Cytosolic ATP Research Articles

Molecular dynamics simulations on apo ADP/ATP carrier shed new lights on the featured motif of the mitochondrial carriers

The ADP/ATP carrier (AAC) is a transporter responsible for the equal molar exchange of cytosolic ADP and ATP synthesized within mitochondrial matrix across the mitochondrial membrane. Its primary structure consists of three homologous repeats, and each repeat contains a conserved motif that is shared by all members of the mitochondrial carrier family (MCF). Although these MCF motif residues cluster together in the crystal structure of AAC, detailed analyses on the interactions among the motif residues are still limited. In the present study, all-atom molecular dynamics (MD) simulations of up to 10 μs have been carried out on AAC, and interactions and structural dynamics of the MCF motif residues have been specifically investigated. Our simulations have revealed: i) a very asymmetrical electrostatic network at the bottom of the pocket of apo AAC, ii) the asymmetrical interactions between the Pro kink region and the [YWF][KR] G motif in three repeats, iii) the role of the conserved Arg residues in stabilizing the C-ends of the odd-numbered helices, iv) the structural change of the [YWF][KR] G motif and its potential involvement in substrate translocation process. Our results highlight the asymmetry of the MCF residues in the three repeats, which might contribute to the ability of the carriers to transport the asymmetrical substrates. Our observations provide microscopic basis for further research on the translocation mechanism of mitochondrial carriers.

P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a variety of external stimuli such as gating of the P2X7 receptor. We used Yac-1 murine lymphoma cells to study P2X7-mediated ATP release. These cells co-express P2X7 and ADP-ribosyltransferase ARTC2, permitting gating of P2X7 by NAD+-dependent ADP-ribosylation without the need to add exogenous ATP. Yac-1 cells released ATP into the extracellular space within minutes after stimulation with NAD+. This was blocked by pre-incubation with the inhibitory P2X7-specific nanobody 13A7. Gating of P2X7 for 3 h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20 h. P2X7-mediated ATP release was dependent on a rise in cytosolic calcium and the depletion of intracellular potassium, but was not blocked by inhibitors of pannexins or connexins. We used genetically encoded FRET-based ATP sensors targeted to the cytosol to image P2X7-mediated changes in the distribution of ATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD+, we observed a marked depletion of ATP in the cytosol. This study demonstrates the potential of ATP sensors as tools to study regulated ATP release by other cell types under other conditions.

SAT-LB057 High-Fructose Corn Syrup Enhances Intestinal Tumor Growth in Mice

An increasing number of studies have suggested a potential link between sugar consumption and cancer. However, a direct relationship between the two remains controversial. Here, we treated APC-mutant mice daily with a modest amount of high-fructose corn syrup (HFCS) via oral gavage to mimic sugar-sweetened beverage (SSB) consumption and evaluated the effects of SSBs on intestinal tumorigenesis. We observed a dramatic increase in tumor size and tumor grade in HFCS-treated mice despite no signs of obesity or metabolic syndrome. A bolus of HFCS spikes the level of fructose and glucose in the intestinal lumen and serum, respectively, allowing tumors to take up both sugars efficiently. Ketohexokinase (KHK) in tumors rapidly converts fructose to fructose-1-phosphate (F1P), lowering cytosolic ATP in the process. Consequently, this leads to the activation of glycolysis as ATP depletion relieves the allosteric inhibition of phosphofructokinase (PFK), the most critical regulatory enzyme in glycolysis. This accelerated glycolysis contributes to the increased de novo lipogenesis pathway that enhances tumor growth in HFCS-treated mice. Importantly, the deletion of KHK or fatty acid synthase (FASN) in APC-mutant mice abolishes the HFCS-induced metabolic changes and tumor growth. Overall, our results provide a mechanistic rationale for restricting SSB consumption to decrease the rate of growth of early stage colorectal cancers. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Uptake and toxicity studies of magnetic TiO2-Based nanophotocatalyst in Arabidopsis thaliana

