-
1.
Functions of Cytochrome c oxidase Assembly Factors.
Watson, SA, McStay, GP
International journal of molecular sciences. 2020;(19)
Abstract
Cytochrome c oxidase is the terminal complex of eukaryotic oxidative phosphorylation in mitochondria. This process couples the reduction of electron carriers during metabolism to the reduction of molecular oxygen to water and translocation of protons from the internal mitochondrial matrix to the inter-membrane space. The electrochemical gradient formed is used to generate chemical energy in the form of adenosine triphosphate to power vital cellular processes. Cytochrome c oxidase and most oxidative phosphorylation complexes are the product of the nuclear and mitochondrial genomes. This poses a series of topological and temporal steps that must be completed to ensure efficient assembly of the functional enzyme. Many assembly factors have evolved to perform these steps for insertion of protein into the inner mitochondrial membrane, maturation of the polypeptide, incorporation of co-factors and prosthetic groups and to regulate this process. Much of the information about each of these assembly factors has been gleaned from use of the single cell eukaryote Saccharomyces cerevisiae and also mutations responsible for human disease. This review will focus on the assembly factors of cytochrome c oxidase to highlight some of the outstanding questions in the assembly of this vital enzyme complex.
-
2.
Quantitative analysis of amino acid metabolism in liver cancer links glutamate excretion to nucleotide synthesis.
Nilsson, A, Haanstra, JR, Engqvist, M, Gerding, A, Bakker, BM, Klingmüller, U, Teusink, B, Nielsen, J
Proceedings of the National Academy of Sciences of the United States of America. 2020;(19):10294-10304
Abstract
Many cancer cells consume glutamine at high rates; counterintuitively, they simultaneously excrete glutamate, the first intermediate in glutamine metabolism. Glutamine consumption has been linked to replenishment of tricarboxylic acid cycle (TCA) intermediates and synthesis of adenosine triphosphate (ATP), but the reason for glutamate excretion is unclear. Here, we dynamically profile the uptake and excretion fluxes of a liver cancer cell line (HepG2) and use genome-scale metabolic modeling for in-depth analysis. We find that up to 30% of the glutamine is metabolized in the cytosol, primarily for nucleotide synthesis, producing cytosolic glutamate. We hypothesize that excreting glutamate helps the cell to increase the nucleotide synthesis rate to sustain growth. Indeed, we show experimentally that partial inhibition of glutamate excretion reduces cell growth. Our integrative approach thus links glutamine addiction to glutamate excretion in cancer and points toward potential drug targets.
-
3.
Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD+ and ATP Depletion.
Kaczara, P, Sitek, B, Przyborowski, K, Kurpinska, A, Kus, K, Stojak, M, Chlopicki, S
Arteriosclerosis, thrombosis, and vascular biology. 2020;(10):2376-2390
-
-
Free full text
-
Abstract
OBJECTIVES Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. CONCLUSIONS The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.
-
4.
Peroxisomal Cofactor Transport.
Plett, A, Charton, L, Linka, N
Biomolecules. 2020;(8)
Abstract
Peroxisomes are eukaryotic organelles that are essential for growth and development. They are highly metabolically active and house many biochemical reactions, including lipid metabolism and synthesis of signaling molecules. Most of these metabolic pathways are shared with other compartments, such as Endoplasmic reticulum (ER), mitochondria, and plastids. Peroxisomes, in common with all other cellular organelles are dependent on a wide range of cofactors, such as adenosine 5'-triphosphate (ATP), Coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD). The availability of the peroxisomal cofactor pool controls peroxisome function. The levels of these cofactors available for peroxisomal metabolism is determined by the balance between synthesis, import, export, binding, and degradation. Since the final steps of cofactor synthesis are thought to be located in the cytosol, cofactors must be imported into peroxisomes. This review gives an overview about our current knowledge of the permeability of the peroxisomal membrane with the focus on ATP, CoA, and NAD. Several members of the mitochondrial carrier family are located in peroxisomes, catalyzing the transfer of these organic cofactors across the peroxisomal membrane. Most of the functions of these peroxisomal cofactor transporters are known from studies in yeast, humans, and plants. Parallels and differences between the transporters in the different organisms are discussed here.
-
5.
Influence of caffeine intake on intravenous adenosine-induced fractional flow reserve.
