Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

NASA Technical Reports Server (NTRS)

Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

1996-01-01

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

NASA Astrophysics Data System (ADS)

Ernst, Stefan; Düser, Monika G.; Zarrabi, Nawid; Börsch, Michael

2012-03-01

The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ɛ subunits of the F1 part. Because γ and ɛ subunits rotate in 120° steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Förster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations.

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia.

PubMed

Martinez-Cruz, O; Calderon de la Barca, A M; Uribe-Carvajal, S; Muhlia-Almazan, A

2012-08-01

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors*

PubMed Central

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D.

2016-01-01

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α3β3γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. PMID:27729450

Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane

NASA Astrophysics Data System (ADS)

Steinberg-Yfrach, Gali; Rigaud, Jean-Louis; Durantini, Edgardo N.; Moore, Ana L.; Gust, Devens; Moore, Thomas A.

1998-04-01

Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.) - an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of FOF1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of ~12kcalmol-1, with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.

SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient- and Exercise-Induced Stress

PubMed Central

Vassilopoulos, Athanassios; Pennington, J. Daniel; Andresson, Thorkell; Rees, David M.; Bosley, Allen D.; Fearnley, Ian M.; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Walker, John E.

2014-01-01

Abstract Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCPK139 directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins. Antioxid. Redox Signal. 21, 551–564. PMID:24252090

Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

PubMed Central

Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

2009-01-01

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors.

PubMed

Sielaff, Hendrik; Martin, James; Singh, Dhirendra; Biuković, Goran; Grüber, Gerhard; Frasch, Wayne D

2016-12-02

The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A 3 B 3 DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor α 3 β 3 γ, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 μs resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A 3 B 3 DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP binding to A 3 B 3 DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40° after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Linking structural features from mitochondrial and bacterial F-type ATP synthases to their distinct mechanisms of ATPase inhibition.

PubMed

Krah, Alexander

2015-10-01

ATP synthases are molecular motors, which synthesize ATP, the ubiquitous energy source in all living cells. They use an electrochemical gradient to drive a rotation in the membrane embedded Fo domain, namely the c-ring, causing a conformational change in the soluble F1 domain which leads to the catalytic event. In the opposite fashion, they can also hydrolyse ATP to maintain the ion gradient across the membrane. To prevent wasteful ATP hydrolysis, bacteria and mammals have developed peculiar mechanistic features in addition to a common one, namely MgADP inhibition. Here I discuss the distinct ATPase inhibition mechanism in mitochondrial (IF1) and bacterial (subunits ε and ζ) F-type ATP synthases, based on available structural, biophysical and biochemical data. Copyright © 2015 Elsevier Ltd. All rights reserved.

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

PubMed Central

Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

2015-01-01

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

PubMed Central

Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

2015-01-01

In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

ATP synthase--a marvellous rotary engine of the cell.

PubMed

Yoshida, M; Muneyuki, E; Hisabori, T

2001-09-01

ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

PubMed Central

Kulish, O.; Wright, A. D.; Terentjev, E. M.

2016-01-01

F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient. PMID:27321713

S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

PubMed

Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

2016-11-01

Mammalian cells can utilize hydrogen sulfide (H 2 S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H 2 S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H 2 S in vitro. The H 2 S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50-300μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H 2 S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H 2 S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE -/- mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H 2 S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H 2 S-producing enzymes and stimulate H 2 S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points

Unidirectional regulation of the F1FO-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria.

PubMed

Zarco-Zavala, Mariel; Mendoza-Hoffmann, Francisco; García-Trejo, José J

2018-06-07

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF 1 subunits, prevent the wasteful CCW F 1 F O -ATPase activity by blocking γ rotation at the α DP /β DP /γ interface of the F 1 portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F 1 -ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF 1 , it also binds to the α DP /β DP /γ interface of the F 1 of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α DP /β DP /γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F 1 F O -ATPase rotation without affecting the CW-F 1 F O -ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as an unidirectional pawl-ratchet PdF 1 F O -ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is an useful model that mimics mitochondrial IF 1 but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF 1 ) of this unique ATP synthase nanomotor, essential for life. Copyright © 2018. Published by Elsevier B.V.

Catalytic and mechanical cycles in F-ATP synthases. Fourth in the Cycles Review Series.

PubMed

Dimroth, Peter; von Ballmoos, Christoph; Meier, Thomas

2006-03-01

Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.

Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

NASA Astrophysics Data System (ADS)

Zarrabi, N.; Ernst, S.; Düser, M. G.; Golovina-Leiker, A.; Becker, W.; Erdmann, R.; Dunn, S. D.; Börsch, M.

2009-02-01

FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ɛ subunits of the F1 sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.

TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F F{sub 1}-ATP synthase and ubiquinone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shertzer, Howard G.; Genter, Mary Beth; Shen, Dongxiao

2006-12-15

Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q wasmore » more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.« less

Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

NASA Astrophysics Data System (ADS)

Heitkamp, Thomas; Deckers-Hebestreit, Gabriele; Börsch, Michael

2016-02-01

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

PubMed

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-10-06

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

PubMed Central

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-01-01

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

Torque generation mechanism of ATP synthase

NASA Astrophysics Data System (ADS)

Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.

2010-03-01

ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping.

PubMed

Hotra, Adam; Suter, Manuel; Biuković, Goran; Ragunathan, Priya; Kundu, Subhashri; Dick, Thomas; Grüber, Gerhard

2016-05-01

The F1 FO -ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the α3 :β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H(+) pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop-encoding sequence (γ166-179 ) in the genome of M. smegmatis. ATP hydrolysis-driven H(+) pumping was observed in IMVs containing the Δγ166-179 mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Δγ166-179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H(+) pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode. © 2016 Federation of European Biochemical Societies.

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation*

PubMed Central

McMillan, Duncan G. G.; Watanabe, Rikiya; Ueno, Hiroshi; Cook, Gregory M.; Noji, Hiroyuki

2016-01-01

F1F0 ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acidin/alkalineout). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2.A1 F1 ATPase (TA2F1) with two forms of single molecule analysis, a magnetic bead duplex and a gold nanoparticle. TA2F1 rotated in a counterclockwise direction in both systems, adhering to Michaelis-Menten kinetics with a maximum rotation rate (Vmax) of 112.4 revolutions/s. TA2F1 displayed 120° unitary steps coupled with ATP hydrolysis. Torque measurements revealed the highest torque (52.4 piconewtons) derived from an F1 molecule using fluctuation theorem. The implications of high torque in terms of extreme environment adaptation are discussed. PMID:27624936

The c15 ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory

PubMed Central

Pogoryelov, Denys; Yu, Jinshu; Meier, Thomas; Vonck, Janet; Dimroth, Peter; Muller, Daniel J

2005-01-01

The oligomeric c ring of the F-ATP synthase from the alkaliphilic cyanobacterium Spirulina platensis was isolated and characterized. Mass spectroscopy analysis indicated a mass of 8,210 Da, reflecting that of a c monomer. The mass increased by 206 Da after treatment with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD), which indicated modification of the ion-binding carboxylate by DCCD. Atomic force microscopy topographs of c rings from S. platensis showed 15 symmetrically assembled subunits. The c15-mer reported here is the largest c ring that is isolated and does not show the classical c-ring mismatch to the three-fold symmetry of the F1 domain. PMID:16170308

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase.

PubMed

Bunney, T D; van Walraven, H S; de Boer, A H

2001-03-27

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F(1) beta-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CF(o)F(1) activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm.

PubMed

Steed, P Ryan; Kraft, Kaitlin A; Fillingame, Robert H

2014-11-25

H(+)-transporting F1F0 ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F0 and F1. H(+) transport at the subunit a-c interface in transmembranous F0 drives rotation of a cylindrical c10 oligomer within the membrane, which is coupled to rotation of subunit γ within the α3β3 sector of F1 to mechanically drive ATP synthesis. F1F0 functions in a reversible manner, with ATP hydrolysis driving H(+) transport. ATP-driven H(+) transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag(+) and/or Cd(+2) with the substituted sulfhydryl groups. The H(+) transport pathway mapped via these Ag(+)(Cd(+2))-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H(+) release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd(+2) directly inhibits passive H(+) transport mediated by F0 reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H(+) transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H(+) transport likely interact in a single structural domain, which then functions in gating H(+) release to the cytoplasm.

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

PubMed

Guo, Hui; Bueler, Stephanie A; Rubinstein, John L

2017-11-17

Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded F O region turns the rotor that drives ATP synthesis in the soluble F 1 region. Although crystal structures of the F 1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the F O region. Using cryo-electron microscopy, we determined the structure of the dimeric F O complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae. Copyright © 2017, American Association for the Advancement of Science.

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

Quantum-mechanical analysis of amino acid residues function in the proton transport during F0F1-ATP synthase catalytic cycle

NASA Astrophysics Data System (ADS)

Ivontsin, L. A.; Mashkovtseva, E. V.; Nartsissov, Ya R.

2017-11-01

Implications of quantum-mechanical approach to the description of proton transport in biological systems are a tempting subject for an overlapping of fundamental physics and biology. The model of proton transport through the integrated membrane enzyme FoF1-ATP synthase responsible for ATP synthesis was developed. The estimation of the mathematical expectation of the proton transfer time through the half-channel was performed. Observed set of proton pathways through the inlet half-channel showed the nanosecond timescale highly dependable of some amino acid residues. There were proposed two types of crucial amino acids: critically localized (His245) and being a part of energy conserving system (Asp119).

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

PubMed

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga; Hochstein, Lawrence I.

1989-01-01

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the

High-Resolution Structure and Mechanism of an F/V-Hybrid Rotor Ring in a Na+-coupled ATP Synthase

PubMed Central

Matthies, Doreen; Zhou, Wenchang; Klyszejko, Adriana L.; Anselmi, Claudio; Yildiz, Özkan; Brandt, Karsten; Müller, Volker; Faraldo-Gómez, José D.; Meier, Thomas

2014-01-01

All rotary ATPases catalyze the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H+ or Na+. Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na+-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits, and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na+ recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring. PMID:25381992

ATP Synthase, a Target for Dementia and Aging?

PubMed

Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

2018-02-01

Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

Twisting and subunit rotation in single FOF1-ATP synthase

PubMed Central

Sielaff, Hendrik; Börsch, Michael

2013-01-01

FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases. PMID:23267178

Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides

PubMed Central

Ahmad, Zulfiqar; Laughlin, Thomas F.

2015-01-01

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented. PMID:20586714

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

PubMed

Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

2017-11-20

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase

PubMed Central

Ahmad, Zulfiqar; Hassan, Sherif S.; Azim, Sofiya

2017-01-01

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phy-tochemicals is based on tradition or word of mouth with few evidence-based studies. Moreo-ver, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become perti-nent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of die-tary phytochemicals are known to inhibit ATP synthase. Structural modifications of phyto-chemicals have been shown to increase the inhibitory potency and extent of inhibition. Site-directed mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can re-sult in selective binding and inhibition of microbial ATP synthase. In this review, the therapeu-tic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective target-ing of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. PMID:28831918

On the Mg(2+) binding site of the ε subunit from bacterial F-type ATP synthases.

PubMed

Krah, Alexander; Takada, Shoji

2015-10-01

F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg(2+) concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg(2+) binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg(2+) ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg(2+) dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg(2+) binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg(2+) binding motif by site directed mutagenesis, which also can be used to crystallize Mg(2+) dependent ε subunits in future. Copyright © 2015 Elsevier B.V. All rights reserved.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

PubMed Central

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

PubMed

Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

2014-04-01

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase.

PubMed

Sellem, Carole H; di Rago, Jean-Paul; Lasserre, Jean-Paul; Ackerman, Sharon H; Sainsard-Chanet, Annie

2016-07-01

Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before.

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

PubMed Central

Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

2008-01-01

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

Resolving the negative potential side (n-side) water-accessible proton pathway of F-type ATP synthase by molecular dynamics simulations.

PubMed

Gohlke, Holger; Schlieper, Daniel; Groth, Georg

2012-10-19

The rotation of F(1)F(o)-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F(o) domain, which is coupled to the ATP-producing F(1) domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the "locked" and "open" conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F(1)F(o)-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

PubMed

Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

2016-05-01

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J

Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

PubMed

Gajadeera, Chathurada S; Weber, Joachim

2013-09-13

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

PubMed

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia

F{sub 1}F{sub o}-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H{sup +} flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H{sup +} within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F{sub 1} andmore » F{sub O} domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.« less

Coupling proton movements to c-ring rotation in F(1)F(o) ATP synthase: aqueous access channels and helix rotations at the a-c interface.

