability. This ability was totally abolished by treatment with the mitochondrial adenosine triphosphate synthase inhibitor oligomycin. Conclusion: Deletion of Hif‐1α gene in T cells aggravates APAP‐induced acute inflammatory responses by enhancing aberrant innate‐like γδ T‐cell recruitment, thereby increasing excessive neutrophil infiltration into the liver. (Hepatology Communications 2018;2:571‐581)

A CTP Synthase Undergoing Stage-Specific Spatial Expression Is Essential for the Survival of the Intracellular Parasite Toxoplasma gondii.

Cytidine triphosphate synthase catalyzes the synthesis of cytidine 5′-triphosphate (CTP) from uridine 5′-triphosphate (UTP), the final step in the production of cytidine nucleotides. CTP synthases also form filamentous structures of different morphologies known as cytoophidia, whose functions in most organisms are unknown. Here, we identified and characterized a novel CTP synthase (TgCTPS) from Toxoplasma gondii. We show that TgCTPS is capable of substituting for its counterparts in the otherwise lethal double mutant (ura7Δ ura8Δ) of Saccharomyces cerevisiae. Equally, recombinant TgCTPS purified from Escherichia coli encodes for a functional protein in enzyme assays. The epitope-tagged TgCTPS under the control of its endogenous promoter displays a punctate cytosolic distribution, which undergoes spatial reorganization to form foci or filament-like structures when the parasite switches from a nutrient-replete (intracellular) to a nutrient-scarce (extracellular) condition. An analogous phenotype is observed upon nutrient stress or after treatment with a glutamine analog, 6-diazo-5-oxo-L-norleucine (DON). The exposure of parasites to DON disrupts the lytic cycle, and the TgCTPS is refractory to a genetic deletion, suggesting an essential requirement of this enzyme for T. gondii. Not least, this study, together with previous studies, supports that CTP synthase can serve as a potent drug target, because the parasite, unlike human host cells, cannot compensate for the lack of CTP synthase activity.

Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5′-triphosphate

While enzyme activity is often regulated by a combination of substrate/effector availability and quaternary structure, many cytosolic enzymes may be further regulated through oligomerization into filaments. Cytidine-5′-triphosphate (CTP) synthase (CTPS) forms such filaments—a process that is promoted by the product CTP. The CTP analog and active chemotherapeutic metabolite gemcitabine-5′-triphosphate (dF-dCTP) is a potent inhibitor of CTPS; however, its effect on the enzyme's ability to form filaments is unknown. Alongside electron microscopy studies, dynamic light scattering showed that dF-dCTP induces Escherichia coli CTPS (EcCTPS) to form filaments in solution with lengths ≥30 nm in the presence of CTP or dF-dCTP. The substrate UTP blocks formation of filaments and effects their disassembly. EcCTPS variants were constructed to investigate the role of CTP-binding determinants in CTP- and dF-dCTP-dependent filament formation. Substitution of Glu 149 (i.e., E149D), which interacts with the ribose of CTP, caused reduced affinity for both CTP and dF-dCTP, and obviated filament formation. Phe 227 appears to interact with CTP through an edge-on interaction with the cytosine ring, yet the F227A and F227L variants bound CTP and dF-dCTP. F227A EcCTPS did not form filaments, while F227L EcCTPS formed shorter filaments in the presence of CTP or dF-dCTP. Hence, Phe 227 plays a role in filament formation, although replacement by a bulky hydrophobic amino acid is sufficient for limited filament formation. That dF-dCTP can induce filament formation highlights the fact that nucleotide analogs employed as chemotherapeutic agents may affect the filamentous states of enzymes and potentially alter their regulation in vivo.

Production of a New Plant-Based Milk from Adenanthera pavonina Seed and Evaluation of Its Nutritional and Health Benefits.

