Physiological and Molecular Responses in the Gill of the Swimming Crab Portunus trituberculatus During Long-Term Ammonia Stress

Ammonia is a common environmental stressor encountered during aquaculture, and is a significant concern due to its adverse biological effects on vertebrate and invertebrate including crustaceans. However, little information is available on physiological and molecular responses in crustaceans under long-term ammonia exposure, which often occurs in aquaculture practices. Here, we investigated temporal physiological and molecular responses in the gills, the main ammonia excretion organ, of the swimming crab Portunus trituberculatus following long-term (4 weeks) exposure to three different ammonia nitrogen concentrations (2, 4, and 8 mg l–1), in comparison to seawater (ammonia nitrogen below 0.03 mg l–1). The results revealed that after ammonia stress, the ammonia excretion and detoxification pathways were initially up-regulated. These processes appear compromised as the exposure duration extended, leading to accumulation of hemolymph ammonia, which coincided with the reduction of adenosine 5′-triphosphate (ATP) and adenylate energy charge (AEC). Considering that ammonia excretion and detoxification are highly energy-consuming, the depression of these pathways are, at least partly, associated with disruption of energy homeostasis in gills after prolonged ammonia exposure. Furthermore, our results indicated that long-term ammonia exposure can impair the antioxidant defense and result in increased lipid peroxidation, as well as induce endoplasmic reticulum stress, which in turn lead to apoptosis through p53-bax pathway in gills of the swimming crab. The findings of the present study further our understanding of adverse effects and underlying mechanisms of long-term ammonia in decapods, and provide valuable information for aquaculture management of P. trituberculatus.

Spatial Probabilistic Mapping of Metabolite Ensembles in Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) vows to enable simultaneous spatially-resolved investigation of hundreds of metabolites in tissue sections, but it still relies on poorly defined ion images for data interpretation. Here, we outline moleculaR, a computational framework in R, that introduces systematic probabilistic mapping and point-for-point statistical testing of metabolites in tissue to MSI. Beyond statistics, moleculaR allows for arithmetic operations within the same MS image and thereby, for instance, analysis and visualization of complex scores like the adenylate energy charge ([ATP]+0.5*[ADP])/ ([ATP]+[ADP]+[AMP]). moleculaR also enables collective molecular projections, for example of all potassium versus all sodium adducts for spatially-resolved investigation of ion milieus, or for surveys of lipid pathways or other user-defined biomolecular ensembles.

Regulatory adenylnucleotide-mediated binding of the PII-like protein SbtB to the cyanobacterial bicarbonate transporter SbtA is controlled by the cellular energy state

ABSTRACTCyanobacteria have evolved one of the most powerful CO2 concentrating mechanisms (CCM), supporting high photosynthetic rates with limiting inorganic carbon (Ci), which makes their CCM a desirable system for integration into higher plant chloroplasts to enhance photosynthetic yield. The CCM is driven by active Ci uptake, facilitated by bicarbonate transporters and CO2 pumps, which locally elevates the CO2 concentration and carboxylation rate of the primary CO2 fixing enzyme, Rubisco, inside cytoplasmic micro-compartments (carboxysomes). Ci uptake responds allosterically to Ci supply and light, but the molecular signals and regulators of protein function are unknowns. Functional analyses of sodium-dependent bicarbonate transporters classified as SbtA in E. coli support the hypothesis that SbtA activity is negatively regulated through association with its cognate PII-like SbtB protein. Here, we demonstrate that the association of SbtA with SbtB from two phylogenetically distant species, Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942, depends on the relative amounts of ATP or cAMP compared to ADP or AMP. Higher ATP over ADP or AMP ratios decreased the formation of SbtA:SbtB complexes, consistent with a sensory response to the cellular adenylate energy charge (AEC=[ATP + 0.5 ADP]/[ATP+ADP+AMP]) and the different binding affinities of these adenylates to SbtB protein trimers. Based on evidence for adenylate ligand-specific conformation changes for the SbtB protein trimer of Cyanobium sp. PCC7001, we propose a role for SbtB as a curfew protein locking SbtA into an inactive state as safe-guard against energetically futile and physiologically disadvantageous activation during prolonged low cellular AEC and photosynthetically unfavourable conditions.

Determination of Adenylate Nucleotides in Amphipod Gammarus fossarum by Ion-Pair Reverse Phase Liquid Chromatography: Possibilities of Positive Pressure Micro-Solid Phase Extraction

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a pH around 11. This method enables separation and accurate detection of adenylates. Our study provides new insight into the complexity of adenylate extraction and quantification that is crucial for the application of adenylate energy charge as a confident physiological measure of environmental stress and as a health index of G. fossarum.