Information on the environmental impact of magnetic TiO2-based nanophotocatalysts is scarce. This study evaluated the potential effects of an innovative magnetic nanophotocatalyst N-TiO2/Fe3O4@SiO2 (NTFS) on plants using Arabidopsis thaliana grown in a hydroponic system. NTFS was detected in the vascular tissues and mesophyll of plants, thus confirming the uptake and upwards transport of NTFS from roots to leaves. Fourier transform infrared spectroscopy was applied to determine compositional and structural alterations in plant tissues exposed to NTFS, or its two main components (N-TiO2 and Fe3O4@SiO2), at concentrations ranging from 0 to 1000 mg/L, but no changes were detected in the lipids, pectins, proteins, cellulose, hemicellulose, and carbohydrates. The morphology and biomass of the plants were not affected by the NTFS or its components either. Biosensors for inorganic phosphate (Pi) and MgATP2− were used to monitor the in vivo Pi and MgATP2− levels in the plant cells. The results showed that NTFS and its components did not induce any adverse effects on the cytosolic Pi level or ATP synthesis, indicating the energy physiology of Arabidopsis was unaffected. In general, NTFS has inconsequential toxic effects on Arabidopsis, but can be taken up by plants, enter the food chain, and cause potential exposure and bioaccumulation in animals and human beings.

3,5-diiodo-L-thyronine increases de novo lipogenesis in liver from hypothyroid rats by SREBP-1 and ChREBP-mediated transcriptional mechanisms.

Hepatic de novo lipogenesis (DNL), the process by which carbohydrates are converted into lipids, is strictly controlled by nutritional and hormonal status. 3,5-Diiodo-L-thyronine (T2), a product of the 3,5,3'-triiodo-L-thyronine (T3) peripheral metabolism, has been shown to mimic some T3 effects on lipid metabolism by a short-term mechanism independent of protein synthesis. Here, we report that T2, administered for 1 week to hypothyroid rats, increases total fatty acid synthesis from acetate in isolated hepatocytes. Studies carried out on liver subcellular fractions demonstrated that T2 not only increases the activity and the expression of acetyl-CoA carboxylase and fatty acid synthase but also of other proteins linked to DNL such as the mitochondrial citrate carrier and the cytosolic ATP citrate lyase. Parallelly, T2 stimulates the activities of enzymes supplying cytosolic NADPH needed for the reductive steps of DNL. With respect to both euthyroid and hypothyroid rats, T2 administration decreases the hepatic mRNA level of SREBP-1, a transcription factor which represents a master regulator of DNL. However, when compared to hypothyroid rats T2 significantly increases, without bringing to the euthyroid value, the content of both mature (nSREBP-1), and precursor (pSREBP-1) forms of the SREBP-1 protein as well as their ratio. Moreover, T2 administration strongly augmented the nuclear content of ChREBP, another crucial transcription factor involved in the regulation of lipogenic genes. Based on these results, we can conclude that in the liver of hypothyroid rats the transcriptional activation by T2 of DNL genes could depend, at least in part, on SREBP-1- and ChREBP-dependent mechanisms. © 2019 IUBMB Life, 2019.

Pioglitazone reversed the fructose-programmed astrocytic glycolysis and oxidative phosphorylation of female rat offspring.

Excessive maternal high-fructose diet (HFD) during pregnancy and lactation has been reported to cause metabolic disorders in the offspring. Whether the infant's brain metabolism is disturbed by maternal HFD is largely unknown. Brain energy metabolism is elevated dramatically during fetal and postnatal development, whereby maternal nutrition is a key factor that determines cellular metabolism. Astrocytes, a nonneuronal cell type in the brain, are considered to support the high-energy demands of neurons by supplying lactate. In this study, the effects of maternal HFD on astrocytic glucose metabolism were investigated using hippocampal primary cultures of female infants. We found that glycolytic capacity and mitochondrial respiration and electron transport chain were suppressed by maternal HFD. Mitochondrial DNA copy number and mitochondrial transcription factor A expression were suppressed by maternal HFD. Western blots and immunofluorescent images further indicated that the glucose transporter 1 was downregulated whereas the insulin receptor-α, phospho-insulin receptor substrate-1 (Y612) and the p85 subunit of phosphatidylinositide 3-kinase were upregulated in the HFD group. Pioglitazone, which is known to increase astrocytic glucose metabolism, effectively reversed the suppressed glycolysis, and lactate release was restored. Moreover, pioglitazone also normalized oxidative phosphorylation with an increase of cytosolic ATP. Together, these results suggest that maternal HFD impairs astrocytic energy metabolic pathways that were reversed by pioglitazone.

