Nucleotide binding domain 1 pharmacophore modeling for visualization and analysis of P-glycoprotein–flavonoid molecular interactions

Background

P-glycoprotein (P-gp) is a 170-kDa membrane protein. It provides a barrier function and help to excrete toxins from the body as a transporter. Some bioflavonoids have been shown to block P-gp activity.

Objective

To evaluate the important amino acid residues within nucleotide binding domain 1 (NBD1) of P-gp that play a key role in molecular interactions with flavonoids using structure-based pharmacophore model.

Methods

In the molecular docking with NBD1 models, a putative binding site of flavonoids was proposed and compared with the site for ATP. The binding modes for ligands were achieved using LigandScout to generate the P-gp–flavonoid pharmacophore models.

Results

The binding pocket for flavonoids was investigated and found these inhibitors compete with the ATP for binding site in NBD1 including the NBD1 amino acid residues identified by the in silico techniques to be involved in the hydrogen bonding and van der Waals (hydrophobic) interactions with flavonoids.

Conclusion

These flavonoids occupy with the same binding site of ATP in NBD1 proffering that they may act as an ATP competitive inhibitor.

     

Glucose metabolism is inhibited by caspases upon the induction of apoptosis

Rapidly proliferating cells, such as cancer cells, have adopted aerobic glycolysis rather than oxidative phosphorylation to supply their energy demand; this phenomenon is known as ‘the Warburg effect''. It is now widely accepted that during apoptosis the loss of energy production, orchestrated by caspases, contributes to the dismantling of the dying cell. However, how this loss of energy production occurs is still only partially known. In the present work, we established that during apoptosis the level of cellular ATP decreased in a caspase-dependent manner. We demonstrated that this decrease in ATP content was independent of any caspase modification of glucose uptake, ATP consumption or reactive oxygen species production but was dependent on a caspase-dependent inhibition of glycolysis. We found that the activity of the two glycolysis-limiting enzymes, phosphofructokinase and pyruvate kinase, were affected by caspases, whereas the activity of phosphoglycerate kinase was not, suggesting specificity of the effect. Finally, using a metabolomic analysis, we observed that caspases led to a decrease in several key metabolites, including phosphoserine, which is a major regulator of pyruvate kinase muscle isozyme activity. Thus, we have established that during apoptosis, caspases can shut down the main energy production pathway in cancer cells, leading to the impairment in the activity of the two enzymes controlling limiting steps of glycolysis.The activation of caspase proteases is fundamental to apoptotic cell death. Once activated, ‘executioner'' caspases, such as caspase-3, orchestrate the rapid dismantling of the cell. Apoptosis is a process that requires energy. ATP is required for caspase activation, enzymatic hydrolysis of molecules, bleb formation and chromatin condensation.¹ However, in contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed ‘the Warburg effect''.² This phenomenon induces an increase of glucose consumption and provides the basis for the most sensitive and specific imaging technique available for the diagnosis and staging of solid cancers: positron emission tomography scan of 2-[¹⁸F] fluoro-2-deoxy-glucose uptake.Glycolysis is a series of metabolic processes, catalyzed by one of ten specific enzymes, by which 1 mole of glucose is catabolized to 2 moles of pyruvate and 2 moles of NADH with a net gain of 2 moles of ATP. Glycolysis is tightly regulated by the three allosteric enzymes, hexokinase (HK), phosphofructokinase-1 (PFK) and pyruvate kinase (PK), which catalyze the irreversible steps. HK, the first enzyme of glycolysis, phosphorylates glucose into glucose-6-phosphate, preventing the molecule from leaking out of the cell.The most complex control over glycolytic flux is attributed to PFK, which catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate using MgATP as the phosphoryl donor.³ PFK1 is stimulated by fructose-2,6-bisphosphate (F-2,6-BP), ADP/AMP and ammonium ions, whereas citrate and ATP act as strong inhibitors.Another limiting step is controlled by the final enzyme of the glycolytic pathway, PK. Four PK isoforms exist in mammals; the L and R isoforms are expressed in liver and red blood cells, respectively, whereas the M1 (muscle) isoform is expressed in most adult tissues, and tumor cells have been shown to mainly express the embryonic M2 isoform.⁴In the presence of oxygen, mitochondria can oxidize pyruvate and NADH, resulting in the production of 36 moles of ATP (OXPHOS). However, even under normoxic conditions, most cancer cells will not perform OXPHOS but will instead reduce pyruvate to lactate. Although, aerobic glycolysis is an inefficient way to generate ATP, aerobic glycolysis seems to confer certain advantages to cancer cells, such as the ability to generate several intermediates that can be used by other metabolic pathways to produce nucleotides or lipids.⁵ However, the exact nature of the benefits conferred by glycolysis is still under debate.It is well established that caspase activation relies on ATP to proceed. However, it has been previously suggested that upon induction of apoptosis, ATP levels dramatically fall in a caspase-dependent manner.⁶ In the present report, we explored the role of caspases on glycolysis, the main energy-producing pathway used by cancer cells.     