Kasumi, I, Fujii, K, Satoru, O, Shin, T, Katsuyuki, H, Hiroto, T, Rui, I, Shingo, Y, Sho, N, Wataru, Y, et al
Journal of cardiology. 2020;(5):472-478
Abstract
BACKGROUND This study evaluated whether caffeine abstention is required before fractional flow reserve (FFR) measurement by intravenous adenosine triphosphate (ATP) administration in Japanese patients. METHODS This study was a subanalysis of a previously published study and a total of 208 intermediate lesions that underwent FFR measurements were enrolled for this analysis. Hyperemia was induced by continuous intravenous ATP infusion at 150 μg/kg/min (IVATP150) and 210 μg/kg/min (IVATP210), and by intracoronary administration of nicorandil 2 mg (ICNIC2mg) as a reference standard. RESULTS The degree of change in the FFR value after ICNIC2mg and IVATP210 was similar between the caffeine and non-caffeine groups (0.00 ± 0.02 vs. 0.01 ± 0.02). In patients who consumed caffeine before the FFR measurement, the degree of FFR change was independent of the time interval (<12 h, 12-24 h, and 24-48 h) between caffeine intake and catheterization both after IVATP150 and ICNIC2mg and after IVATP210 and ICNIC2mg. CONCLUSION When compared with the FFR value after ICNIC2mg, the degree of change in the FFR value after IVATP210 were similar regardless of caffeine intake. Strict caffeine abstention before intravenous ATP-induced FFR measurement may not be required in clinical practice.
-
6.
ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival.
Gilbert, SM, Oliphant, CJ, Hassan, S, Peille, AL, Bronsert, P, Falzoni, S, Di Virgilio, F, McNulty, S, Lara, R
Oncogene. 2019;(2):194-208
-
-
Free full text
-
Abstract
The ATP-gated receptor P2X7 is expressed in multiple malignant tumours including neuroblastoma, melanoma, prostate, lung and breast. P2X7 has a significant role in mediating diverse cell responses, which upon dysregulation are associated with tumour initiation and development. The rapid, ATP-mediated activation of P2X7 induces a fast-inward cation current in cells. However, prolonged ATP-mediated activation of P2X7 leads to formation of a pore that increases membrane permeability and eventually causes cell death. This presents a potential paradox, as the tumour microenvironment contains extracellular ATP at levels sufficient to activate the P2X7 pore and trigger cell death. However, P2X7 expression is associated with enhanced cancer cell survival, proliferation and metastatic potential. At least one distinct conformational form of P2X7, termed non-pore functional P2X7 (nfP2X7), has been described, which is not able to form a functional pore. We demonstrate for the first time in this study that exposure to a high ATP concentration, equivalent to those measured in the tumour microenvironment, drives nfP2X7 expression and also that nfP2X7 is essential for tumour cell survival. We show that monoclonal antibodies raised against a P2X7 amino acid sequence (200-216), whose conformation is distinct from that of wild-type (WT) P2X7, bind specifically to nfP2X7 expressed on the surface of tumour cells. We also show that nfP2X7 is broadly expressed in patient-derived tumour sections from a wide range of cancers. Therefore, antibodies raised against E200 provide tools that can differentiate between forms of the P2X7 receptor that have a key role in cancer.
-
7.
Effect of gummy candy containing ubiquinol on secretion of saliva: A randomized, double-blind, placebo-controlled parallel-group comparative study and an in vitro study.
Ushikoshi-Nakayama, R, Ryo, K, Yamazaki, T, Kaneko, M, Sugano, T, Ito, Y, Matsumoto, N, Saito, I
PloS one. 2019;(4):e0214495
Abstract
A randomized, double-blind, placebo-controlled, parallel-group comparative clinical study was conducted to examine the effects of ubiquinol (the reduced form of Coenzyme Q10) on secretion of saliva. This interventional study enrolled 40 subjects aged 65 years or younger who were healthy, but noted slight dryness of the mouth. Subjects were randomized with stratification according to gender and age to ingestion of gummy candy containing 50 mg of ubiquinol or placebo twice daily for 8 weeks. At the end of study, along with a significant increase of the CoQ10 level in saliva (p = 0.025*, d = 0.65), there was a significant increase of the saliva flow rate (p = 0.048*, d = 0.66) in the ubiquinol candy group (n = 18; 47.4±6.2 years; 6 men and 12 women) compared to the placebo group (n = 20; 52.2±7.7 years; 4 men and 16 women). The strength of the stomatognathic muscles was not significantly enhanced by ingestion of ubiquinol candy. Compared with baseline, significant improvement of the following four questionnaire items was observed in the ubiquinol group at the end of the study: feeling tired (p = 0.00506, d = -0.726), dryness of the mouth (p = 0.04799, d = -0.648), prone to catching a cold (p = 0.00577, d = -0.963), and diarrhea (p = 0.0166, d = -0.855). There were no serious adverse events. An in vitro study revealed that ubiquinol stimulated a significant and concentration-dependent increase of ATP production by a cell line derived from human salivary gland epithelial cells (p<0.05), while 1 nM ubiquinol significantly suppressed (p = 0.028) generation of malondialdehyde by cells exposed to FeSO4-induced oxidative stress. These findings suggest that ubiquinol increases secretion of saliva by suppressing oxidative stress in the salivary glands and by promoting ATP production. Trial Registration: UMIN-CTR UMIN000024406.