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

In pancreatic β-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in β-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

Non-Canonical Control of Neuronal Energy Status by the Na+ Pump

Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors invitro and invivo. A short theta burst triggered immediate Na+ entry, followed by a delayed stimulation of the Na+/K+ ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na+ pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na+ pump stimulation. Experiments revealed that the Na+ pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca2+, but by a mechanism commanded by the Na+ pump.

A Unifying Hypothesis Linking Hepatic Adaptations for Ethanol Metabolism to the Proinflammatory and Profibrotic Events of Alcoholic Liver Disease.

The pathogenesis of alcoholic liver disease (ALD) remains poorly understood but is likely a multihit pathophysiological process. Here, we propose a hypothesis of how early mitochondrial adaptations for alcohol metabolism lead to ALD pathogenesis. Acutely, ethanol (EtOH) feeding causes a near doubling of hepatic EtOH metabolism and oxygen consumption within 2 to 3hours. This swift increase in alcohol metabolism (SIAM) is an adaptive response to hasten metabolic elimination of both EtOH and its more toxic metabolite, acetaldehyde (AcAld). In association with SIAM, EtOH causes widespread hepatic mitochondrial depolarization (mtDepo), which stimulates oxygen consumption. In parallel, voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane close. Together, VDAC closure and respiratory stimulation promote selective and more rapid oxidation of EtOH first to AcAld in the cytosol and then to nontoxic acetate in mitochondria, since membrane-permeant AcAld does not require VDAC to enter mitochondria. VDAC closure also inhibits mitochondrial fatty acid oxidation and ATP release, promoting steatosis and a decrease in cytosolic ATP. After acute EtOH, these changes revert as EtOH is eliminated with little hepatocellular cytolethality. mtDepo also stimulates mitochondrial autophagy (mitophagy). After chronic high EtOH exposure, the capacity to process depolarized mitochondria by mitophagy becomes compromised, leading to intra- and extracellular release of damaged mitochondria, mitophagosomes, and/or autolysosomes containing mitochondrial damage-associated molecular pattern (mtDAMP) molecules. mtDAMPs cause inflammasome activation and promote inflammatory and profibrogenic responses, causing hepatitis and fibrosis. We propose that persistence of mitochondrial responses to EtOH metabolism becomes a tipping point, which links initial adaptive EtOH metabolism to maladaptive changes initiating onset and progression of ALD.

Dysfunction of SERCA pumps as novel mechanism of methylglyoxal cytotoxicity

A novel pathway of methylglyoxal (MGX)-induced apoptosis via sarcoplasmic reticulum Ca2+-ATPase (SERCA) is presented. Interaction of SERCA1 with MGX was investigated by molecular docking and experimentally in a cell-free system. MGX concentration- and time-dependently decreased SERCA1 activity. A significant increase of sarcoplasmic reticulum (SR) carbonylation was found in the concentration range of 1–10 mM caused by MGX and a decrease of thiol groups at the concentrations of 5 and 40 mM. Affinities of SERCA1 to ATP and Ca2+ were not influenced by MGX, however decreases of Vmax related to both binding sites were observed. Molecular docking indicated binding of MGX at the cytosolic region of SERCA1, inducing conformational changes in the cytosolic-transmembrane interface. This interaction resulted in conformational changes in the cytosolic region (FITC fluorescence decrease) as well as in the transmembrane region of SERCA1 (Trp fluorescence decrease) without direct binding to the cytosolic ATP or transmembrane Ca2+ binding sites.Regarding the MGX inhibitory effect in a cell-free system and similarities of SERCA1 to its other isoforms, proapoptotic properties of MGX may be expected in cellular systems. At cellular level, MGX induced a decrease of SERCA2b expression in the pancreatic INS-1E β-cell line. This was accompanied by cell viability decrease, increase in apoptosis, impaired insulin secretion and elevation of basal intracellular Ca2+ levels. Decreased expression of SERCA2b may contribute to induction of apoptosis of pancreatic β-cells.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect

Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Cytosolic ATP Relieves Voltage-Dependent Inactivation of T-Type Calcium Channels and Facilitates Excitability of Neurons in the Rat Central Medial Thalamus

The central medial nucleus (CeM) is a part of the intralaminar thalamus, which is involved in the control of arousal and sensory processing. However, ionic conductances and mechanisms that regulate the activity of the CeM are not well studied. Here, we used in vitro electrophysiology in acute brain slices from adolescent rats to demonstrate that T-type calcium currents (T-currents) are prominent in the majority of the studied CeM neurons and are critical determinants of low-threshold calcium spikes (LTSs), which in turn regulate excitability of these neurons. Using an ATP-free internal solution decreased T-current density and induced a profound hyperpolarizing shift in steady-state inactivation curves while voltage-dependent activation kinetics were spared. Furthermore, selective pharmacological blockade of T-channels or use of an ATP-free solution reduced both tonic action potential (AP) frequency and rebound burst firing in CeM neurons. Our results indicate that T-channels are critical regulators of a thalamocortical circuit output and suggest that cytosolic ATP could be an endogenous regulatory mechanism in which T-channels may functionally gate sensory transmission and arousal in vivo.

The Vacuolar ATPase - A Nano-scale Motor That Drives Cell Biology.

The vacuolar H+-ATPase (V-ATPase) is a ~1MDa membrane protein complex that couples the hydrolysis of cytosolic ATP to the transmembrane movement of protons. In essentially all eukaryotic cells, this acid pumping function plays critical roles in the acidification of endosomal/lysosomal compartments and hence in transport, recycling and degradative pathways. It is also important in acid extrusion across the plasma membrane of some cells, contributing to homeostatic control of cytoplasmic pH and maintenance of appropriate extracellular acidity. The complex, assembled from up to 30 individual polypeptides, operates as a molecular motor with rotary mechanics. Historically, structural inferences about the eukaryotic V-ATPase and its subunits have been made by comparison to the structures of bacterial homologues. However, more recently, we have developed a much better understanding of the complete structure of the eukaryotic complex, in particular through advances in cryo-electron microscopy. This chapter explores these recent developments, and examines what they now reveal about the catalytic mechanism of this essential proton pump and how its activity might be regulated in response to cellular signals.

Patterns of Microvascular Response to Refractory Shock and Their Modulation by Vasoactive Resuscitations