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO₃⁻/H⁺ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.Nitrate is among the major nitrogen sources for plants in aerobic soils. It is taken up by root cells through plasma membrane transporters of nitrate-nitrite transporter and peptide transporter families. Once in the cytoplasm it can enter the amino acid biosynthesis pathway (1) or be accumulated in the vacuolar lumen via tonoplast transporters (2).The vacuolar nitrate transporter of the model plant Arabidopsis thaliana, AtCLCa, has been shown to work as an anion/proton antiporter (3, 4), similarly to the bacterial CLCec-1 (5) and human hCLC-4 (6) as well as hCLC-5 (7). However, whereas bacterial and animal CLCs² transport chloride ions, the AtCLCa antiporter is more selective for nitrate, and therefore, it is able to mediate the accumulation of nitrate into the plant vacuole.Little is known on the modulation of CLC-proteins by nucleotides. The effects of ATP on the ion channel hCLC-1 are a matter of debate (8). Indeed, some reports have shown that ATP inhibits hCLC-1 currents, probably interacting with the C terminus of the protein (9–11). Conversely, other reports indicate that ATP does not modify the properties of hCLC-1 current (12). This discrepancy has been attributed to the oxidation state of the channel, as ATP would be effective only in the presence of reducing agents (13).The C terminus domain of all eukaryotic CLC proteins has two cystathionine β-synthetase motifs (CBS (14, 15)), each one characterized by a βαββα topology (16, 17). A structural and biochemical study of the hCLC-5 C-terminal part demonstrates that this region binds nucleotides (14). However, the effect of ATP binding on the transport activity of hCLC-5 is still unknown.The presence of analogous CBS domains in the C terminus of the AtCLCa antiporter suggested the hypothesis that ATP binds to this plant transporter and modulates its transport activity. Hence, we undertook a functional analysis of the effect of adenosine nucleotides on AtCLCa and found that ATP inhibits the AtCLCa-mediated transport. Based on a homology model of the C terminus of the channel, we identified two residues that would be putatively involved in the protein-nucleotide interaction.     

Regulation of the Ecto-Nucleotidase Pathway in Rat Hippocampal Nerve Terminals

Ecto-nucleotidases play a pivotal role in terminating the signalling via ATP and in producing adenosine, a neuromodulator in the nervous system. We have now investigated the pattern of adenosine formation with different concentrations of extracellular ATP in rat hippocampal nerve terminals. It was found that adenosine formation is delayed with increasing concentrations of ATP. Also, the rate of adenosine formation increased sharply when the extracellular concentrations of ATP + ADP decrease below 5 M, indicating that ATP/ADP feed-forwardly inhibit ecto-5-nucleotidase allowing a burst-like formation of adenosine possibly designed to activate facilitatory A_2A receptors. Initial rate measurements of ecto-5-nucleotidase in hippocampal nerve terminals, using IMP as substrate, showed that ATP and ADP are competitive inhibitors (apparent K_i of 14 and 4 M). In contrast, in hippocampal immunopurified cholinergic nerve terminals, a burst-like formation of adenosine is not apparent, suggesting that channelling processes may overcome the feed-forward inhibition of ecto-5-nucleotidase, thus favouring A₁ receptor activation.     