A new plant milk was discovered from the seed of Adenanthera pavonina. The physicochemical and nutritional properties of the new pro-milk extract were assessed, and their biochemical effects were compared with those of soy bean extracts. Eleven groups of three albino rats each were used to assess the health benefits of the pro-milk. Groups were separately administered 3.1, 6.1, and 9.2 µl/g animal wt. pro-milk extract from A. pavonina seed, 6.1 µl/g animal wt. milk extract from soybean, and 6.1 µl/g animal wt. normal saline for 7 or 14 days. The “baseline” group consisted of those sacrificed on day 0. Among the physical properties considered, the pro-milk from A. pavonina had significantly higher (P < 0.05) hue color value and significantly lower (P < 0.05) L* than that from soy bean did. The pro-milk from A. pavonina had a significantly higher (P < 0.05) level of protein (36.14 ± 0.12%), Ca (440.99 ± 0.93 mg/l), Mg (96.69 ± 0.03 mg/l), K (190.41 ± 0.11 mg/l), Na (64.24 ± 0.24 mg/l), and Cu (0.55 ± 0.24 mg/l), and a significantly lower (P < 0.05) level of Mn (0.04 ± 0.01 mg/l) and vitamins A (undetectable), C (1.87 ± 0.01 mg/100 g), and E (0.12 ± 0.01 mg/100 g) compared to those of soy milk. The daily consumption of the pro-milk extract from A. pavonina for 14 days significantly reduced (P < 0.05) Ca2+-adenosine triphosphate synthase (Ca2+-ATPase) at low dose (3.1 µl/g animal wt.), but significantly increased (P < 0.05) Mg2+-ATPase at high dose (9.2 µl/g animal wt.). Daily administration of the A. pavonina extract for 14 days caused a significant reduction (P < 0.05) in acetylcholinesterase activity in the liver, intestine, heart, and kidney, suggesting that the pro-milk may facilitate ions transportation across the membrane. The pro-milk offers promising beneficial effects for patients with neurological diseases, as well as supporting general health owing to the high protein and mineral content. Vitamins fortification is recommended during production.

Supramolecular Assembly of Photosystem II and Adenosine Triphosphate Synthase in Artificially Designed Honeycomb Multilayers for Photophosphorylation.

Plant thylakoids have a typical stacking structure, which is the site of photosynthesis, including light-harvesting, water-splitting, and adenosine triphosphate (ATP) production. This stacking structure plays a key role in exchange of substances with extremely high efficiency and minimum energy consumption through photosynthesis. Herein we report an artificially designed honeycomb multilayer for photophosphorylation. To mimic the natural thylakoid stacking structure, the multilayered photosystem II (PSII)-ATP synthase-liposome system is fabricated via layer-by-layer (LbL) assembly, allowing the three-dimensional distributions of PSII and ATP synthase. Under light illumination, PSII splits water into protons and generates a proton gradient for ATP synthase to produce ATP. Moreover, it is found that the ATP production is extremely associated with the numbers of PSII layers. With such a multilayer structure assembled via LbL, one can better understand the mechanism of PSII and ATP synthase integrated in one system, mimicking the photosynthetic grana structure. On the other hand, such an assembled system can be considered to improve the photophosphorylation.

Bioinspired Assembly of Hierarchical Light‐Harvesting Architectures for Improved Photophosphorylation

AbstractMolecular assembly offers a bottom‐up way to construct biomimetic architectures with unique structures and properties. Although artificial photophosphorylation systems have long been developed, their microstructures have yet to achieve the sophisticated order and hierarchy of natural organisms. Herein, by utilizing principles in the natural plant leaves, it is shown that a biomimetic system with hierarchically ordered and compartmentalized structures, combining photosystem II (PSII) and adenosine triphosphate (ATP) synthase, can be obtained through template‐directed layer‐by‐layer assembly. Under light illumination, PSII in such a highly ordered light‐harvesting array, splits water to produce protons and electrons. Furthermore, a remarkable proton gradient is created across the covering ATP synthase‐reconstituted lipid membrane. As a consequence, highly efficient photophosphorylation is achieved. Outstandingly, the rate of ATP production in this hierarchical light‐harvesting architecture is enhanced 14 times, compared to that in the nature. This study paves a new way to assemble bioinspired systems with enhanced solar‐to‐chemical energy conversion efficiency.

Mitochondrial Dysfunction Causes Oxidative Stress and Tapetal Apoptosis in Chemical Hybridization Reagent-Induced Male Sterility in Wheat.