Objective: Progressive hemorrhagic shock (HS) causes splanchnic vasoconstriction and hypo-perfusion together with profound depletions of cellular cytosolic energy phosphates (ATP). Cellular energy failure and the splanchnic hypo-perfusion are critical to the pathogenesis of shock decompensation and the subsequent death from cardiocirculatory arrest. We recently demonstrated post-resuscitation survival benefit for cellular cytosolic ATP replenishment but not for vasopressors use in a rat model of refractory HS. The aim of this study was to determine the effects of progressive HS on the splanchnic microvasculature, and to compare the effects of adjuvant resuscitations with either norepinephrine, vasopressin or lipid vesicles encapsulating ATP (ATPv) on this microvasculature. Methods: 40-male Sprague-Dawley rats were randomized into 4 groups of 10 each: HS/conventionalresuscitation (CR), HS/CR+Norepinephrine, HS/CR+Vasopressin, and HS/CR+ATPv. HS=initial removal of 30% of the calculated blood volume plus subsequent transection of the spleen for uncontrolled haemorrhage until a predefined intervention set-point was achieved; CR=shed blood returned+double the shed blood volume as lactated Ringer’s solution. Four-level intestinal microvascular A1 through A4 arterioles (100–8 μm diameter) in the terminalileum were continuously monitored with intravital-microscopy, and their diameters were measured at baseline, during shock, after completion of resuscitation, and during a 2 h post-resuscitation observation period. In addition, we recorded measurements of the mean arterial pressure, shock index, blood-gas profiles, and complete metabolic panels. Results: Progressive HS caused bimodal arteriole responses with progressive vasoconstriction from baseline of first-order A1 and second-order A2 (-22.1 ± 1.9%), a progressive vasodilation of the third-order A3 and fourth-order A4 arterioles (+22.2 ± 2.8%). Resuscitation initially restored A1 and A2 diameters to near baseline. This was followed by post-resuscitation A1 and A2 vasoconstriction in all groups, except the ATPv group (-8.1 ± 3.4%). The hemorrhage-induced vasodilation of the A3 and A4 arterioles was maintained during the post-resuscitation observation period in the CR (+55.3 ± 6.4%) and the ATPv groups (+39.5 ± 5.2%), but remarkably attenuated in the norepinephrine (+9.6 ± 5.8%) and vasopressin (+9.4 ± 8.8) groups. Conclusions: Severe hemorrhagic shock causes specific splanchnic microvascular bimodal response that is dependent on arteriolar level. First-order A1 and second-order A2 arterioles exhibit progressive vasoconstriction, whereas, third-order A3 and fourth-order A4 arterioles show progressive vasodilation. Temporary administration of either norepinephrine or vasopressin along with conventional resuscitation exerted deleterious effects at all splanchnic microvascular levels by virtue of their peripheral vascular actions. The cellular cytosolic ATP replenishment maintained a better microvascular profile when compared with the two vasopressors.

Carnosic Acid, a Natural Diterpene, Attenuates Arsenic-Induced Hepatotoxicity via Reducing Oxidative Stress, MAPK Activation, and Apoptotic Cell Death Pathway.

The present studies have been executed to explore the protective mechanism of carnosic acid (CA) against NaAsO2-induced hepatic injury. CA exhibited a concentration dependent (1–4 μM) increase in cell viability against NaAsO2 (12 μM) in murine hepatocytes. NaAsO2 treatment significantly enhanced the ROS-mediated oxidative stress in the hepatic cells both in in vitro and in vivo systems. Significant activation of MAPK, NF-κB, p53, and intrinsic and extrinsic apoptotic signaling was observed in NaAsO2-exposed hepatic cells. CA could significantly counteract with redox stress and ROS-mediated signaling and thereby attenuated NaAsO2-mediated hepatotoxicity. NaAsO2 (10 mg/kg) treatment caused significant increment in the As bioaccumulation, cytosolic ATP level, DNA fragmentation, and oxidation in the liver of experimental mice (n = 6). The serum biochemical and haematological parameters were significantly altered in the NaAsO2-exposed mice (n = 6). Simultaneous treatment with CA (10 and 20 mg/kg) could significantly reinstate the NaAsO2-mediated toxicological effects in the liver. Molecular docking and dynamics predicted the possible interaction patterns and the stability of interactions between CA and signal proteins. ADME prediction anticipated the drug-likeness characteristics of CA. Hence, there would be an option to employ CA as a new therapeutic agent against As-mediated toxic manifestations in future.

Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis.

Glioblastoma multiforme (GBM) is the most common and malignant of the primary adult brain cancers. Ultrastructural and biochemical evidence shows that GBM cells exhibit mitochondrial abnormalities incompatible with energy production through oxidative phosphorylation (OxPhos). Under such conditions, the mitochondrial F0-F1 ATP synthase operates in reverse at the expense of ATP hydrolysis to maintain a moderate membrane potential. Moreover, expression of the dimeric M2 isoform of pyruvate kinase in GBM results in diminished ATP output, precluding a significant ATP production from glycolysis. If ATP synthesis through both glycolysis and OxPhos was impeded, then where would GBM cells obtain high-energy phosphates for growth and invasion? Literature is reviewed suggesting that the succinate-CoA ligase reaction in the tricarboxylic acid cycle can substantiate sufficient ATP through mitochondrial substrate-level phosphorylation (mSLP) to maintain GBM growth when OxPhos is impaired. Production of high-energy phosphates would be supported by glutaminolysis—a hallmark of GBM metabolism—through the sequential conversion of glutamine → glutamate → alpha-ketoglutarate → succinyl CoA → succinate. Equally important, provision of ATP through mSLP would maintain the adenine nucleotide translocase in forward mode, thus preventing the reverse-operating F0-F1 ATP synthase from depleting cytosolic ATP reserves. Because glucose and glutamine are the primary fuels driving the rapid growth of GBM and most tumors for that matter, simultaneous restriction of these two substrates or inhibition of mSLP should diminish cancer viability, growth, and invasion.