The reception of Eduard Buchner's discovery of cell-free fermentation

Conclusions What general conclusions can be drawn about the reception of zymase, its relation to the larger shift from a protoplasm to an enzyme theory of life, and its status as a social phenomenon?The most striking and to me unexpected pattern is the close correlation between attitude toward zymase and professional background. The disbelief of the fermentation technologists, Will, Delbrück, Wehmer, and even Stavenhagen, was as sharp and unanimous as the enthusiasm of the immunologists and enzymologists, Duclaux, Roux, Fernback, and Bertrand, and of Pfeffer, the experimental plant physiologist. Other skeptics—Voit, Kupffer, and Fischer—were conservatives in traditional fields. In all these cases it seems clear that professional commitments and outlook profoundly influenced the reception of zymase.In some cases there is a correlation with a specific earlier statement favoring the protoplasm theory or predicting a fermentation enzyme. Reynolds Green is a striking case of both. In 1893 he stated his belief that life processes in higher plants and fungi were identical, both being mediated by protoplasm.¹⁰⁹ Thus he claimed that digestion was not carried out by enzymes, as in higher animals, but by the whole protoplasm, and so too fermentation in yeast: All the metabolic processes must be carried out in the unicellular organism in the same mass of protoplasm.¹¹⁰ But Green also insisted that there was no essential difference between organized and unorganized ferments; enzymes were simply more highly differentiated and specialized forms of primitive protoplasm. He spoke of the alcoholic ferment (that is, enzyme), and of intracellular enzymes—very advanced beliefs for 1893. Thus it is understandable why Green saw no reason to doubt his first negative results with yeast juice, and rushed into print. But it is also understandable that he persisted and soon became an outspoken advocate of Buchner's view; like Wroblewski and Pfeffer, he had already accepted the idea of an intracellular fermentation enzyme.The physiological chemists also fit the pattern in a special way. Physiological chemistry is generally regarded as the source of biochemistry; indeed, many early biochemists were trained in physiological chemistry, and imbibed there a progressive interest in enzymes and metabolism. It is not surprising, therefore, that on the whole Neumeister, Madfadyen, Wroblewski, and Loew accepted cell-free fermentation. But what is surprising, and significant, is the diversity of opinion in this group, from Abeles' living protoplasm to Wroblewski's organized array of active proteins. The feelings of the physiological chemists were mixed, and there was enough of the old protoplasm view in Macfadyen's and even Wroblewski's views to allow Buchner to see them as attacks on zymase. The ideas of physiological chemistry were also changing; the idea of protoplasm was already adjusting to the new interest in enzymes in the early 1890's. But in contrast to immunochemistry, the change in physiological chemistry was gradual, uneven, undramatic, and relatively invisible. For the new biochemistry, the tradition of physiological chemistry was the source of traditional conservatism as well as of new ideas of enzymes.In general, then, I claim that it was not experimental facts that determined attitudes toward zymase so much as previous commitments, experience, and expectations. Initially, at least, zymase was less a determinant of opinion than a touchstone of pre-existing opinion. Like a prism, it revealed the spectrum of existing attitudes toward vital phenomena.Also relevant here is the fact that the experimental evidence was entirely ambiguous; the interpretation of Buchner's experiments with antiseptics or dried yeast depended entirely on how far one was willing to stretch the scope of the terms protoplasm and enzyme. Likewise, how important one judged the instability of zymase to be, or the low level of in vitro activity, depended largely on one's point of view. There was no crucial experiment, no certain proof. Logically zymase could be regarded as protoplasm. A. Fischer did so in 1900;¹¹¹ Macfadyen was still wavering in 1907;¹¹² and Beijerinck was holding out as late as 1916.