PubMed

Fillingame, Robert H; Angevine, Christine M; Dmitriev, Oleg Y

2002-09-10

F(1)F(o) ATP synthases generate ATP by a rotary catalytic mechanism in which H(+) transport is coupled to rotation of a ring of c subunits within the transmembrane sector of the enzyme. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Proton access channels to and from aspartyl-61 are thought to form in subunit a of the F(o) sector. Here, we summarize new information on the structural organization of subunit a and the mapping of aqueous accessible residues in the fourth and fifth transmembrane helices (TMHs). Cysteine substituted residues, lying on opposite faces of aTMH-4, preferentially react with either N-ethyl-maleimide (NEM) or Ag(+). We propose that aTMH-4 rotates to alternately expose each helical face to aspartyl-61 of subunit c during the proton transport cycle. The concerted helical rotation of aTMH-4 and cTMH-2 are proposed to be coupled to the stepwise mechanical movement of the c-rotor.

How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

PubMed

Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

2013-07-01

A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

Ecto-F1-ATPase: A moonlighting protein complex and an unexpected apoA-I receptor

PubMed Central

Vantourout, Pierre; Radojkovic, Claudia; Lichtenstein, Laeticia; Pons, Véronique; Champagne, Eric; Martinez, Laurent O

2010-01-01

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F1-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F1-ATPase. Particularly, the role of the F1-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions. PMID:21157968

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

PubMed

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

PubMed Central

Nishio, Kazuaki; Iwamoto-Kihara, Atsuko; Yamamoto, Akitsugu; Wada, Yoh; Futai, Masamitsu

2002-01-01

ATP synthase FoF1 (α3β3γδɛab2c10–14) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F1 β or membrane sector Fo a subunit when the Fo c subunit ring was immobilized; a biotin-tag was introduced onto the β or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the β or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the α or β subunit. These results established that the γɛc10–14 and α3β3δab2 complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis. PMID:12357031

Macromolecular organization of ATP synthase and complex I in whole mitochondria

PubMed Central

Davies, Karen M.; Strauss, Mike; Daum, Bertram; Kief, Jan H.; Osiewacz, Heinz D.; Rycovska, Adriana; Zickermann, Volker; Kühlbrandt, Werner

2011-01-01

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F1 heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis. PMID:21836051

REVIEWS OF TOPICAL PROBLEMS Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor

NASA Astrophysics Data System (ADS)

Romanovsky, Yurii M.; Tikhonov, Alexander N.

2010-12-01

The free energy released upon the enzymatic hydrolysis of adenosine triphosphate (ATP) is the main source of energy for the functioning of the living cell and all multicellular organisms. The overwhelming majority of ATP molecules are formed by proton ATP synthases, which are the smallest macromolecular electric motors in Nature. This paper reviews the modern concepts of the molecular structure and functioning of the proton ATP synthase, and real-time biophysical experiments on the rotation of the 'rotor' of this macromolecular motor. Some mathematical models describing the operation of this nanosized macromolecular machine are described.

The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology

PubMed Central

Bernardi, Paolo; Rasola, Andrea; Forte, Michael; Lippe, Giovanna

2015-01-01

The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca2+-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology. PMID:26269524

Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

NASA Astrophysics Data System (ADS)

Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael

2016-02-01

Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

PubMed Central

Ludtmann, Marthe H.R.; Angelova, Plamena R.; Ninkina, Natalia N.; Gandhi, Sonia

2016-01-01

Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance

Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

NASA Astrophysics Data System (ADS)

Su, Bertram; Düser, Monika G.; Zarrabi, Nawid; Heitkamp, Thomas; Starke, Ilka; Börsch, Michael

2015-03-01

To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Förster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle more than 1000-fold. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.

Mechanics of coupling proton movements to c-ring rotation in ATP synthase.

PubMed

Fillingame, Robert H; Angevine, Christine M; Dmitriev, Oleg Y

2003-11-27

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.

The prokaryotic thermophilic TF1-ATPase is functionally compatible with the eukaryotic CFo-part of the chloroplast ATP-synthase.

PubMed

Galmiche, J M; Pezennec, S; Zhao, R; Girault, G; Baeuerlein, E

1994-01-31

The ATP synthase from chloroplasts, CFo.F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP-dependent proton uptake was restored with these CFo-liposomes even better by the addition of the bacterial TF1-than of the related CF1-part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1-ATPase, in these CFo.F1-liposomes, but not in the hybrid CFo.TF1-liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo.TF1-liposomes and reconstituted CFo.F1-liposomes. These results indicate that the bacterial TF1-part binds to the eukaryotic CFo-part of four subunits forming a functional CFo.TF1-ATPase.

ATP Synthase Diseases of Mitochondrial Genetic Origin

PubMed Central

Dautant, Alain; Meier, Thomas; Hahn, Alexander; Tribouillard-Tanvier, Déborah; di Rago, Jean-Paul; Kucharczyk, Roza

2018-01-01

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures. PMID:29670542

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4

PubMed Central

Preiss, Laura; Klyszejko, Adriana L.; Hicks, David B.; Liu, Jun; Fackelmayer, Oliver J.; Yildiz, Özkan; Krulwich, Terry A.; Meier, Thomas

2013-01-01

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles. PMID:23613590

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity.

PubMed

Milgrom, Y M; Ehler, L L; Boyer, P D

1990-11-05

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

The Chloroplast ATP Synthase Features the Characteristic Redox Regulation Machinery

PubMed Central

Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-01-01

Abstract Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854. PMID:23145525

Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

NASA Astrophysics Data System (ADS)

Hammann, Eva; Zappe, Andrea; Keis, Stefanie; Ernst, Stefan; Matthies, Doreen; Meier, Thomas; Cook, Gregory M.; Börsch, Michael

2012-02-01

Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans*

PubMed Central

Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W. V.; Sivaraman, J.

2015-01-01

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

PubMed

Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

2017-01-01

, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented. Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions.

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

PubMed Central

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

2017-01-01

, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented. Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions. PMID:28791032

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE PAGES

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; ...

2017-07-24

, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.« less

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.

, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.« less

Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F0-ATP synthase.

PubMed

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

2013-09-01

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria.

The structure of subunit E of the Pyrococcus horikoshii OT3 A-ATP synthase gives insight into the elasticity of the peripheral stalk.

PubMed

Balakrishna, Asha Manikkoth; Hunke, Cornelia; Grüber, Gerhard

2012-07-13

A(1)A(O) ATP synthases are the major energy converters of archaea. They are composed of an A(1) region that synthesizes ATP and an integral part A(O) that conducts ions. Subunit E is a component of the peripheral stalk that links the A(1) with the A(O) part of the A-ATP synthase. We have determined the crystal structure of the entire subunit E (PhE) of the Pyrococcus horikoshii OT3 A-ATP synthase at 3.6 Å resolution. The structure reveals an extended S-shaped N-terminal α-helix with 112.29 Å in length, followed by a globular head group. The S-shaped feature, common in elastic connectors and spacers, would facilitate the storage of transient elastic energy during rotary motion in the enzyme. The structure has been superimposed into the asymmetric peripheral stalks of the three-dimensional reconstruction of the Pyrococcus furiosus enzyme, revealing that the S-shaped subunit PhE fits well into the bent peripheral stalk, whereas the previously solved E subunit structure (3.1 Å resolution) of Thermus thermophilus A-ATP synthase is well accommodated in the density of the straight stator domain. The different features of the two stalk subunits are discussed in light of a novel coupling mechanism in A-ATP synthases proposed to differ from the Wankel engine of F-ATP synthases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit

PubMed Central

Tran, Lee; Hanavan, Paul D.; Campbell, Latoya E.; De Filippis, Elena; Lake, Douglas F.; Coletta, Dawn K.; Roust, Lori R.; Mandarino, Lawrence J.; Carroll, Chad C.; Katsanos, Christos S.

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = – 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish

Prolonged Exposure of Primary Human Muscle Cells to Plasma Fatty Acids Associated with Obese Phenotype Induces Persistent Suppression of Muscle Mitochondrial ATP Synthase β Subunit.

PubMed

Tran, Lee; Hanavan, Paul D; Campbell, Latoya E; De Filippis, Elena; Lake, Douglas F; Coletta, Dawn K; Roust, Lori R; Mandarino, Lawrence J; Carroll, Chad C; Katsanos, Christos S

2016-01-01

Our previous studies show reduced abundance of the β-subunit of mitochondrial H+-ATP synthase (β-F1-ATPase) in skeletal muscle of obese individuals. The β-F1-ATPase forms the catalytic core of the ATP synthase, and it is critical for ATP production in muscle. The mechanism(s) impairing β-F1-ATPase metabolism in obesity, however, are not completely understood. First, we studied total muscle protein synthesis and the translation efficiency of β-F1-ATPase in obese (BMI, 36±1 kg/m2) and lean (BMI, 22±1 kg/m2) subjects. Both total protein synthesis (0.044±0.006 vs 0.066±0.006%·h-1) and translation efficiency of β-F1-ATPase (0.0031±0.0007 vs 0.0073±0.0004) were lower in muscle from the obese subjects when compared to the lean controls (P<0.05). We then evaluated these same responses in a primary cell culture model, and tested the specific hypothesis that circulating non-esterified fatty acids (NEFA) in obesity play a role in the responses observed in humans. The findings on total protein synthesis and translation efficiency of β-F1-ATPase in primary myotubes cultured from a lean subject, and after exposure to NEFA extracted from serum of an obese subject, were similar to those obtained in humans. Among candidate microRNAs (i.e., non-coding RNAs regulating gene expression), we identified miR-127-5p in preventing the production of β-F1-ATPase. Muscle expression of miR-127-5p negatively correlated with β-F1-ATPase protein translation efficiency in humans (r = - 0.6744; P<0.01), and could be modeled in vitro by prolonged exposure of primary myotubes derived from the lean subject to NEFA extracted from the obese subject. On the other hand, locked nucleic acid inhibitor synthesized to target miR-127-5p significantly increased β-F1-ATPase translation efficiency in myotubes (0.6±0.1 vs 1.3±0.3, in control vs exposure to 50 nM inhibitor; P<0.05). Our experiments implicate circulating NEFA in obesity in suppressing muscle protein metabolism, and establish

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

PubMed

Joon, Shin; Ragunathan, Priya; Sundararaman, Lavanya; Nartey, Wilson; Kundu, Subhashri; Manimekalai, Malathy S S; Bogdanović, Nebojša; Dick, Thomas; Grüber, Gerhard

2018-03-01

Mycobacterium tuberculosis (Mt) F 1 F 0 ATP synthase (α 3 :β 3 :γ:δ:ε:a:b:b':c 9 ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal β-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD. The Mtε structure provides a novel mechanistic model of coupling c-ring- and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α 3 β 3 -headpiece. To test our model, genome site-directed mutagenesis was employed to introduce amino acid changes in these two parts of the epsilon subunit. Inverted vesicle assays show that these mutations caused an increase in ATP hydrolysis activity and a reduction in ATP synthesis. The structural and enzymatic data are discussed in light of the transition mechanism of a compact and extended state of Mtε, which provides the inhibitory effects of this coupling subunit inside the rotary engine. Finally, the employment of these data with molecular docking shed light into the second binding site of the drug Bedaquiline. Structural data are available in the PDB under the accession number 5YIO. © 2018 Federation of European Biochemical Societies.

Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: effect of repositioning within helix 4 of subunit a and helix 2 of subunit c.

PubMed

Langemeyer, Lars; Engelbrecht, Siegfried

2007-07-01

FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((alphabeta)3gammadeltaepsilon) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10gammaepsilon relative to (alphabeta)3deltaab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but "horizontal" repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of "vertical" repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen[OA

PubMed Central

Rott, Markus; Martins, Nádia F.; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M.; Schöttler, Mark A.