-
8.
TRPP2 dysfunction decreases ATP-evoked calcium, induces cell aggregation and stimulates proliferation in T lymphocytes.
Magistroni, R, Mangolini, A, Guzzo, S, Testa, F, Rapanà, MR, Mignani, R, Russo, G, di Virgilio, F, Aguiari, G
BMC nephrology. 2019;(1):355
Abstract
BACKGROUND Autosomal dominant polycystic kidney disease (ADPKD) is mainly characterised by the development and enlargement of renal cysts that lead to end-stage renal disease (ESRD) in adult patients. Other clinical manifestations of this pathology include hypertension, haematuria, abdominal pain, cardiovascular system alterations and intracranial aneurysms. ADPKD is linked to mutations in either PKD1 or PKD2 that codifies polycystin-1 (PC1) and polycystin-2 (PC2 or TRPP2), respectively. PC1 and TRPP2 are membrane proteins that function as receptor-channel elements able to regulate calcium homeostasis. The function of polycystins has been mainly studied in kidney cells; but the role of these proteins in T lymphocytes is not well defined. METHODS T lymphocytes were produced from ADPKD1 and ADPKD2 patients as well as from non-ADPKD subjects undergoing renal replacement therapy (RRT) and healthy controls. Protein expression and phosphorylation levels were analysed by western blotting, cell proliferation was calculated by direct counting using trypan blue assay and intracellular calcium concentration was measured by Fura-2 method. RESULTS PKD2 mutations lead to the significant reduction of TRPP2 expression in T lymphocytes derived from ADPKD patients. Furthermore, a smaller TRPP2 truncated protein in T lymphocytes of patients carrying the mutation R872X in PKD2 was also observed, suggesting that TRPP2 mutated proteins may be stably expressed. The silencing or mutation of PKD2 causes a strong reduction of ATP-evoked calcium in Jurkat cells and ADPKD2 T lymphocytes, respectively. Moreover, T lymphocytes derived from both ADPKD1 and ADPKD2 patients show increased cell proliferation, basal chemotaxis and cell aggregation compared with T lymphocytes from non-ADPKD subjects. Similarly to observations made in kidney cells, mutations in PKD1 and PKD2 dysregulate ERK, mTOR, NFkB and MIF pathways in T lymphocytes. CONCLUSIONS Because the alteration of ERK, mTOR, NFkB and MIF signalling found in T lymphocytes of ADPKD patients may contribute to the development of interstitial inflammation promoting cyst growth and kidney failure (ESRD), the targeting of inflammasome proteins could be an intriguing option to delay the progression of ADPKD.
-
9.
ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
Strickland, KM, Stock, G, Cui, G, Hwang, H, Infield, DT, Schmidt-Krey, I, McCarty, NA, Gumbart, JC
The journal of physical chemistry. B. 2019;(15):3177-3188
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 μs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.
-
10.
A Single Dose of Oral ATP Supplementation Improves Performance and Physiological Response During Lower Body Resistance Exercise in Recreational Resistance-Trained Males.
Freitas, MC, Cholewa, JM, Gerosa-Neto, J, Gonçalves, DC, Caperuto, EC, Lira, FS, Rossi, FE
Journal of strength and conditioning research. 2019;(12):3345-3352
Abstract
Freitas, MC, Cholewa, JM, Gerosa-Neto, J, Gonçalves, DC, Caperuto, EC, Lira, FS, and Rossi, FE. A single dose of oral ATP supplementation improves performance and physiological response during lower body resistance exercise in recreational resistance-trained males. J Strength Cond Res 33(12): 3345-3352, 2019-The aim of this study was to investigate the acute effect of adenosine-5'-triphosphate (ATP) supplementation on performance and physiological responses during resistance exercise in recreationally resistance-trained males. Eleven men (age = 27.5 ± 5.5 years, mass = 83.4 ± 9.8 kg, height = 182 ± 0.04 cm) completed 2 randomized, double-blind trials: ATP supplement condition (ATP = 400 mg) or a placebo condition. Thirty minutes after supplement consumption, subjects performed 4 sets of half-squats until momentary muscular failure at 80% of the 1 repetition maximum with 2 minutes of recovery between sets. The total number of repetitions, blood pressure, heart rate, blood lactate, and oxygen consumption were evaluated. The total weight lifted were higher for the ATP condition compared with placebo (Placebo = 3,995.7 ± 1,137.8, ATP = 4,967.4 ± 1,497.9 kg; p = 0.005). Heart rate was higher at set-4 for ATP compared with placebo (p < 0.001) and oxygen consumption during exercise was greater for ATP (p = 0.021). There were no differences between conditions for lactate and blood pressure. In summary, a single oral dose of ATP supplementation improved lower-body resistance training performance and energy expenditure in recreational resistance-trained males.