¹¹³ There were undoubtedly others. Besides the special case of Reynolds Green and the technologists, there are no recorded cases of conversion.But if there was no evidence to convert a dedicated protoplasmist, there was plenty to encourage a dedicated belief in the enzyme theory, and that, I believe, is precisely what happened. The arguments of Buchner et al in support of zymase had the greatest effect not on the protoplasmists but on those who were already predisposed toward the biochemical view. The zymase debate enabled the emerging group of biochemists to recognizethemselves as a group with a community of out-look and objectives; it brought the issue of protoplasm vs. enzyme into the open and gave it a specific and concrete issue on which to turn. The debate sharpened the biochemists' awareness that their point of view was becoming the new mainstream of biochemical thought.All this of course had an important secondary effect on those who opposed the new view, or who were not immediately concerned with it. After the zymase debate, it would have been almost impossible to ignore the new ideas; the dramatic and wide publicity the debate enjoyed would have made it difficult not to take sides. Buchner's successful defense of zymase itself became an important influence on opinion; the new movement had to be looked at with respect. But the primary effect of the debate, I assert, was on those who were already inclined to the new view. The outcome was not a mass conversion of individuals, nor the ejection of one idea by another. The sides probably did not change much; the real change was in the way each side regarded itself and its place in history. The zymase debate may perhaps best be regarded as a process of selection. Buchner's party became aware of itself as the new progressive point of view, while those holding the traditional view could no longer expound it publicly without appearing obstinate or old-fashioned. Thus the way was clear for the gradually wider establishment of the new ideas, especially among the new generation of biochemists. In this specific sense, the acceptance of zymase was a social phenomenon.This way of looking at the zymase debate also illuminates its coincidence with the emergence of biochemistry as a profession. The new professional trappings and organizations were the social manifestation of the new self-awareness gained from the zymase controversy. One of the most important effects on those disposed toward the new view was the awareness of an intellectual community and of the need to give that community social forms. The almost mythical importance of Buchner's victory over the old protoplasm theory that later became current is readily understandable. It was a useful ideology for a group anxious to assert the novelty of their approach, a novelty which they in fact felt quite keenly.But if the primary effect of the zymase controversy was on those predisposed to accept zymase, then obviously the success of zymase depended on the fact that change had already begun to occur. Indeed, one of the most striking, and again unexpected, results of this study is the extent to which the importance of enzymes was already recognized in the early 1890's. The new immunology clearly pointed the way. Macfadyen and Hans Buchner were looking for active proteins inside the cell in 1893. Reynolds Green, Wroblewski, and Pfeffer had all anticipated the discovery of a fermentation enzyme. Other discoveries in enzymology in the 1890's pointed the same way.¹¹⁴ Moreover, it is clear from this study that the old protoplams theory was adapting to the new discoveries concerning the importance of enzymes. The sidechains of the old protoplasm molecule were simply reinterpreted as enzymes, as in Wroblewski's theory, for example, or in Paul Ehrlich's extremely influential sidechain theory of antibody formation (1897). Despite the later views of Hopkins et al., the old protoplasm and the new enzyme theories were not irreconcilable; a continuity of development had already begun when zymase came so dramatically on the scene. Buchner himself observed that the difference between the two views had become largely verbal; in retrospect we might say, not verbal, but social and historical. The gap between the continuous change in ideas and the contemporary feeling of sharply discontnuous change is a measure of the elusive but real social and historical implications of the zymase debate, of the demise of an old establishment, and the emergence of a new.In retrospect, then, it is clear that a trend was already under way in various quarters when zymase appeared. But it was still a collection of isolated and dispersed events. The zymase controversy made the trend visible and gave it a dramatic unity. The debate influenced those who had already begun to change. It was so effective because things had begun to change and because it coincided with the direction of that trend.     