2011-01-01

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. PMID:21278125

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

PubMed

Chin, Randall M; Fu, Xudong; Pai, Melody Y; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S; Monsalve, Gabriela C; Hu, Eileen; Whelan, Stephen A; Wang, Jennifer X; Jung, Gwanghyun; Solis, Gregory M; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S; Godwin, Hilary A; Chang, Helena R; Faull, Kym F; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R; Clarke, Catherine F; Teitell, Michael A; Petrascheck, Michael; Reue, Karen; Jung, Michael E; Frand, Alison R; Huang, Jing

2014-06-19

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing

Cardioprotective Effect of Danshensu against Ischemic/Reperfusion Injury via c-Subunit of ATP Synthase Inhibition

PubMed Central

Zhao, JingYi; Fan, Zixuan; Bao, Jiadi; Sun, Dawei; Sun, Chun

2017-01-01

Mitochondrial permeability transition pore (MPTP) opening is the main culprit of ischemic/reperfusion (IR) injury. It is reported that c-subunit of ATP synthase is the core component of MPTP. Danshensu (DSS), a monomer isolated from the traditional Chinese herb Danshen, has showed cardioprotective effect against IR injury through unknown mechanism. In this study, rat hearts were suspended in Langendorff instrument and perfused with Krebs-Henseleit (KH) buffer containing DSS for 60 minutes, followed by 30 minutes of global ischemia. Parameters including heart rate, left ventricular developed pressure, and the rate of left ventricle diastolic pressure change were recorded to assess their cardiac function. All these indexes were improved in DSS group. The rate of cardiomyocytes apoptosis and MPTP opening were both inhibited in DSS group. In addition, DSS administration leads to downregulation of c-subunit of ATP synthase in both mRNA and protein levels. Consistently, when c-subunit of ATP synthase was overexpressed in H9C2 cells through pcDNA3/5G1 plasmid transfection, MPTP opening was enhanced when the cardioprotective effect of DSS also tapers. In conclusion, DSS could alleviate cardiac IR injury via inhibiting c-subunit of ATP synthase expression. PMID:29250127

IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice.

PubMed

Nakamura, Junji; Fujikawa, Makoto; Yoshida, Masasuke

2013-09-17

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation

PubMed Central

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-01-01

Background: The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)– and mitochondrial DNA (mtDNA)–encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. Objectives: We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. Methods: We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. Results: TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. Conclusions: In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. Citation: López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399–1405;�

Effects of Tributyltin Chloride on Cybrids with or without an ATP Synthase Pathologic Mutation.

PubMed

López-Gallardo, Ester; Llobet, Laura; Emperador, Sonia; Montoya, Julio; Ruiz-Pesini, Eduardo

2016-09-01

The oxidative phosphorylation system (OXPHOS) includes nuclear chromosome (nDNA)- and mitochondrial DNA (mtDNA)-encoded polypeptides. Many rare OXPHOS disorders, such as striatal necrosis syndromes, are caused by genetic mutations. Despite important advances in sequencing procedures, causative mutations remain undetected in some patients. It is possible that etiologic factors, such as environmental toxins, are the cause of these cases. Indeed, the inhibition of a particular enzyme by a poison could imitate the biochemical effects of pathological mutations in that enzyme. Moreover, environmental factors can modify the penetrance or expressivity of pathological mutations. We studied the interaction between mitochondrially encoded ATP synthase 6 (p.MT-ATP6) subunit and an environmental exposure that may contribute phenotypic differences between healthy individuals and patients suffering from striatal necrosis syndromes or other mitochondriopathies. We analyzed the effects of the ATP synthase inhibitor tributyltin chloride (TBTC), a widely distributed environmental factor that contaminates human food and water, on transmitochondrial cell lines with or without an ATP synthase mutation that causes striatal necrosis syndrome. Doses were selected based on TBTC concentrations previously reported in human whole blood samples. TBTC modified the phenotypic effects caused by a pathological mtDNA mutation. Interestingly, wild-type cells treated with this xenobiotic showed similar bioenergetics when compared with the untreated mutated cells. In addition to the known genetic causes, our findings suggest that environmental exposure to TBTC might contribute to the etiology of striatal necrosis syndromes. López-Gallardo E, Llobet L, Emperador S, Montoya J, Ruiz-Pesini E. 2016. Effects of tributyltin chloride on cybrids with or without an ATP synthase pathologic mutation. Environ Health Perspect 124:1399-1405; http://dx.doi.org/10.1289/EHP182.

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

DOE PAGES

Kanazawa, Atsuko; Ostendorf, Elisabeth; Kohzuma, Kaori; ...

2017-05-03

In wild type plants, decreasing CO 2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b 6f complex. Here in this paper, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO 2. The increased thylakoid proton conductivitymore » (g H +) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO 2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.« less

The chemo-mechanical coupled model for F(1)F(0)-motor.

PubMed

Xu, Lizhong; Liu, Fang

2012-04-01

F(1)F(0)-motor (ATP synthase) is the universal enzyme in biological energy conversion that is present in the membranes of mitochondria, chloroplasts and bacteria. It uses the energy of the proton gradient across the membrane to synthesize ATP. Previous theory and model about rotation of the ATP synthase is reviewed, then a novel chemo-mechanical coupled model for rotation of the F(1)F(0)-motor is proposed. In the model, more events are considered simultaneously that includes the movement of F(1), the movement of F(0), reactions at F(1) and reactions at F(0). Using the model, the possible substep modes of the rotation for F(1)F(0) are predicted, the dependence of the motor efficiency and its rotation rate on the rigidity of the γ shaft is investigated. We conclude that the γ shaft has a large rotation rate for a limited driving potential because two ends of the γ shaft can rotate alternately for its flexibility. The flexibility also makes the efficiency of F(1)F(0) drop because elastic twisting deformation power is needed during alternate rotation of the γ shaft at two ends. Copyright © 2012 Elsevier Ltd. All rights reserved.

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

PubMed Central

Hong, Sangjin; Pedersen, Peter L.

2008-01-01

Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea.

PubMed

Daum, Bertram; Nicastro, Daniela; Austin, Jotham; McIntosh, J Richard; Kühlbrandt, Werner

2010-04-01

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

Converting conformational changes to electrostatic energy in molecular motors: The energetics of ATP synthase.

PubMed

Strajbl, Marek; Shurki, Avital; Warshel, Arieh

2003-12-09

F1-ATPase is the catalytic component of the ATP synthase molecular machine responsible for most of the uphill synthesis of ATP in living systems. The enormous advances in biochemical and structural studies of this machine provide an opportunity for detailed understanding of the nature of its rotary mechanism. However, further quantitative progress in this direction requires development of reliable ways of translating the observed structural changes to the corresponding energies. This requirement is particularly challenging because we are dealing with a large system that couples major structural changes with a chemical process. The present work provides such a structure-function correlation by using the linear response approximation to describe the rotary mechanism. This approach allows one to evaluate the energy of transitions between different conformational states by considering only the changes in the corresponding electrostatic energies of the ligands. The relevant energetics are also obtained by calculating the linear response approximation-based free energies of transferring the ligands from water to the different sites of F1-ATPase in their different conformational states. We also use the empirical valence bond approach to evaluate the actual free-energy profile for the ATP synthesis in the different conformational states of the system. Integrating the information from the different approaches provides a semiquantitative structure-function correlation for F1-ATPase. It is found that the conformational changes are converted to changes in the electrostatic interaction between the protein and its ligands, which drives the ATP synthesis.

PGC-1α/ERRα-Sirt3 Pathway Regulates DAergic Neuronal Death by Directly Deacetylating SOD2 and ATP Synthase β

PubMed Central

Zhang, Xuefei; Ren, Xiaoqing; Zhang, Qi; Li, Zheyi; Ma, Shuaipeng; Bao, Jintao; Li, Zeyang; Bai, Xue; Zheng, Liangjun; Zhang, Zhong; Shang, Shujiang; Zhang, Chen; Wang, Chuangui; Cao, Liu

2016-01-01

Abstract Aims: Parkinson's disease (PD) heavily affects humans and little is known about its cause and pathogenesis. Sirtuin 3 (Sirt3) plays a key role in regulating mitochondrial dysfunction, which is the main cause of DAergic neuronal loss in PD. We investigated the mechanisms of neuroprotective role of Sirt3 in DAergic neuronal survival. Results: Sirt3 was reduced in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-treated neurons with its overexpression being neuroprotective. We identified that Sirt3 interacted with manganese superoxide dismutase (SOD2) and adenosine triphosphate (ATP) synthase β and modulated their activities by deacetylating SOD2 (K130) and ATP synthase β (K485) to prevent reactive oxygen species accumulation and ATP depletion, and to alleviate DAergic neuronal death upon MPTP treatment. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) interacted with estrogen-related receptor alpha (ERRα) that bound to the Sirt3 promoter as its transcription factor to regulate Sirt3 expression and DAergic neuronal death. In the mouse midbrain, MPTP administration led to the loss of PGC-1α and Sirt3, high acetylation level of SOD2 and ATP synthase β, and the specific loss of DAergic neurons, while Sirt3 overexpression could protect against DAergic neuronal loss. Sirt3 knockout mice exhibited more sensitive and more DAergic neuronal loss to MPTP treatment. Innovation: The study provides new insights into a critical PGC-1α/ERRα-Sirt3 pathway, linking regulation of mitochondrial protein acetylation and DAergic neuronal death in PD pathogenesis, which provide a potential therapeutic strategy and target in PD treatment. Conclusion: These results provide a vital PGC-1α/ERRα-Sirt3 pathway that protects against DAergic neuronal death by directly deacetylating SOD2 (K130) and ATP synthase β (K485) in PD. Antioxid. Redox Signal. 24, 312–328. PMID:26421366

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

PubMed

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

PubMed Central

Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2013-01-01

ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1992-01-01

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

PubMed Central

Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra

2012-01-01

The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10–15 c-subunits is commonly thought to drive rotation of the rotor moiety (c10–14γε) relative to stator moiety (α3β3δab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 Å. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase. PMID:18515064

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila.

PubMed

Sawyer, Eric M; Brunner, Elizabeth C; Hwang, Yihharn; Ivey, Lauren E; Brown, Olivia; Bannon, Megan; Akrobetu, Dennis; Sheaffer, Kelsey E; Morgan, Oshauna; Field, Conroy O; Suresh, Nishita; Gordon, M Grace; Gunnell, E Taylor; Regruto, Lindsay A; Wood, Cricket G; Fuller, Margaret T; Hales, Karen G

2017-03-23

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase.

PubMed

Rögner, M; Gräber, P

1986-09-01

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.

Introducing the human Leigh syndrome mutation T9176G into Saccharomyces cerevisiae mitochondrial DNA leads to severe defects in the incorporation of Atp6p into the ATP synthase and in the mitochondrial morphology.

PubMed

Kucharczyk, Roza; Salin, Bénédicte; di Rago, J-P

2009-08-01

The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane alpha-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.

Insights into the Molecular Mechanism of Rotation in the Fo Sector of ATP Synthase

PubMed Central

Aksimentiev, Aleksij; Balabin, Ilya A.; Fillingame, Robert H.; Schulten, Klaus

2004-01-01

F1Fo-ATP synthase is a ubiquitous membrane protein complex that efficiently converts a cell's transmembrane proton gradient into chemical energy stored as ATP. The protein is made of two molecular motors, Fo and F1, which are coupled by a central stalk. The membrane unit, Fo, converts the transmembrane electrochemical potential into mechanical rotation of a rotor in Fo and the physically connected central stalk. Based on available data of individual components, we have built an all-atom model of Fo and investigated through molecular dynamics simulations and mathematical modeling the mechanism of torque generation in Fo. The mechanism that emerged generates the torque at the interface of the a- and c-subunits of Fo through side groups aSer-206, aArg-210, and aAsn-214 of the a-subunit and side groups cAsp-61 of the c-subunits. The mechanism couples protonation/deprotonation of two cAsp-61 side groups, juxtaposed to the a-subunit at any moment in time, to rotations of individual c-subunit helices as well as rotation of the entire c-subunit. The aArg-210 side group orients the cAsp-61 side groups and, thereby, establishes proton transfer via aSer-206 and aAsn-214 to proton half-channels, while preventing direct proton transfer between the half-channels. A mathematical model proves the feasibility of torque generation by the stated mechanism against loads typical during ATP synthesis; the essential model characteristics, e.g., helix and subunit rotation and associated friction constants, have been tested and furnished by steered molecular dynamics simulations. PMID:14990464

Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii.

PubMed

Gayen, Shovanlal; Balakrishna, Asha M; Biuković, Goran; Yulei, Wu; Hunke, Cornelia; Grüber, Gerhard

2008-04-01

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.

ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

PubMed Central

Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

2015-01-01

F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

Two nuclear life cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina.

PubMed

Déquard-Chablat, Michelle; Sellem, Carole H; Golik, Pawel; Bidard, Frédérique; Martos, Alexandre; Bietenhader, Maïlis; di Rago, Jean-Paul; Sainsard-Chanet, Annie; Hermann-Le Denmat, Sylvie; Contamine, Véronique

2011-07-01

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

PubMed Central

Gutiérrez‐Sanz, Óscar; Natale, Paolo; Márquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Inês A. C.