Male sterility in plants has been strongly linked to mitochondrial dysfunction. Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. Therefore, it is important to better understand the relationship between mitochondria and CHA-induced male sterility in wheat. This study reports on the impairment of mitochondrial function duo to CHA-SQ-1, which occurs by decreasing cytochrome oxidase and adenosine triphosphate synthase protein levels and theirs activities, respiratory rate, and in turn results in the inhibition of the mitochondrial electron transport chain (ETC), excessive production of reactive oxygen species (ROS) and disruption of the alternative oxidase pathway. Subsequently, excessive ROS combined with MnSOD defects results in damage to the mitochondrial membrane, followed by ROS release into the cytoplasm. The microspores underwent severe oxidative stress during pollen development. Furthermore, chronic oxidative stress, together with the overexpression of type II metacaspase, triggered premature tapetal apoptosis, which resulted in pollen abortion. Accordingly, we propose a metabolic pathway for mitochondrial-mediated male sterility in wheat, which provides information on the molecular events underlying CHA-SQ-1-induced abortion of anthers and may serve as an additional guide to the practical application of hybrid breeding.

Purification and Reconstitution of Ilyobacter tartaricus ATP Synthase.

F-type adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to power ATP synthesis by a unique rotary mechanism and can also operate in reverse, ATP hydrolysis, to generate ion gradients across membranes. The F1Fo-ATP synthases from bacteria consist of eight different structural subunits, forming a complex of ∼550kDa in size. In the bacterium Ilyobacter tartaricus the ATP synthase has the stoichiometry α3β3γδεab2c11. This chapter describes a wet-lab working protocol for the purification of several tens of milligrams of pure, heterologously (E. coli-)produced I. tartaricus Na+-driven F1Fo-ATP synthase and its subsequent efficient reconstitution into proteoliposomes. The methods are useful for a broad range of subsequent biochemical and biotechnological applications.

Atypical Evolution of the F1Fo Adenosine Triphosphate Synthase Regulatory ATP6 subunit in Glacier Ice Worms (Annelida: Clitellata: Mesenchytraeus).

The glacier ice worm, Mesenchytraeus solifugus, is among a few animals that reside permanently in glacier ice. Their adaptation to cold temperature has been linked to relatively high intracellular adenosine triphosphate (ATP) levels, which compensate for reductions in molecular motion at low physiological temperatures. Here, we show that ATP6—the critical regulatory subunit of the F1Fo-ATP synthase and primary target of mitochondrial disease—acquired an unprecedented histidine-rich, 18-amino acid carboxy-terminal extension, which counters the strong evolutionary trend of mitochondrial genome compaction. Furthermore, sequence analysis suggests that this insertion is not of metazoan origin, but rather is a product of horizontal gene transfer from a microbial dietary source, and may act as a proton shuttle to accelerate the rate of ATP synthesis.

Notes on The Energy Equivalence of Information.

Maxwell's demon makes observations and thereby collects information. As Brillouin points out such information is the negative of entropy (negentropy) and is the equivalent of a cost in energy. The energy cost of information can be quantified in the relationship E = kT ln 2, where k is the Boltzmann constant, T is the absolute temperature, and the factor ln 2 arises from the existence of two possibilities for a "yes/no" circumstance, as, for example, in the passage of a proton through a barrier controlled by a Maxwell's demon. This paper considers further conclusions that follow from the quantification of the energy cost of information. First, consideration of the minimum uncertainty in the measurement of energy cost of information leads to an expression for the uncertainty in the corresponding time of the measurement, which depends inversely upon temperature at which the measurements occur. Second, because of the universal connection between energy and mass, an almost imperceptible mass accompanies the accumulation of information. And third, to account for the total free energy change that describes the action of adenosine triphosphate (ATP) synthase, an additional term is suggested to be appended to the Mitchell chemiosmotic equation, which describes this process. The additional term accounts for the energy cost of sorting away from background ions those protons allowed to enter the ATP synthase.

Atomic model for the dimeric FO region of mitochondrial ATP synthase.

Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded FO region turns the rotor that drives ATP synthesis in the soluble F1 region. Although crystal structures of the F1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the FO region. Using cryo-electron microscopy, we determined the structure of the dimeric FO complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae.

A rare clinical presentation of neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) accompanied with spinal cord atrophy: A case report

Background: Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) is caused predominantly by m.8993T>G/C mutations in the mitochondrial adenosine triphosphate synthase subunit 6 gene (MT-ATP6). While absence of the signs of mitochondrial dysfunction in muscle fibers and presence of neurogenic changes due to neuropathy are well described as the characteristic features related to its weakness, spinal cord involvement has rarely been reported.