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms. It is a key substrate and vital cofactor in many biochemical reactions and is thus conserved by all cells. However, in addition to its localization and functions inside cells, ATP is actively secreted to the extracellular matrix where it forms a halo around the external cell surface. The existence of this extracellular ATP (eATP)¹ has been reported in several organisms including bacteria (1), primitive eukaryotes (2), animals (3), and plants (4–6). This eATP is not wasted, but harnessed at the cell surface as a potent signaling molecule enabling cells to communicate with their neighbors and regulate crucial growth and developmental processes.In animals, eATP is a crucial signal molecule in several physiological processes such as neurotransmission (7, 8), regulation of blood pressure (9), enhanced production of reactive oxygen species (ROS) (10), protein translocation (11), and apoptosis (12). Extracellular ATP signal perception at the animal cell surface is mediated by P2X and P2Y receptors, which bind ATP extracellularly and recruit intracellular second messengers (13, 14). P2X receptors are ligand-gated ion channels that provide extracellular Ca²⁺ a corridor for cell entry after binding eATP, facilitating a surge in cytosolic [Ca²⁺] that is essential in activating down-stream signaling. P2Y receptors transduce the eATP signal by marshalling heteromeric G-proteins on the cytosolic face of the plasma membrane and activating appropriate downstream effectors.Although eATP exists in plants, homologous P2X/P2Y receptors for eATP signal perception have not yet been identified, even in plant species with fully sequenced genomes. Notwithstanding the obscurity of plant eATP signal sensors, some of the key downstream messengers recruited by eATP-mediated signaling are known. For example, eATP triggers a surge in cytosolic Ca²⁺ concentration (15–17) and a heightened production of nitric oxide (18–20) and reactive oxygen species (17, 21, 22). Altering eATP levels is attended by activation of plant gene expression (16, 21) and changes in protein abundance (5, 23), indicating that eATP-mediated signaling impacts on plant physiology. Indeed eATP has been demonstrated to regulate plant growth (20, 24–26), gravitropic responses (27), xenobiotic resistance (4), plant-symbiont interactions (28), and plant-pathogen interactions (23, 29). However, the mechanism by which eATP regulates these processes remains unclear, largely because the eATP signal sensors and downstream signal regulatory genes and proteins have not been identified.We previously reported that eATP plays a central regulatory role in plant cell death processes (5). Therefore, an understanding of the signaling components galvanized by eATP in cell death regulation might serve a useful purpose in providing mechanistic detail of how eATP signals in plant physiological processes. We found that eATP-mediated signaling negatively regulates cell death as its removal by application of ATP-degrading enzymes to the apoplast activates plant cell death (5). Remarkably, fumonisin B1 (FB1), a pathogen-derived molecule that activates defense gene expression in Arabidopsis (30), commandeers this eATP-regulated signaling to trigger programmed cell death (5). FB1 is a mycotoxin secreted by fungi in the genus Fusarium and initiates programmed cell death in both animal and plant cells (31, 32). In Arabidopsis, FB1 inaugurates cell death by inactivating eATP-mediated signaling via triggering a drastic collapse in the levels of eATP (5). FB1-induced Arabidopsis programmed cell death is dependent on the plant signaling hormone salicylic acid (33), which is a key regulator of eATP levels (29). Because concurrent application of FB1 and exogenous ATP to remedy the FB1-induced eATP deficit blocks death, FB1 and exogenous ATP treatments can therefore be used as probes to identify the key signal regulators downstream of eATP in cell death control. This is vital for achieving the global objective of elucidating the mechanism of eATP signaling in plant physiology.Gel-based proteomic analyses have been previously applied to successfully identify the novel role of eATP in the regulation of plant defense gene expression and disease resistance (23, 29). We have now employed FB1 and ATP treatments together with two-dimensional difference in-gel electrophoresis (DIGE) and matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS) to identify the changes in Arabidopsis protein profiles associated with a shift from normal to cell death-inception metabolism. Additional reverse genetic analyses enabled us to definitively identify a putative ATP synthase β-subunit as a target for eATP-mediated signaling with an unexpected function in the regulation of plant programmed cell death.     

Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the β subunit: regulation during proliferation and differentiation

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism. (Mol Cell Biochem 118: 49–60, 1992)Abbreviations CK Casein Kinase - TLCK N--p Tosyl Lysil-Chloromethylketone - SDS Sodium Dodecyl Sulphate - PAGE Polyacrylamide Gel Electrophoresis - R₃E₃TE₃ Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu - TCA Trichloroacetic Acid     

Metabolic interaction between purine nucleotide cycle and oxypurine cycle during skeletal muscle contraction of different intensities: a biochemical reappraisal

Background

A substrate cycle is a metabolic transformation in which a substrate A is phosphorylated to A?P at the expense of ATP (or another “high energy” compound), and A?P is converted back to A by a nucleotidase or a phosphatase. Many biochemists resisted the idea of such an ATP waste. Why a non-phosphorylated metabolite should be converted into a phosphorylated form, and converted back to its non-phosphorylated form through a “futile cycle”?

Aim of review

In this Review we aim at presenting our present knowledge on the biochemical features underlying the interrelation between the muscle purine nucleotide cycle and the oxypurine cycle, and on the metabolic responses of the two cycles to increasing intensities of muscle contraction.

Key scientific concepts of review

Nowadays it is widely accepted that the substrate cycles regulate many vital functions depending on the expense of large amounts of ATP, including skeletal muscle contraction, so that the expense of some extra ATP and “high energy” compounds, such as GTP and PRPP via substrate cycles, is not surprising. The Review emphasizes the strict metabolic interrelationship between the purine nucleotide cycle and the oxipurine cycle.

     

Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5′-phosphate-dependent exonuclease in <Emphasis Type="Italic">Candida albicans</Emphasis> and other yeasts

Background

Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5´-phosphate-dependent, 5´→3´ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5´-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell.

Results

We have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior.

Conclusions

Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.

     

Concept of virus species

The species concept is applicable in virology because viruses have genomes, replicate, evolve, and occupy particular ecological niches. The following definition of virus species was accepted in 1991 by the International Committee on Taxonomy of Viruses: A virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche. This definition does not provide a list of diagnostic properties for recognizing members of particular virus species. Furthermore, since a virus species is a polythetic class, it is impossible to use a single property such as a certain level of genome homology as defining property of the species. The implications of this new definition of virus species for future virus classification are discussed.     

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase

Background

There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.

Methodology/Principal Findings

Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.

Conclusions/Significance

Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.     

The release of adenosine at the electric organ ofTorpedo. A study using a continuous chemiluminescent method

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ ofTorpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H₂O₂ by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction ofTorpedo electric organ. The presence of , -methylene ADP, an inhibitor of 5-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.     

Regulation of Proton Flow and ATP Synthesis in Chloroplasts

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF₁ play key roles in both regulation of activity and proton translocation.     