2016-01-01

Abstract ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. PMID:26991333

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases.

PubMed

Matsuhashi, Tetsuro; Sato, Takeya; Kanno, Shin-Ichiro; Suzuki, Takehiro; Matsuo, Akihiro; Oba, Yuki; Kikusato, Motoi; Ogasawara, Emi; Kudo, Tai; Suzuki, Kosuke; Ohara, Osamu; Shimbo, Hiroko; Nanto, Fumika; Yamaguchi, Hiroaki; Saigusa, Daisuke; Mukaiyama, Yasuno; Watabe, Akiko; Kikuchi, Koichi; Shima, Hisato; Mishima, Eikan; Akiyama, Yasutoshi; Oikawa, Yoshitsugu; Hsin-Jung, H O; Akiyama, Yukako; Suzuki, Chitose; Uematsu, Mitsugu; Ogata, Masaki; Kumagai, Naonori; Toyomizu, Masaaki; Hozawa, Atsushi; Mano, Nariyasu; Owada, Yuji; Aiba, Setsuya; Yanagisawa, Teruyuki; Tomioka, Yoshihisa; Kure, Shigeo; Ito, Sadayoshi; Nakada, Kazuto; Hayashi, Ken-Ichiro; Osaka, Hitoshi; Abe, Takaaki

2017-06-01

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

DOE PAGES

Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; ...

2015-09-01

The proton-driven ATP synthase (F OF 1) is comprised of two rotary, stepping motors (F O and F 1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other F O subunits (ab 2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure.more » Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.

The proton-driven ATP synthase (F OF 1) is comprised of two rotary, stepping motors (F O and F 1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other F O subunits (ab 2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure.more » Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

PubMed

de Kloet, S R

2001-08-01

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes.

PubMed

Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O

2017-09-01

Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

PubMed

Sgarbi, G; Barbato, S; Costanzini, A; Solaini, G; Baracca, A

2018-02-01

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF 1 , that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O 2 ) unexpectedly showed that Δψ m is preserved independently of the presence of IF 1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ Η + with the FCCP uncoupler, the IF 1 -silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF 1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF 1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction. Copyright © 2017 Elsevier B.V. All rights reserved.

An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts.

PubMed

Pateraki, Irini; Renato, Marta; Azcón-Bieto, Joaquín; Boronat, Albert

2013-04-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ-subunit present in tomato leaf and green fruit chloroplasts by the atypical γ-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Tomatidine Is a Lead Antibiotic Molecule That Targets Staphylococcus aureus ATP Synthase Subunit C.

PubMed

Lamontagne Boulet, Maxime; Isabelle, Charles; Guay, Isabelle; Brouillette, Eric; Langlois, Jean-Philippe; Jacques, Pierre-Étienne; Rodrigue, Sébastien; Brzezinski, Ryszard; Beauregard, Pascale B; Bouarab, Kamal; Boyapelly, Kumaraswamy; Boudreault, Pierre-Luc; Marsault, Éric; Malouin, François

2018-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10 5 -fold for FC04-100. Copyright © 2018 American Society for Microbiology.

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

PubMed Central

Tetaud, Emmanuel; Godard, François; Giraud, Marie-France; Ackerman, Sharon H.; di Rago, Jean-Paul

2014-01-01

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1 and the proton translocation channel in F0 for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0 subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0. PMID:24451261

Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F₀-ATP synthase

PubMed Central

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

2013-01-01

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53+/+ cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria. PMID:23966169

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

PubMed Central

Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

2013-01-01

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

Mitochondrial flashes regulate ATP homeostasis in the heart

PubMed Central

Wang, Xianhua; Zhang, Xing; Wu, Di; Huang, Zhanglong; Hou, Tingting; Jian, Chongshu; Yu, Peng; Lu, Fujian; Zhang, Rufeng; Sun, Tao; Li, Jinghang; Qi, Wenfeng; Wang, Yanru; Gao, Feng; Cheng, Heping

2017-01-01

The maintenance of a constant ATP level (‘set-point’) is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart. DOI: http://dx.doi.org/10.7554/eLife.23908.001 PMID:28692422

Atomistic simulations indicate the c-subunit ring of the F1Fo ATP synthase is not the mitochondrial permeability transition pore

PubMed Central

Zhou, Wenchang; Marinelli, Fabrizio; Nief, Corrine; Faraldo-Gómez, José D

2017-01-01

Pathological metabolic conditions such as ischemia induce the rupture of the mitochondrial envelope and the release of pro-apoptotic proteins, leading to cell death. At the onset of this process, the inner mitochondrial membrane becomes depolarized and permeable to osmolytes, proposedly due to the opening of a non-selective protein channel of unknown molecular identity. A recent study purports that this channel, referred to as Mitochondrial Permeability Transition Pore (MPTP), is formed within the c-subunit ring of the ATP synthase, upon its dissociation from the catalytic domain of the enzyme. Here, we examine this claim for two c-rings of different lumen width, through calculations of their ion conductance and selectivity based on all-atom molecular dynamics simulations. We also quantify the likelihood that the lumen of these c-rings is in a hydrated, potentially conducting state rather than empty or blocked by lipid molecules. These calculations demonstrate that the structure and biophysical properties of a correctly assembled c-ring are inconsistent with those attributed to the MPTP. DOI: http://dx.doi.org/10.7554/eLife.23781.001 PMID:28186490

Oligomycin frames a common drug-binding site in the ATP synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric

We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100%more » conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.« less

Thermodynamics of proton transport coupled ATP synthesis.

PubMed

Turina, Paola; Petersen, Jan; Gräber, Peter

2016-06-01

The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Surface-Enhanced Raman Spectroscopy Study of 4-ATP on Gold Nanoparticles for Basal Cell Carcinoma Fingerprint Detection

NASA Astrophysics Data System (ADS)

Quynh, Luu Manh; Nam, Nguyen Hoang; Kong, K.; Nhung, Nguyen Thi; Notingher, I.; Henini, M.; Luong, Nguyen Hoang

2016-05-01

The surface-enhanced Raman signals of 4-aminothiophenol (4-ATP) attached to the surface of colloidal gold nanoparticles with size distribution of 2 to 5 nm were used as a labeling agent to detect basal cell carcinoma (BCC) of the skin. The enhanced Raman band at 1075 cm-1 corresponding to the C-S stretching vibration in 4-ATP was observed during attachment to the surface of the gold nanoparticles. The frequency and intensity of this band did not change when the colloids were conjugated with BerEP4 antibody, which specifically binds to BCC. We show the feasibility of imaging BCC by surface-enhanced Raman spectroscopy, scanning the 1075 cm-1 band to detect the distribution of 4-ATP-coated gold nanoparticles attached to skin tissue ex vivo.

The γ-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli

PubMed Central

Omote, Hiroshi; Sambonmatsu, Noriko; Saito, Kiwamu; Sambongi, Yoshihiro; Iwamoto-Kihara, Atsuko; Yanagida, Toshio; Wada, Yoh; Futai, Masamitsu

1999-01-01

The rotation of the γ-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (FOF1). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the γ-subunit of the F1 sector rotated on addition of ATP. This progress enabled us to analyze the γM23K (the γ-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the γM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa. PMID:10393898

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

PubMed Central

Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

2014-01-01

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

PubMed Central

Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

1998-01-01

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

PubMed

Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

PubMed Central

Okuno, Daichi; Nishiyama, Masayoshi; Noji, Hiroyuki

2013-01-01

F1-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F1-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F1 from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F1 actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F1 has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F1(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å3 and +88 Å3 for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F1 and pressure-induced protein unfolding. PMID:24094404

Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

PubMed Central

Uhlemann, Eva-Maria E; Pierson, Hannah E; Fillingame, Robert H; Dmitriev, Oleg Y

2012-01-01

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes. PMID:22162071

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

PubMed

Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E

2015-05-15

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species

PubMed Central

Liu, Sidong; Charlesworth, Thomas J.; Bason, John V.; Montgomery, Martin G.; Harbour, Michael E.; Fearnley, Ian M.; Walker, John E.

2015-01-01

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex. PMID:25759169

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

PubMed

Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

2013-12-01

Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Ethanol Dose- and Time-dependently Increases α and β Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

PubMed

Mashimo, Keiko; Arthur, Peter G; Ohno, Youkichi

2015-01-01

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

The nonlinear chemo-mechanic coupled dynamics of the F 1 -ATPase molecular motor.

PubMed

Xu, Lizhong; Liu, Fang

2012-03-01

The ATP synthase consists of two opposing rotary motors, F0 and F1, coupled to each other. When the F1 motor is not coupled to the F0 motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F1-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F1-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F1-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F1 motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F1 motor, which increases the motor's rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F1 motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F1 motor and provide insight about the stochastic wave mechanics of F1-ATPase.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Energy Equivalence of Information in the Mitochondrion and the Thermodynamic Efficiency of ATP Synthase.

PubMed

Matta, Chérif F; Massa, Lou

2015-09-01

Half a century ago, Johnson and Knudsen resolved the puzzle of the apparent low efficiency of the kidney (∼ 0.5%) compared to most other bodily organs (∼ 40%) by taking into account the entropic cost of ion sorting, the principal function of this organ. Similarly, it is shown that the efficiency of energy transduction of the chemiosmotic proton-motive force by ATP synthase is closer to 90% instead of the oft-quoted textbook value of only 60% when information theoretic considerations are applied to the mitochondrion. This high efficiency is consistent with the mechanical energy transduction of ATP synthase known to be close to the 100% thermodynamic limit. It would have been wasteful for evolution to maximize the mechanical energy transduction to 100% while wasting 40% of the chemiosmotic free energy in the conversion of the proton-motive force into mechanical work before being captured as chemical energy in adenosine 5'-triphosphate.

Effects of an ATP analogue, adenosine 5'-[α-thio]-triphosphate, on F1-ATPase rotary catalysis, torque generation, and inhibited intermediated formation.

PubMed

Yukawa, Ayako; Watanabe, Rikiya; Noji, Hiroyuki

2015-03-13

F1-ATPase (F1), an important rotary motor protein, converts the chemical energy of ATP hydrolysis into mechanical energy using rotary motion with extremely high efficiency. The energy-conversion mechanism for this molecular motor has been extensively clarified by previous studies, which indicate that the interactions between the catalytic residues and the β- and γ-phosphates of ATP are indispensable for efficient catalysis and torque generation. However, the role of α-phosphate is largely unknown. In this study, we observed the rotation of F1 fuelled with an ATP analogue, adenosine 5'-[α-thio]-triphosphate (ATPαS), in which the oxygen has been substituted with a sulfur ion to perturb the α-phosphate/F1 interactions. In doing so, we have revealed that ATPαS does not appear to have any impact on the kinetic properties of the motor or on torque generation compared to ATP. On the other hand, F1 was observed to lapse into the ADP-inhibited intermediate states when in the presence of ATPαS more severely than in the presence of ATP, suggesting that the α-phosphate group of ATP contributes to the avoidance of ADP-inhibited intermediate formation. Copyright © 2015 Elsevier Inc. All rights reserved.

ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

PubMed Central

Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

2016-01-01

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

PubMed Central

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-01-01

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

PubMed

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, K.K.; Sanduja, R.; Tsai, A.L.

Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarlymore » low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.« less

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ϵ Subunit*

PubMed Central

Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Yano, Shio; Futai, Masamitsu

2014-01-01

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer108 in loop 2 and ϵTyr114 in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits. PMID:25228697

Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe

PubMed Central

Furuike, Shou; Nakano, Masahiro; Adachi, Kengo; Noji, Hiroyuki; Kinosita, Kazuhiko; Yokoyama, Ken

2011-01-01

Vacuole-type ATPases (VoV1) and FoF1 ATP synthases couple ATP hydrolysis/synthesis in the soluble V1 or F1 portion with proton (or Na+) flow in the membrane-embedded Vo or Fo portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V1 and the whole VoV1 from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V1 made 120° steps, commensurate with the presence of three catalytic sites. Dwells between the steps involved at least two events other than ATP binding, one likely to be ATP hydrolysis. VoV1 exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the Vo rotor in T. thermophilus. Unlike F1 that undergoes 80°–40° substepping, chemo-mechanical checkpoints in isolated V1 are all at the ATP-waiting position, and Vo adds further bumps through stator–rotor interactions outside and remote from V1. PMID:21407199

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

PubMed Central

Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

2014-01-01

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na+ and uptake of K+ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression. PMID:24452105

Epigenetic silencing of Na,K-ATPase β 1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma.