Reconstruction of Oryza sativa indica Genome Scale Metabolic Model and Its Responses to Varying RuBisCO Activity, Light Intensity, and Enzymatic Cost Conditions.

To combat decrease in rice productivity under different stresses, an understanding of rice metabolism is needed. Though there are different genome scale metabolic models (GSMs) of Oryza sativa japonica, no GSM with gene-protein-reaction association exist for Oryza sativa indica. Here, we report a GSM, OSI1136 of O.s. indica, which includes 3602 genes and 1136 metabolic reactions and transporters distributed across the cytosol, mitochondrion, peroxisome, and chloroplast compartments. Flux balance analysis of the model showed that for varying RuBisCO activity (Vc/Vo) (i) the activity of the chloroplastic malate valve increases to transport reducing equivalents out of the chloroplast under increased photorespiratory conditions and (ii) glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase can act as source of cytosolic ATP under decreased photorespiration. Under increasing light conditions we observed metabolic flexibility, involving photorespiration, chloroplastic triose phosphate and the dicarboxylate transporters of the chloroplast and mitochondrion for redox and ATP exchanges across the intracellular compartments. Simulations under different enzymatic cost conditions revealed (i) participation of peroxisomal glutathione-ascorbate cycle in photorespiratory H2O2 metabolism (ii) different modes of the chloroplastic triose phosphate transporters and malate valve, and (iii) two possible modes of chloroplastic Glu–Gln transporter which were related with the activity of chloroplastic and cytosolic isoforms of glutamine synthetase. Altogether, our results provide new insights into plant metabolism.

The Glycolytic Pyruvate Kinase Is Recruited Directly into the Viral Replicase Complex to Generate ATP for RNA Synthesis

Viruses accomplish their replication by exploiting many cellular resources, including metabolites and energy. Similarly to other (+)RNA viruses, tomato bushy stunt virus (TBSV) induces major changes in infected cells. However, the source of energy required to fuel TBSV replication is unknown. We find that TBSV co-opts the cellular glycolytic ATP-generating pyruvate kinase (PK) directly into the viral replicase complex to boost progeny RNA synthesis. The co-opted PK generates high levels of ATP within the viral replication compartment at the expense of a reduction in cytosolic ATP pools. The ATP generated by the co-opted PK is used to promote the helicase activity of recruited cellular DEAD-box helicases, which are involved in the production of excess viral (+)RNA progeny. Altogether, recruitment of PK and local production of ATP within the replication compartment allow the virus replication machinery an access to plentiful ATP, facilitating robust virus replication.

General Anesthetics Regulate Autophagy via Modulating the Inositol 1,4,5-Trisphosphate Receptor: Implications for Dual Effects of Cytoprotection and Cytotoxicity

General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms. General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). DT40 or SH-SY5Y cells with only or over 99% expression of InsP3R-1 were treated with isoflurane or propofol. Cell viability was determined by MTT reduction or LDH release assays. Apoptosis was determined by measuring Caspase-3 or by TUNEL assay. Autophagy activity was determined by measuring LC3 II and P62. We evaluated mitochondrial integrity using MitoTracker Green and cytosolic ATP levels. Fura2-AM was used to measure the concentrations of cytosolic calcium ([Ca2+]c). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R. Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. Short exposure to propofol adequately activated InsP3-1 to provide sufficient autophagy for cytoprotection, while prolonged exposure to propofol induced cell apoptosis via impairment of autophagy flux through over activation of InsP3-1. Propofol damaged mitochondria and decreased cytosolic ATP. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R.

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes.

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.