Characterization of the molecular form of cardiac phospholamban

The native form of phospholamban is not known and it is presently under debate whether this protein exists as a monomer or an oligomer in cardiac sarcoplasmic reticulum. The currently accepted model for phospholamban is pentameric, based primarily on its behavior in SDS-polyacrylamide gel electrophoresis. In this study, sucrose density gradient centrifugation and gel filtration chromatography were used to determine the form of phospholamban under nondenaturing conditions. Purified phospholamban or phospholamban present in solubilized cardiac sarcoplasmic reticulum was centrifuged through 5–20% sucrose density gradients in the absence or presence ofn-octylgucoside. The sucrose density gradient fractions were assayed for acid precipitable³²P-incorporation in the presence of [-³²P]ATP and cAMP-dependent protein kinase catalytic subunit.³²P-containing peak fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis, using a phospholamban-polyclonal antibody, to confirm the presence of phospholamban. Purified phosphoblamban migrated with an apparent molecular weight of 25,000 daltons in the sucrose gradients in either the absence or presence of detergent. Phospholamban present in solubilized cardiac sarcoplasmic reticulum migrated with a similar apparent molecular weight when detergent was included in the sucrose gradients. In addition, solubilized cardiac sarcoplasmic reticulum was subjected to gel filtration chromatography in the presence of deoxycholate. Under these conditions phospholamban migrated with an apparent molecular weight of 24,500 daltons. These data suggest that phospholamban prefers an oligomeric assembly and this may be the form present in cardiac sarcoplasmic reticulum membranes.     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis.Faithful segregation of low copy number plasmids in bacteria depends on partition loci, named Par. Such loci are composed of two genes, generically termed parA and parB, encoding an ATPase and a DNA-binding protein, respectively, and a cis-acting centromeric site parS (reviewed in Ref. 1). These three essential elements are sufficient for the partition process. ParBs assemble on parS to form nucleoprotein structures called partition complexes (2–6). ParA ATPases are considered to be motors that direct displacement and positioning of partition complexes inside the cell.Partition systems have been classified into two major types, distinguished by the nature of their ATPase proteins (7). Type I is characterized by Walker box ATPases, which are specified by many plasmids and most bacterial chromosomes. In some (Type Ia) the nucleotide-binding P-loop is preceded by an N-terminal regulatory domain, in the others (Type Ib) it is not. Type II specifies actin-like ATPases and is present on relatively few plasmids. It is presently the best understood system at the molecular level (8–10). However, the underlying mechanism that drives partition still remains elusive for both systems. Our work aims at the understanding of an archetypal representative of Type Ia, namely SopABC of the Escherichia coli plasmid F.The several activities of Type Ia ParA proteins are regulated by binding of adenine nucleotides (11, 12), which induce conformational changes in the proteins (13, 14). In their apo and/or ADP-bound forms these proteins display site-specific DNA binding activity, recognizing their cognate promoters through their N-terminal domains. Such activity is involved in the autoregulation of par operon expression (15, 16). In the ATP-bound form, they specifically interact with cognate partition complexes through contact with ParB proteins. The ATP-bound form of type I ParAs spontaneously forms polymers, which appear as bundled filaments in electron micrographs (12, 17–19). The role of these filaments is not understood but they could be related to the rapid movement of partition complexes in the cell. In vivo, ParA proteins form dynamic assemblies that move back and forth in the cell if the cognate ParB protein and parS centromere are present (20–23). The link between this oscillatory behavior and the segregation of partition complexes is not clear. They both require the ATPase activity of ParA proteins but the role of ATP hydrolysis in the partition process is not understood.It has long been known that ParA partition proteins exhibit low intrinsic ATPase activity (24, 25). ATP hydrolysis is modestly stimulated by either DNA or the cognate ParB alone but is strongly activated (up to 35-fold) when both DNA and ParBs are present (12, 24, 25). The lack of major stimulation of ATPase by DNA in the absence of ParB proteins has been taken to mean that the DNA-bound form of ParB is the effective activator (26). However, incorporation of centromere sites in the DNA added to ParB did not increase stimulation of ATPase (24, 25), leaving doubts as to the role of the partition complex in ATPase activation.The mechanism by which ATP hydrolysis acts in the partition process is not known for type I systems. This is in marked contrast to actin-based partition ATPases whose ATPase activity is stimulated in growing filaments (8), where it provokes the rapid disassembly of filaments unless these are capped by the cognate partition complex (9). Therefore, for the type II partition system, ATP hydrolysis ensures discrimination between unproductive filaments that are rapidly disassembled and productive filaments that drive partition complexes to opposite ends of the cell. This dynamic instability, which ensures elongation of actin-like filaments only between two partition complexes to be segregated, thus provides regulation of the partition process.Recently, it has been shown that two members of the type I ParA family, Soj of Thermus thermophilus and SopA of plasmid F, bind nonspecific DNA in the presence of ATP (12, 26). Two studies revealed that this DNA binding activity is essential for partition (27, 28). Importantly, it has been shown that a SopA mutant deficient in DNA binding no longer stimulates ATP hydrolysis efficiently, suggesting that DNA could play a direct role in the regulation of the ATPase activity (28). This finding raises the issue of the interactions required for activation of the type I partition ATPase activity by cognate proteins and DNA.In this study, we have investigated the mechanism of activation of ATP hydrolysis by SopA. First, we have found that the formation of the F partition complex is required for strong stimulation of the SopA intrinsic ATPase activity. We have also found that the partition complex and DNA stimulate ATP hydrolysis independently but that these two independent interactions act in synergy to amplify SopA ATPase activity. Lastly, we have identified an arginine finger motif in SopB responsible for the stimulation of SopA ATPase activity.