PubMed

Selvakumar, Ponniah; Owens, Tori A; David, Justin M; Petrelli, Nicholas J; Christensen, Brock C; Lakshmikuttyamma, Ashakumary; Rajasekaran, Ayyappan K

2014-04-01

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na(+) and uptake of K(+) across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β 1 (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients' tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2'-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.

Astakine LvAST binds to the β subunit of F1-ATP synthase and likely plays a role in white shrimp Litopeneaus vannamei defense against white spot syndrome virus.

PubMed

Liang, Gao-Feng; Liang, Yan; Xue, Qinggang; Lu, Jin-Feng; Cheng, Jun-Jun; Huang, Jie

2015-03-01

Cytokines play a critical role in innate and adaptive immunity. Astakines represent a group of invertebrate cytokines that are related to vertebrate prokineticin and function in promoting hematopoiesis in crustaceans. We have identified an astakine from the white shrimp Litopeneaus vannamei and named it LvAST in a previous research. In the present research, we investigated the interactions among LvAST, the envelope protein VP37 of white spot syndrome virus (i.e., WSSV), and the β subunit of F1-ATP synthase (ATPsyn-β) of the white shrimp (i.e., BP53) using binding assays and co-precipitations. We also examined the effects of LvAST on shrimp susceptibility to WSSV. We found that LvAST and VP37 competitively bound to BP53, but did not bind to each other. Shrimps that had been injected with recombinant LvAST exhibited significantly lower mortality and longer survival time in experimental infections by WSSV. In contrast, shrimps whose LvAST gene expression had been inhibited by RNA interference showed significantly higher WSSV infection intensity and shorter survival time following viral challenges. These results suggested that LvAST and WSSV both likely use ATPsyn-β as a receptor and LvAST plays a role in shrimp defense against WSSV infection. This represented the first research showing the involvement of astakines in host antiviral immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of the relationship between the Adenosine Triphosphate (ATP) bioluminescence assay and the presence of Bacillus anthracis spores and vegetative cells.

PubMed

Gibbs, Shawn G; Sayles, Harlan; Colbert, Erica M; Hewlett, Angela; Chaika, Oleg; Smith, Philip W

2014-05-28

The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.

Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation.

PubMed

Huang, Wei; Tikkanen, Mikko; Cai, Yan-Fei; Wang, Ji-Hua; Zhang, Shi-Bao

2018-06-11

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (g H + ) than the mature leaves. Furthermore, g H + was significantly and positively correlated with CO 2 assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that g H + was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in g H + . In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO 2 assimilation and photoprotection during leaf maturation. Copyright © 2018. Published by Elsevier B.V.

Predicted Structures of the Proton-Bound Membrane-Embedded Rotor Rings of the Saccharomyces cerevisiae and Escherichia coli ATP Synthases.

PubMed

Zhou, Wenchang; Leone, Vanessa; Krah, Alexander; Faraldo-Gómez, José D

2017-04-20

Recent years have witnessed a renewed interest in the ATP synthase as a drug target against human pathogens. Indeed, clinical, biochemical, and structural data indicate that hydrophobic inhibitors targeting the membrane-embedded proton-binding sites of the c-subunit ring could serve as last-resort antibiotics against multidrug resistant strains. However, because inhibition of the mitochondrial ATP synthase in humans is lethal, it is essential that these inhibitors be not only potent but also highly selective for the bacterial enzyme. To this end, a detailed understanding of the structure of this protein target is arguably instrumental. Here, we use computational methods to predict the atomic structures of the proton-binding sites in two prototypical c-rings: that of the ATP synthase from Saccharomyces cerevisiae, which is a model system for mitochondrial enzymes, and that from Escherichia coli, which can be pathogenic for humans. Our study reveals the structure of these binding sites loaded with protons and in the context of the membrane, that is, in the state that would mediate the recognition of a potential inhibitor. Both structures reflect a mode of proton coordination unlike those previously observed in other c-ring structures, whether experimental or modeled.

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

PubMed Central

Ostap, E. M.; Thomas, D. D.

1991-01-01

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin. PMID:1651780

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

PubMed

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

Evidence for rotation of V1-ATPase

PubMed Central

Imamura, Hiromi; Nakano, Masahiro; Noji, Hiroyuki; Muneyuki, Eiro; Ohkuma, Shoji; Yoshida, Masasuke; Yokoyama, Ken

2003-01-01

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft. PMID:12598655

Inhibition of F1-ATPase rotational catalysis by the carboxyl-terminal domain of the ϵ subunit.

PubMed

Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Yano, Shio; Futai, Masamitsu

2014-10-31

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer(108) in loop 2 and ϵTyr(114) in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

PubMed Central

Vahidi, Siavash; Bi, Yumin; Dunn, Stanley D.; Konermann, Lars

2016-01-01

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling. PMID:26884184

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

PubMed Central

Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

2011-01-01

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID

ATP6V1H regulates the growth and differentiation of bone marrow stromal cells.

PubMed

Li, Lin; Yang, Shaoqing; Zhang, Yanli; Ji, Dongrui; Jin, Zuolin; Duan, Xiaohong

2018-05-18

ATP6V1H encodes subunit H of vacuolar ATPase (V-ATPase) and may regulate osteoclastic function. The deficiency of ATP6V1H caused bone loss in human, mouse and zebrafish. In this report, we identified the mechanisms by which ATP6V1H regulates proliferation and differentiation of bone marrow stromal cells (BMSCs). We found that ATP6V1H was expressed in BMSCs, andAtp6v1h +/- BMSCs exhibited the lower proliferation rate, cell cycle arrest and reduced osteogenic differentiation capacity, as well as the increased adipogenic potentials. Histologic analysis confirmed less bone formation and more fatty degeneration in Atp6v1h +/- mice in the different age groups. Q-PCR analysis revealed that loss of ATP6V1H function downregulated the mRNA level of TGF-β1 receptor, and its binding molecule, subunit β of adaptor protein complex 2 (AP-2), suggesting ATP6V1H regulates the proliferation and differentiation of BMSCs by interacting with TGF-β receptor I and AP-2 complex. Copyright © 2018. Published by Elsevier Inc.

Asymmetry in the F1-ATPase and Its Implications for the Rotational Cycle

PubMed Central

Sun, Sean X.; Wang, Hongyun; Oster, George

2004-01-01

ATP synthase uses a rotary mechanism to carry out its cellular function of manufacturing ATP. The centralγ-shaft rotates inside a hexameric cylinder composed of alternating α- and β-subunits. When operating in the hydrolysis direction under high frictional loads and low ATP concentrations, a coordinated mechanochemical cycle in the three catalytic sites of the β-subunits rotates the γ-shaft in three 120° steps. At low frictional loads, the 120° steps alternate with three ATP-independent substeps separated by ∼30°. We present a quantitative model that accounts for these substeps and show that the observed pauses are due to 1), the asymmetry of the F1 hexamer that produces a propeller-like motion of the power-stroke and 2), the relatively tight binding of ADP to the catalytic sites. PMID:14990467

Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells.

PubMed

Bertoni, Ana Paula Santin; de Campos, Rafael Paschoal; Tsao, Marisa; Braganhol, Elizandra; Furlanetto, Tania Weber; Wink, Márcia Rosângela

2018-02-17

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

PubMed

Tantry, Subramanyam J; Markad, Shankar D; Shinde, Vikas; Bhat, Jyothi; Balakrishnan, Gayathri; Gupta, Amit K; Ambady, Anisha; Raichurkar, Anandkumar; Kedari, Chaitanyakumar; Sharma, Sreevalli; Mudugal, Naina V; Narayan, Ashwini; Naveen Kumar, C N; Nanduri, Robert; Bharath, Sowmya; Reddy, Jitendar; Panduga, Vijender; Prabhakar, K R; Kandaswamy, Karthikeyan; Saralaya, Ramanatha; Kaur, Parvinder; Dinesh, Neela; Guptha, Supreeth; Rich, Kirsty; Murray, David; Plant, Helen; Preston, Marian; Ashton, Helen; Plant, Darren; Walsh, Jarrod; Alcock, Peter; Naylor, Kathryn; Collier, Matthew; Whiteaker, James; McLaughlin, Robert E; Mallya, Meenakshi; Panda, Manoranjan; Rudrapatna, Suresh; Ramachandran, Vasanthi; Shandil, Radha; Sambandamurthy, Vasan K; Mdluli, Khisi; Cooper, Christopher B; Rubin, Harvey; Yano, Takahiro; Iyer, Pravin; Narayanan, Shridhar; Kavanagh, Stefan; Mukherjee, Kakoli; Balasubramanian, V; Hosagrahara, Vinayak P; Solapure, Suresh; Ravishankar, Sudha; Hameed P, Shahul

2017-02-23

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

The Barley Genome Sequence Assembly Reveals Three Additional Members of the CslF (1,3;1,4)-β-Glucan Synthase Gene Family

PubMed Central

Schreiber, Miriam; Wright, Frank; MacKenzie, Katrin; Hedley, Pete E.; Schwerdt, Julian G.; Little, Alan; Burton, Rachel A.; Fincher, Geoffrey B.; Marshall, David; Waugh, Robbie; Halpin, Claire

2014-01-01

An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls. PMID:24595438

Identification of Novel Mitochondrial Protein Components of Chlamydomonas reinhardtii. A Proteomic Approach1

PubMed Central

van Lis, Robert; Atteia, Ariane; Mendoza-Hernández, Guillermo; González-Halphen, Diego

2003-01-01

Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F1F0-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F1F0-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii. PMID:12746537

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

PubMed Central

Shoji, Kenji F; Sáez, Pablo J; Harcha, Paloma A; Aguila, Hector L; Sáez, Juan C

2014-01-01

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X7 receptors (P2X7Rs). However, a link between P2X7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1−/− mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1−/− mice, in which levels of P2X7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP. PMID:24590064

ATP7B detoxifies silver in ciliated airway epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ibricevic, Aida, E-mail: aidaibricevic@hotmail.co; Brody, Steven L., E-mail: sbrody@dom.wustl.ed; Youngs, Wiley J., E-mail: youngs@uakron.ed

2010-03-15

Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compoundsmore » but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.« less

The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

PubMed

Nath, Sunil

2016-12-01

As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

Protection of ATP-Depleted Cells by Impermeant Strychnine Derivatives

PubMed Central

Dong, Zheng; Venkatachalam, Manjeri A.; Weinberg, Joel M.; Saikumar, Pothana; Patel, Yogendra

2001-01-01

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine. PMID:11238050

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

PubMed

Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

2007-01-01

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures

[P4-ATP-ase Atp8b1/FIC1: structural properties and (patho)physiological functions].

PubMed

Korneenko, T V; Pestov, N B; Okkelman, I A; Modyanov, N N; Shakhparonov, M I

2015-01-01

P4-ATP-ases comprise an interesting family among P-type ATP-ases, since they are thought to play a major role in the transfer of phospholipids such as phosphatydylserine from the outer leaflet to the inner leaflet. Isoforms of P4-ATP-ases are partially interchangeable but peculiarities of tissue-specific expression of their genes, intracellular localization of proteins, as well as regulatory pathways lead to the fact that, on the organismal level, serious pathologies may develop in the presence of structural abnormalities in certain isoforms. Among P4-ATP-ases a special place is occupied by ATP8B1, for which several mutations are known that lead to serious hereditary diseases: two forms of congenital cholestasis (PFIC1 or Byler disease and benign recurrent intrahepatic cholestasis) with extraliver symptoms such as sensorineural hearing loss. The physiological function of the Atp8b1/FIC1 protein is known in general outline: it is responsible for transport of certain phospholipids (phosphatydylserine, cardiolipin) for the outer monolayer of the plasma membrane to the inner one. It is well known that perturbation of membrane asymmetry, caused by the lack of Atp8B1 activity, leads to death of hairy cells of the inner ear, dysfunction of bile acid transport in liver-cells that causes cirrhosis. It is also probable that insufficient activity of Atp8b1/FIC1 increases susceptibility to bacterial pneumonia.Regulatory pathways of Atp8b1/FIC1 activity in vivo remain to be insufficiently studied and this opens novel perspectives for research in this field that may allow better understanding of molecular processes behind the development of certain pathologies and to reveal novel therapeutical targets.

Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha

by the strikingly similar makeups of the active sites at the interfaces between {alpha}/{beta} heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.« less

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

PubMed Central

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells*

PubMed Central

Dai, Feihan F.; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B.