Changes in the Concentration of Purine and Pyridine as a Response to Single Whole-Body Cryostimulation

To our knowledge, this is the first study in which we provide evidence that a single whole-body cryostimulation treatment leads to changes associated with erythrocyte energy metabolism. These changes are beneficial from the point of view of cellular bioenergetics, because they are associated with an increase in ATP concentration and erythrocyte energy potential expressed by an increase in the ATP/ADP and ATP/AMP ratios and the value of adenylate energy charge (AEC). In addition, as affected by cryogenic temperatures, there is a decrease in the concentration of purine catabolism products, i.e., inosine and hypoxanthine in the blood.

Cytosolic 5′-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator

Cytosolic 5′-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

Enzyme activities predicted by metabolite concentrations and solvent capacity in the cell

Experimental measurements or computational model predictions of the post-translational regulation of enzymes needed in a metabolic pathway is a difficult problem. Consequently, regulation is mostly known only for well-studied reactions of central metabolism in various model organisms. In this study, we use two approaches to predict enzyme regulation policies and investigate the hypothesis that regulation is driven by the need to maintain the solvent capacity in the cell. The first predictive method uses a statistical thermodynamics and metabolic control theory framework while the second method is performed using a hybrid optimization–reinforcement learning approach. Efficient regulation schemes were learned from experimental data that either agree with theoretical calculations or result in a higher cell fitness using maximum useful work as a metric. As previously hypothesized, regulation is herein shown to control the concentrations of both immediate and downstream product concentrations at physiological levels. Model predictions provide the following two novel general principles: (1) the regulation itself causes the reactions to be much further from equilibrium instead of the common assumption that highly non-equilibrium reactions are the targets for regulation; and (2) the minimal regulation needed to maintain metabolite levels at physiological concentrations maximizes the free energy dissipation rate instead of preserving a specific energy charge. The resulting energy dissipation rate is an emergent property of regulation which may be represented by a high value of the adenylate energy charge. In addition, the predictions demonstrate that the amount of regulation needed can be minimized if it is applied at the beginning or branch point of a pathway, in agreement with common notions. The approach is demonstrated for three pathways in the central metabolism of E. coli (gluconeogenesis, glycolysis-tricarboxylic acid (TCA) and pentose phosphate-TCA) that each require different regulation schemes. It is shown quantitatively that hexokinase, glucose 6-phosphate dehydrogenase and glyceraldehyde phosphate dehydrogenase, all branch points of pathways, play the largest roles in regulating central metabolism.

Adenylate kinase 1 deficiency disrupts mouse sperm motility under conditions of energy stress†

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

Modulation of adenine phosphoribosyltransferase-mediated salvage to promote diabetic wound healing

Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. The up-regulated APRT found in wound skin correlated with the demands of repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. LC-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. These data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. Our study provided an explanation for the up- regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation.

Machine Learning and Optimal Control of Enzyme Activities to Preserve Solvent Capacity in the Cell

ABSTRACTExperimental measurements or computational model predictions of the post-translational regulation of enzymes needed in a metabolic pathway is a difficult problem. Consequently, regulation is mostly known only for well-studied reactions of central metabolism in various model organisms. In this study, we utilize two approaches to predict enzyme regulation policies and investigate the hypothesis that regulation is driven by the need to maintain the solvent capacity in the cell. The first predictive method uses a statistical thermodynamics and metabolic control theory framework while the second method is performed using a hybrid optimization-reinforcement learning approach. Efficient regulation schemes were learned from experimental data that either agree with theoretical calculations or result in a higher cell fitness using maximum useful work as a metric. Model predictions provide the following novel general principles: (1) the regulation itself causes the reactions to be much further from equilibrium instead of the common assumption that highly non-equilibrium reactions are the targets for regulation; (2) regulation is used to maintain the concentrations of both immediate and downstream product concentrations rather than to maintain a specific energy charge; and (3) the minimal regulation needed to maintain metabolite levels at physiological concentrations also results in the maximal energy production rate that can be obtained at physiological conditions. The resulting energy production rate is an emergent property of regulation which may be represented by a high value of the adenylate energy charge. In addition, the predictions demonstrate that the amount of regulation needed can be minimized if it is applied at the beginning or branch point of a pathway, in agreement with common notions. The approach is demonstrated for three pathways in the central metabolism of E. coli (gluconeogenesis, glycolysis-TCA and Pentose Phosphate-TCA) that each require different regulation schemes. It is shown quantitatively that hexokinase, glucose 6-phosphate dehydrogenase and glyceraldehyde phosphate dehydrogenase, all branch points of pathways, play the largest roles in regulating central metabolism.