2015-01-01

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H+-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. PMID:26272612

ATP Synthesis in the Extremely Halophilic Bacteria

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

1994-01-01

The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

PubMed Central

Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

2010-01-01

Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

PubMed

Niimi, Kenta; Ueda, Mizuha; Fukumoto, Moe; Kohara, Misaki; Sawano, Toshinori; Tsuchihashi, Ryo; Shibata, Satoshi; Inagaki, Shinobu; Furuyama, Tatsuo

2017-08-05

Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

A Single Nucleotide Polymorphism Associates With the Response of Muscle ATP Synthesis to Long-Term Exercise Training in Relatives of Type 2 Diabetic Humans

PubMed Central

Kacerovsky-Bielesz, Gertrud; Kacerovsky, Michaela; Chmelik, Marek; Farukuoye, Michaela; Ling, Charlotte; Pokan, Rochus; Tschan, Harald; Szendroedi, Julia; Schmid, Albrecht Ingo; Gruber, Stephan; Herder, Christian; Wolzt, Michael; Moser, Ewald; Pacini, Giovanni; Smekal, Gerhard; Groop, Leif; Roden, Michael

2012-01-01

OBJECTIVE Myocellular ATP synthesis (fATP) associates with insulin sensitivity in first-degree relatives of subjects with type 2 diabetes. Short-term endurance training can modify their fATP and insulin sensitivity. This study examines the effects of moderate long-term exercise using endurance or resistance training in this cohort. RESEARCH DESIGN AND METHODS A randomized, parallel-group trial tested 16 glucose-tolerant nonobese relatives (8 subjects in the endurance training group and 8 subjects in the resistance training group) before and after 26 weeks of endurance or resistance training. Exercise performance was assessed from power output and oxygen uptake (Vo2) during incremental tests and from maximal torque of knee flexors (MaxTflex) and extensors (MaxText) using isokinetic dynamometry. fATP and ectopic lipids were measured with 1H/31P magnetic resonance spectroscopy. RESULTS Endurance training increased power output and Vo2 by 44 and 30%, respectively (both P < 0.001), whereas resistance training increased MaxText and MaxTflex by 23 and 40%, respectively (both P < 0.001). Across all groups, insulin sensitivity (382 ± 90 vs. 389 ± 40 mL ⋅ min−1 ⋅ m−2) and ectopic lipid contents were comparable after exercise training. However, 8 of 16 relatives had 26% greater fATP, increasing from 9.5 ± 2.3 to 11.9 ± 2.4 μmol ⋅ mL−1 ⋅ m−1 (P < 0.05). Six of eight responders were carriers of the G/G single nucleotide polymorphism rs540467 of the NDUFB6 gene (P = 0.019), which encodes a subunit of mitochondrial complex I. CONCLUSIONS Moderate exercise training for 6 months does not necessarily improve insulin sensitivity but may increase ATP synthase flux. Genetic predisposition can modify the individual response of the ATP synthase flux independently of insulin sensitivity. PMID:22190678

Variable effects of the mitoK(ATP) channel modulators diazoxide and 5-HD in ATP-depleted renal epithelial cells.

PubMed

Nilakantan, Vani; Liang, Huanling; Mortensen, Jordan; Taylor, Erin; Johnson, Christopher P

2010-02-01

The role of mitochondrial K(ATP) (mitoK(ATP)) channels in renal ischemia-reperfusion injury is controversial with studies showing both protective and deleterious effects. In this study, we compared the effects of the putative mitoK(ATP) opener, diazoxide, and the mitoK(ATP) blocker, 5-hydroxydecanoate (5-HD) on cytotoxicity and apoptosis in tubular epithelial cells derived from rat (NRK-52E) and pig (LLC-PK1) following in vitro ischemic injury. Following ATP depletion-recovery, there was a significant increase in cytotoxicity in both NRK cells and LLC-PK1 cells although NRK cells were more sensitive to the injury. Diazoxide treatment attenuated cytotoxicity in both cell types and 5-HD treatment-increased cytotoxicity in the sensitive NRK cells in a superoxide-dependant manner. The protective effect of diazoxide was also reversed in the presence of 5-HD in ATP-depleted NRK cells. The ATP depletion-mediated increase in superoxide was enhanced by both diazoxide and 5-HD with the effect being more pronounced in the cells undergoing 5-HD treatment. Further, ATP depletion-induced activation of caspase-3 was decreased by diazoxide in NRK cells. In order to determine the signaling pathways involved in apoptosis, we examined the activation of Erk and JNK in ATP-depleted NRK cells. Diazoxide-activated Erk in ATP-depleted cells, but did not have any effect on JNK activation. In contrast, 5-HD did not impact Erk levels but increased JNK activation even under controlled conditions. Further, the use of a JNK inhibitor with 5-HD reversed the deleterious effects of 5-HD. This study demonstrates that in cells that are sensitive to ATP depletion-recovery, mitoK(ATP) channels protect against ATP depletion-mediated cytotoxicity and apoptosis through Erk- and JNK-dependant mechanisms.

Cell Selective Cardiovascular Biology of Microsomal Prostaglandin E Synthase-1

PubMed Central

Chen, Lihong; Yang, Guangrui; Xu, Xiufeng; Grant, Gregory; Lawson, John A.; Bohlooly-Y, Mohammad; FitzGerald, Garret A.

2013-01-01

Background Global deletion of microsomal prostaglandin E synthase (mPGES) -1 in mice attenuates the response to vascular injury without a predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell specific differential utilization by prostaglandin (PG) synthases of the accumulated PGH2 substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and myeloid cells further to elucidate the cardiovascular function of this enzyme. Methods and Results VSMC and EC mPGES-1 deletion did not alter blood pressure at baseline or in response to a high salt diet. The propensity to evoked macrovascular and microvascular thrombogenesis was also unaltered. However, both VSMC and EC mPGES-1 deficient mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery compared to their littermate controls. The hyperplasia was associated with increased proliferating cell nuclear antigen (PCNA) and tenascin-C (TN-C) expression. In contrast, the response to injury was markedly suppressed by myeloid cell depletion of mPGES-1 with decreased hyperplasia, leukocyte infiltration and expression of PCNA and TN-C. Conditioned medium derived from mPGES-1 deficient macrophages less potently induced VSMC proliferation and migration than that from wild type macrophages. Conclusion Deletion of mPGES-1 in the vasculature and myeloid cells differentially modulates the response to vascular injury, implicating macrophage mPGES-1 as a cardiovascular drug target. PMID:23204105

ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis.

PubMed Central

Stefanelli, C; Bonavita, F; Stanic', I; Farruggia, G; Falcieri, E; Robuffo, I; Pignatti, C; Muscari, C; Rossoni, C; Guarnieri, C; Caldarera, C M

1997-01-01

In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some

Renal epithelial cells can release ATP by vesicular fusion

PubMed Central

Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

2013-01-01

Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

PubMed

Martin, Gregory M; Rex, Emily A; Devaraneni, Prasanna; Denton, Jerod S; Boodhansingh, Kara E; DeLeon, Diva D; Stanley, Charles A; Shyng, Show-Ling

2016-10-14

ATP-sensitive potassium (K ATP ) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of K ATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by K ATP channel openers. Cross-linking experiments showed that K ATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the K ATP channel opener diazoxide. Our study expands the list of K ATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of the αβ Conformational Change in F1-ATPase after ATP Hydrolysis: Free-Energy Simulations

PubMed Central

Ito, Yuko; Ikeguchi, Mitsunori

2015-01-01

One of the motive forces for F1-ATPase rotation is the conformational change of the catalytically active β subunit due to closing and opening motions caused by ATP binding and hydrolysis, respectively. The closing motion is accomplished in two steps: the hydrogen-bond network around ATP changes and then the entire structure changes via B-helix sliding, as shown in our previous study. Here, we investigated the opening motion induced by ATP hydrolysis using all-atom free-energy simulations, combining the nudged elastic band method and umbrella sampling molecular-dynamics simulations. Because hydrolysis requires residues in the α subunit, the simulations were performed with the αβ dimer. The results indicate that the large-scale opening motion is also achieved by the B-helix sliding (in the reverse direction). However, the sliding mechanism is different from that of ATP binding because sliding is triggered by separation of the hydrolysis products ADP and Pi. We also addressed several important issues: 1), the timing of the product Pi release; 2), the unresolved half-closed β structure; and 3), the ADP release mechanism. These issues are fundamental for motor function; thus, the rotational mechanism of the entire F1-ATPase is also elucidated through this αβ study. During the conformational change, conserved residues among the ATPase proteins play important roles, suggesting that the obtained mechanism may be shared with other ATPase proteins. When combined with our previous studies, these results provide a comprehensive view of the β-subunit conformational change that drives the ATPase. PMID:25564855

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase.

PubMed

Ezaki, J; Takeda-Ezaki, M; Kominami, E

2000-09-01

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.

Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

PubMed

Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

2017-09-08

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proton gradients produced by glucose oxidase microcapsules containing motor F0F1-ATPase for continuous ATP biosynthesis.

PubMed

Duan, Li; Qi, Wei; Yan, Xuehai; He, Qiang; Cui, Yue; Wang, Kewei; Li, Dongxiang; Li, Junbai

2009-01-15

Glucose oxidase (GOD) microcapsules held together by cross-linker, glutaraldehyde (GA), are fabricated by the layer-by-layer (LbL) assembly technique. The lipid bilayer containing CF(0)F(1)-ATPase was coated on the outer shell of GOD microcapsules. Driven under the proton gradients produced by catalysis of GOD microcapsules for glucose, ATP is synthesized from ADP and inorganic phosphate catalyzed by the ATPase rotary catalysis. The results show here that ATPase reconstituted on the GOD microcapsules retains its catalytic activity.

Rotation of Subunits During Catalysis by Escherichia coli F_1-ATPase

NASA Astrophysics Data System (ADS)

Duncan, Thomas M.; Bulygin, Vladimir V.; Zhou, Yuantai; Hutcheon, Marcus L.; Cross, Richard L.

1995-11-01

During oxidative and photo-phosphorylation, F_0F_1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F_0F_1. Guided by a recent, high-resolution structure for bovine F_1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central γ subunit relative to the three catalytic β subunits in soluble F_1 from Escherichia coli. In the bovine F_1 structure, a specific point of contact between the γ subunit and one of the three catalytic β subunits includes positioning of the homolog of E. coli γ-subunit C87 (γC87) close to the β-subunit 380DELSEED386 sequence. A βD380C mutation allowed us to induce formation of a specific disulfide bond between β and γC87 in soluble E. coli F_1. Formation of the crosslink inactivated βD380C-F_1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked βD380C-F_1, we incorporated radiolabeled β subunits into the two noncrosslinked β-subunit positions of F_1. After reduction of the initial nonradio-active β-γ crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled β subunits with γC87 upon reoxidation. The results demonstrate that γ subunit rotates relative to the β subunits during catalysis.

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

PubMed

Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

2015-08-01

F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1. © 2015 FEBS.

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase.

PubMed

Nagatoishi, Satoru; Yamaguchi, Sou; Katoh, Etsuko; Kajita, Keita; Yokotagawa, Takane; Kanai, Satoru; Furuya, Toshio; Tsumoto, Kouhei

2018-05-01

19 F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19 F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19 F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19 F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yun

quantification mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca 2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca 2+ signaling through gap junctions seems more effective.« less

Light and Fungal Elicitor Induce 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase mRNA in Suspension Cultured Cells of Parsley (Petroselinum crispum L.) 1

PubMed Central

Henstrand, John M.; McCue, Kent F.; Brink, Kent; Handa, Avtar K.; Herrmann, Klaus M.; Conn, Eric E.

1992-01-01

Light and fungal elicitor induce mRNA encoding 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension cultured cells of parsley (Petroselinum crispum L.). The kinetics and dose response of mRNA accumulation were similar for DAHP synthase and phenylalanine ammonia-lyase (PAL). Six micrograms of elicitor from Phytophthora megasperma f. glycinia gave a detectable induction within 1 hour. Induction of DAHP synthase and PAL mRNAs by light was transient, reaching maximal levels at 4 hours and returning to pretreatment levels after 24 hours. Our data suggest that either light or fungal elicitor transcriptionally activate DAHP synthase. A coordinate regulation for key enzymes in the synthesis of primary and secondary metabolites is indicated. ImagesFigure 1 PMID:16668708

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

PubMed

Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

2018-04-10

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes1[W][OA

PubMed Central

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T.; Galway, Moira E.; Mansfield, Shawn D.; Hocart, Charles H.; Wasteneys, Geoffrey O.

2013-01-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1’s permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature. PMID:23532584

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

PubMed Central

Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Lam, Hon-Ming

2016-01-01

G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein’s ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure–function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

Inhibitory effect of protopine on K(ATP) channel subunits expressed in HEK-293 cells.

PubMed

Jiang, Bo; Cao, Kun; Wang, Rui

2004-12-15

Protopine is an isoquinoline alkaloid purified from Corydalis tubers and other families of medicinal plants. The purpose of the present study was to investigate the effects of protopine on K(ATP) channels and big conductance (BKCa) channels. Protopine concentration-dependently inhibited K(ATP) channel currents in human embryonic kidney cells (HEK-293) which were cotransfected with Kir6.1 and sulfonylurea receptor 1 (SUR1) subunits, but not that with Kir6.1 cDNA transfection alone. At 25 muM, protopine reversibly decreased Kir6.1/SUR1 currents densities from -17.4+/-3 to -13.2+/-2.4 pA/pF at -60 mV (n=5, P<0.05). The heterologously expressed mSlo-encoded BK(Ca) channel currents in HEK-293 cells were not affected by protopine (25 muM), although iberiotoxin (100 nM) significantly inhibited the expressed BK(Ca) currents (n=5, P<0.05). In summary, protopine selectively inhibited K(ATP) channels by targeting on SUR1 subunit. This discovery may help design specific agents to selectively modulate the function of Kir6.1/SUR1 channel complex and facilitate the understanding of the structure-function relationship of specific subtype of K(ATP) channels.

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

PubMed

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T; Galway, Moira E; Mansfield, Shawn D; Hocart, Charles H; Wasteneys, Geoffrey O

2013-05-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

Chaperones of F[subscript 1]-ATPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas

2009-09-25

Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11pmore » for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.« less

Silencing the Menkes Copper-Transporting ATPase (Atp7a) Gene in Rat Intestinal Epithelial (IEC-6) Cells Increases Iron Flux via Transcriptional Induction of Ferroportin 1 (Fpn1)123

PubMed Central

Gulec, Sukru; Collins, James F.

2014-01-01

The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron (59Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished. PMID:24174620

Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

PubMed

Spaans, F; Melgert, B N; Chiang, C; Borghuis, T; Klok, P A; de Vos, P; van Goor, H; Bakker, W W; Faas, M M

2014-08-01

Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

PubMed

Masha, Roland T; Houreld, Nicolette N; Abrahamse, Heidi

2013-02-01

Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Programmed death-1 controls T cell survival by regulating oxidative metabolism1

PubMed Central

Tkachev, Victor; Goodell, Stefanie; Opipari, Anthony W.; Hao, Ling-Yang; Franchi, Luigi; Glick, Gary D.; Ferrara, James L.M.; Byersdorfer, Craig A.

2015-01-01

The co-inhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly up-regulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1HiROSHi phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels and PD-1 driven increases in ROS were dependent upon the oxidation of fatty acids, as treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by anti-oxidants. Furthermore, PD-1 driven changes in ROS were fundamental to establishing a cell’s susceptibility to subsequent metabolic inhibition, as blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing reactive oxygen species in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic. PMID:25972478

Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

PubMed

Guntur, Anyonya R; Gerencser, Akos A; Le, Phuong T; DeMambro, Victoria E; Bornstein, Sheila A; Mookerjee, Shona A; Maridas, David E; Clemmons, David E; Brand, Martin D; Rosen, Clifford J

2018-06-01

Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

PubMed

Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

2012-12-01

Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

Phosphate release coupled to rotary motion of F1-ATPase

PubMed Central

Okazaki, Kei-ichi; Hummer, Gerhard

2013-01-01

F1-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three αβ-dimers creates torque on its central γ-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation. In response to torque-driven rotation of the γ-subunit in the hydrolysis direction, the nucleotide-free αβE interface forming the “empty” E site loosens and singly charged Pi readily escapes to the P loop. By contrast, the interface stays closed with doubly charged Pi. The γ-rotation tightens the ATP-bound αβTP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the αβE interface 120° after ATP hydrolysis closely matches the ∼1-ms functional timescale. Conversely, Pi release from the ADP-bound αβDP interface postulated in earlier models would occur through a kinetically infeasible inward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of Pi exit, its pathway and kinetics, associated changes in Pi protonation, and changes of the F1-ATPase structure in the 40° substep. Important elements of the molecular mechanism of Pi release emerging from our simulations appear to be conserved in myosin despite the different functional motions. PMID:24062450

Structure and mechanism of the ATP synthase membrane motor inferred from quantitative integrative modeling.

PubMed

Leone, Vanessa; Faraldo-Gómez, José D

2016-12-01

Two subunits within the transmembrane domain of the ATP synthase-the c-ring and subunit a-energize the production of 90% of cellular ATP by transducing an electrochemical gradient of H + or Na + into rotational motion. The nature of this turbine-like energy conversion mechanism has been elusive for decades, owing to the lack of definitive structural information on subunit a or its c-ring interface. In a recent breakthrough, several structures of this complex were resolved by cryo-electron microscopy (cryo-EM), but the modest resolution of the data has led to divergent interpretations. Moreover, the unexpected architecture of the complex has cast doubts on a wealth of earlier biochemical analyses conducted to probe this structure. Here, we use quantitative molecular-modeling methods to derive a structure of the a-c complex that is not only objectively consistent with the cryo-EM data, but also with correlated mutation analyses of both subunits and with prior cross-linking and cysteine accessibility measurements. This systematic, integrative approach reveals unambiguously the topology of subunit a and its relationship with the c-ring. Mapping of known Cd 2+ block sites and conserved protonatable residues onto the structure delineates two noncontiguous pathways across the complex, connecting two adjacent proton-binding sites in the c-ring to the space on either side of the membrane. The location of these binding sites and of a strictly conserved arginine on subunit a, which serves to prevent protons from hopping between them, explains the directionality of the rotary mechanism and its strict coupling to the proton-motive force. Additionally, mapping of mutations conferring resistance to oligomycin unexpectedly reveals that this prototypical inhibitor may bind to two distinct sites at the a-c interface, explaining its ability to block the mechanism of the enzyme irrespective of the direction of rotation of the c-ring. In summary, this study is a stepping stone toward

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

PubMed

Liu, Yan-Yan; Sun, Ling-Cong; Wei, Jing-Jing; Li, Dong; Yuan, Ye; Yan, Bin; Liang, Zhi-Hui; Zhu, Hui-Fen; Xu, Yong; Li, Bo; Song, Chuan-Wang; Liao, Sheng-Jun; Lei, Zhang; Zhang, Gui-Mei; Feng, Zuo-Hua

2010-09-01

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

Anatomy of F1-ATPase powered rotation.

PubMed

Martin, James L; Ishmukhametov, Robert; Hornung, Tassilo; Ahmad, Zulfiqar; Frasch, Wayne D

2014-03-11

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank).

Anatomy of F1-ATPase powered rotation

PubMed Central

Martin, James L.; Ishmukhametov, Robert; Hornung, Tassilo; Ahmad, Zulfiqar; Frasch, Wayne D.

2014-01-01

F1-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F1 structures 2HLD1 and 1H8E (Protein Data Bank). PMID:24567403

Mechanism of action of minoxidil in the treatment of androgenetic alopecia is likely mediated by mitochondrial adenosine triphosphate synthase-induced stem cell differentiation.

PubMed

Goren, A; Naccarato, T; Situm, M; Kovacevic, M; Lotti, T; McCoy, J

2017-01-01

Topical minoxidil is the only topical drug approved by the US Food and Drug Administration (FDA) for the treatment of androgenetic alopecia. However, the exact mechanism by which minoxidil stimulates anagen phase and promotes hair growth is not fully understood. In the late telegen phase of the hair follicle growth cycle, stem cells located in the bulge region differentiate and re-enter anagen phase, a period of growth lasting 2-6 years. In androgenetic alopecia, the anagen phase is shortened and a progressive miniaturization of hair follicles occurs, eventually leading to hair loss. Several studies have demonstrated that minoxidil increases the amount of intracellular Ca2+, which has been shown to up-regulate the enzyme adenosine triphosphate (ATP) synthase. A recent study demonstrated that ATP synthase, independent of its role in ATP synthesis, promotes stem cell differentiation. As such, we propose that minoxidil induced Ca2+ influx can increase stem cell differentiation and may be a key factor in the mechanism by which minoxidil facilitates hair growth. Based on our theory, we provide a roadmap for the development of a new class of drugs for the treatment of androgenetic alopecia.

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

PubMed Central

Garg, Abhishek D; Krysko, Dmitri V; Verfaillie, Tom; Kaczmarek, Agnieszka; Ferreira, Gabriela B; Marysael, Thierry; Rubio, Noemi; Firczuk, Malgorzata; Mathieu, Chantal; Roebroek, Anton J M; Annaert, Wim; Golab, Jakub; de Witte, Peter; Vandenabeele, Peter; Agostinis, Patrizia

2012-01-01

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC-IIhigh) and functional stimulation (NOhigh, IL-10absent, IL-1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress. PMID:22252128

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

PubMed

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Neurospora crassa 1,3-α-glucan synthase, AGS-1, is required for cell wall biosynthesis during macroconidia development

PubMed Central

Fu, Ci; Tanaka, Asuma

2014-01-01

The Neurospora crassa genome encodes two 1,3-α-glucan synthases. One of these 1,3-α-glucan synthase genes, ags-1, was shown to be required for the synthesis of 1,3-α-glucan in the aerial hyphae and macroconidia cell walls. 1,3-α-Glucan was found in the conidia cell wall, but was absent from the vegetative hyphae cell wall. Deletion of ags-1 affected conidial development. Δags-1 produced only 5 % as many conidia as the WT and most of the conidia produced by Δags-1 were not viable. The ags-1 upstream regulatory elements were shown to direct cell-type-specific expression of red fluorescent protein in conidia and aerial hyphae. A haemagglutinin-tagged AGS-1 was found to be expressed in aerial hyphae and conidia. The research showed that 1,3-α-glucan is an aerial hyphae and conidia cell wall component, and is required for normal conidial differentiation. PMID:24847001

Persistence of biomarker ATP and ATP-generating capability in bacterial cells and spores contaminating spacecraft materials under earth conditions and in a simulated martian environment.

PubMed

Fajardo-Cavazos, Patricia; Schuerger, Andrew C; Nicholson, Wayne L

2008-08-01

Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5'-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m(-2) of UV-C (200 to 280 nm), -10 degrees C, and a Mars gas mix of CO(2) (95.54%), N(2) (2.7%), Ar (1.6%), O(2) (0.13%), and H(2)O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.

Palmitoleic acid (16:1n7) increases oxygen consumption, fatty acid oxidation and ATP content in white adipocytes.

PubMed

Cruz, Maysa M; Lopes, Andressa B; Crisma, Amanda R; de Sá, Roberta C C; Kuwabara, Wilson M T; Curi, Rui; de Andrade, Paula B M; Alonso-Vale, Maria I C

2018-03-20

We have recently demonstrated that palmitoleic acid (16:1n7) increases lipolysis, glucose uptake and glucose utilization for energy production in white adipose cells. In the present study, we tested the hypothesis that palmitoleic acid modulates bioenergetic activity in white adipocytes. For this, 3 T3-L1 pre-adipocytes were differentiated into mature adipocytes in the presence (or absence) of palmitic (16:0) or palmitoleic (16:1n7) acid at 100 or 200 μM. The following parameters were evaluated: lipolysis, lipogenesis, fatty acid (FA) oxidation, ATP content, oxygen consumption, mitochondrial mass, citrate synthase activity and protein content of mitochondrial oxidative phosphorylation (OXPHOS) complexes. Treatment with 16:1n7 during 9 days raised basal and isoproterenol-stimulated lipolysis, FA incorporation into triacylglycerol (TAG), FA oxidation, oxygen consumption, protein expression of subunits representing OXPHOS complex II, III, and V and intracellular ATP content. These effects were not observed in adipocytes treated with 16:0. Palmitoleic acid, by concerted action on lipolysis, FA esterification, mitochondrial FA oxidation, oxygen consumption and ATP content, does enhance white adipocyte energy expenditure and may act as local hormone.

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

PubMed

Belcik, J Todd; Davidson, Brian P; Xie, Aris; Wu, Melinda D; Yadava, Mrinal; Qi, Yue; Liang, Sherry; Chon, Chae Ryung; Ammi, Azzdine Y; Field, Joshua; Harmann, Leanne; Chilian, William M; Linden, Joel; Lindner, Jonathan R

2017-03-28

Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 8 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of

Emodin Inhibits ATP-Induced Proliferation and Migration by Suppressing P2Y Receptors in Human Lung Adenocarcinoma Cells.

PubMed

Wang, Xia; Li, Long; Guan, Ruijuan; Zhu, Danian; Song, Nana; Shen, Linlin

2017-01-01

Extracellular ATP performs multiple important functions via activation of P2 receptors on the cell surface. P2Y receptors play critical roles in ATP evoked response in human lung adenocarcinoma cells (A549 cells). Emodin is an anthraquinone derivative originally isolated from Chinese rhubarb, possesses anticancer properties. In this study we examined the inhibiting effects of emodin on proliferation, migration and epithelial-mesenchymal transition (EMT) by suppressing P2Y receptors-dependent Ca2+ increase and nuclear factor-κB (NF-KB) signaling in A549 cells. A549 cells were pretreated with emodin before stimulation with ATP for the indicated time. Then, intracellular Ca2+ concentration ([Ca2+]i) was measured by Fluo-8/AM staining. Cell proliferation and cell cycle progression were tested by CCK8 assay and flow cytometry In addition, wound healing and western blot were performed to determine cell migration and related protein levels (Bcl-2, Bax, claudin-1, NF-κB). Emodin blunted ATP/UTP-induced increase of [Ca2+]i and cell proliferation concentration-dependently Meanwhile, it decreased ATP-induced cells accumulation in the S phase. Furthermore, emodin altered protein abundance of Bcl-2, Bax and claudin-1 and attenuated EMT caused by ATP. Such ATP-induced cellular reactions were also inhibited by a nonselective P2Y receptors antagonist, suramin, in a similar way to emodin. Besides, emodin could inhibit activation of NF-κB, thus suppressed ATP-induced proliferation, migration and EMT. Our results demonstrated that emodin inhibits ATP-induced proliferation, migration, EMT by suppressing P2Y receptors-mediated [Ca2+]i increase and NF-κB signaling in A549 cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Use of luciferase probes to measure ATP in living cells and animals.

PubMed

Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

2017-08-01

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

PubMed

Dong, Z; Venkatachalam, M A; Weinberg, J M; Saikumar, P; Patel, Y

2001-03-01

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

PubMed

Sunggip, Caroline; Shimoda, Kakeru; Oda, Sayaka; Tanaka, Tomohiro; Nishiyama, Kazuhiro; Mangmool, Supachoke; Nishimura, Akiyuki; Numaga-Tomita, Takuro; Nishida, Motohiro

2018-01-01

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca 2+ -dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca 2+ -mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca 2+ signaling requires inositol 1,4,5-trisphosphate (IP 3 ) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca 2+ /NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP 3 -mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

PubMed Central

Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

2016-01-01

Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

Biophysical comparison of ATP synthesis mechanisms shows a kinetic advantage for the rotary process.

PubMed

Anandakrishnan, Ramu; Zhang, Zining; Donovan-Maiye, Rory; Zuckerman, Daniel M

2016-10-04

The ATP synthase (F-ATPase) is a highly complex rotary machine that synthesizes ATP, powered by a proton electrochemical gradient. Why did evolution select such an elaborate mechanism over arguably simpler alternating-access processes that can be reversed to perform ATP synthesis? We studied a systematic enumeration of alternative mechanisms, using numerical and theoretical means. When the alternative models are optimized subject to fundamental thermodynamic constraints, they fail to match the kinetic ability of the rotary mechanism over a wide range of conditions, particularly under low-energy conditions. We used a physically interpretable, closed-form solution for the steady-state rate for an arbitrary chemical cycle, which clarifies kinetic effects of complex free-energy landscapes. Our analysis also yields insights into the debated "kinetic equivalence" of ATP synthesis driven by transmembrane pH and potential difference. Overall, our study suggests that the complexity of the F-ATPase may have resulted from positive selection for its kinetic advantage.

Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA.

PubMed

Sgarbi, Gianluca; Baracca, Alessandra; Lenaz, Giorgio; Valentino, Lucia M; Carelli, Valerio; Solaini, Giancarlo

2006-05-01

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyler, Andreas, E-mail: andreas.tyler@medbio.umu.se; Johansson, Anders; Karlsson, Terese

Background: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expressionmore » of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance. Methods: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analyzed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72 h on expression and cisplatin cytotoxicity was tested. Results: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells. Conclusions: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin

AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase*

PubMed Central

Gregg, Christina M.; Goetzl, Sebastian; Jeoung, Jae-Hun

2016-01-01

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO, CoA, and a methyl-cation to form acetyl-CoA at a unique Ni,Ni-[4Fe4S] cluster (the A-cluster). However, it was unknown which proteins support the assembly of the A-cluster. We analyzed the product of a gene from the cluster containing the ACS gene, cooC2 from Carboxydothermus hydrogenoformans, named AcsFCh, and showed that it acts as a maturation factor of ACS. AcsFCh and inactive ACS form a stable 2:1 complex that binds two nickel ions with higher affinity than the individual components. The nickel-bound ACS-AcsFCh complex remains inactive until MgATP is added, thereby converting inactive to active ACS. AcsFCh is a MinD-type ATPase and belongs to the CooC protein family, which can be divided into homologous subgroups. We propose that proteins of one subgroup are responsible for assembling the Ni,Ni-[4Fe4S] cluster of ACS, whereas proteins of a second subgroup mature the [Ni4Fe4S] cluster of carbon monoxide dehydrogenases. PMID:27382049

A genetic polymorphism in the sex-linked ATP5A1 gene is associated with individual fitness in Ovenbirds (Seiurus aurocapilla)

Treesearch

Judith D. Toms; Lori S. Eggert; Wayne J. Arendt; John Faaborg

2012-01-01

While testing genetic sexing techniques in Ovenbirds (Seiurus aurocapilla),we found a genetic polymorphism in the ATP5A1 gene in 38% of individuals. The Z ' allele included changes in both intronic and exonic portions of the sequenced region, but there was no evidence that this changed the resulting ATP synthase product. Males that had one or more copies of...

An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad.

PubMed Central

Ugur, M; Drummond, R M; Zou, H; Sheng, P; Singer, J J; Walsh, J V

1997-01-01

1. Whole-cell and single-channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP-gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP-gated channel does not mediate the formation of large non-specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole-cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl- did not. Ratiometric fura-2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell-attached or excised patches elicited inwardly rectifying single-channel currents with a 22 pS conductance. Cl- did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of -70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP-induced whole-cell currents. Mg2+ decreased both the amplitude and the activity of ATP-activated single-channel currents. 6. ADP, UTP, P1, P5-di-adenosine pentaphosphate (AP5A), adenosine and alpha, beta-methylene ATP (alpha, beta-Me-ATP) did not induce significant whole-cell current. ATP-gamma-S and 2-methylthio ATP (2-Me-S-ATP) were significantly less effective than ATP in inducing whole-cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP

Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells.

PubMed

Yee, Karen K; Sukumaran, Sunil K; Kotha, Ramana; Gilbertson, Timothy A; Margolskee, Robert F

2011-03-29

Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium-glucose cotransporter (SGLT1), and two components of the ATP-gated K(+) (K(ATP)) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K(ATP) channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K(ATP) channels constitute a major portion of K(+) channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K(ATP) may serve as mediators of the T1r-independent sweet taste of sugars.

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

PubMed

Liu, Jun; Liu, Wenjing; Yang, Jun

2016-02-11

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

PubMed Central

Liu, Jun; Liu, Wenjing; Yang, Jun

2016-01-01

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

PubMed

Xu, Pei; Yang, Yuwen; Zhang, Zhengzhi; Chen, Weihua; Zhang, Caiqin; Zhang, Lixia; Zou, Sixiang; Ma, Zhengqiang

2008-01-01

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

[Change in concentration of cytosolic Ca2+ caused by extracellular ATP and ecto-ATP-ase activity in thymocytes and transformed MT-4 cells].

PubMed

Hrebinyk, S M; Artemenko, O Iu; Hryniuk, I I; Perepelitsyna, O M; Matyshevs'ka, O P

2009-01-01

The comparative study of extracellular ATP (ATP0) effect on free cytosolic calcium concentration ([Ca2+]i) in normal (isolated rat thymocytes) and transformed (leukosis MT-4 line) T-cells was carried out. Addition of 1 mM ATP to Ca-free incubation medium of both types of cells, loaded with indo-1, had no effect on [Ca2+]i level. Upon subsequent addition of 1 mM CaCl2 to the incubation medium the rapid and significant increase of [Ca2+]i in MT-4 cells was registered. This effect was maintained within 10 min and was not inhibited by phospholipase C inhibitor 0.2 mM neomycin, that was induced by cation entry into the cells from the extracellular medium. Both types of cells were shown to demonstrate ecto-ATPase activity in the presence of 1 mM MgCl2 or CaC12 in the incubation medium. Estimation of kinetic parameters has indicated that the maximum rate of extracellular ATP hydrolysis by MT-4 cells is higher and Mg2+ and Ca2+ activation constants are lower as compared to respective parameters of ATP hydrolysis by thymocytes. The possible functional significance of the increased level of ecto-ATPase activity in malignantly transformed cells is discussed.

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cellmore » cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.« less

Regulation of necrotic cell death: p53, PARP1 and cyclophilin D-overlapping pathways of regulated necrosis?

PubMed

Ying, Yuan; Padanilam, Babu J

2016-06-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator and the phosphate carrier are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury.

Propofol restores TRPV1 sensitivity via a TRPA1-, nitric oxide synthase-dependent activation of PKCε

PubMed Central

Sinharoy, Pritam; Zhang, Hongyu; Sinha, Sayantani; Prudner, Bethany C; Bratz, Ian N; Damron, Derek S

2015-01-01

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε. PMID:26171233

ATP and microfilaments in cellular oxidant injury.

PubMed Central

Hinshaw, D. B.; Armstrong, B. C.; Burger, J. M.; Beals, T. F.; Hyslop, P. A.

1988-01-01

Oxidant injury produces dramatic changes in cytoskeletal organization and cell shape. ATP synthetic pathways are major targets of oxidant injury resulting in rapid depletion of cellular ATP following oxidant exposure. The relation of ATP depletion to the changes in microfilament organization seen following H2O2 exposure were examined in the P388D1 cell line. Three hours of glucose depletion alone resulted in a decline in cellular ATP levels to less than 10% of controls, which was comparable to ATP levels in cells 30 to 60 minutes after exposure to 5 mM H2O2 in the presence of glucose. Adherent cells stained with rhodamine phalloidin, a probe specific for polymerized (F) actin, revealed a progressive shortening of microfilaments into globular aggregates within cells depleted of glucose over 3 hours, a pattern similar to earlier observations of H2O2-injured cells after 1 hour. The changes in cellular ATP associated with glucose depletion or H2O2 exposure were then correlated with G actin content measured by the DNAse 1 inhibition assay. No real differences in G actin content as a percentage of total actin were seen in P388D1 cells following 3 hours of glucose depletion or 30 to 60 minutes after exposure to 5 mM H2O2. But 2 to 3 hours after exposure to H2O2 there was a progressive decrease in G actin as a percentage of total actin within the cells. Transmission electron microscopy of cells depleted of glucose for 3 h or 1 hour after exposure to H2O2 revealed the presence of side-to-side aggregates or bundles of microfilaments within the cells. These observations suggest that declining levels of ATP either from metabolic inhibition or H2O2 injury are correlated with the fragmentation and shortening of microfilaments into aggregates. No net change in monomeric or polymeric actin was necessary for this to occur. However, at later time points after H2O2 exposure some actin assembly did occur. Images p[484]-a p481-a p482-a Figure 2 Figure 3 PMID:3414780

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2015-10-15

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

PubMed Central

Ferguson, Scott A.; Cook, Gregory M.; Montgomery, Martin G.; Leslie, Andrew G. W.

2016-01-01

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. PMID:27621435

ATP synthase β-subunit abnormality in pancreas islets of rats with polycystic ovary syndrome and type 2 diabetes mellitus.

PubMed

Li, Wei; Li, Sai-Jiao; Yin, Tai-Lang; Yang, Jing; Cheng, Yan

2017-04-01

This study investigated the abnormal expression of ATP synthase β-subunit (ATPsyn-β) in pancreas islets of rat model of polycystic ovary syndrome (PCOS) with type 2 diabetes mellitus (T2DM), and the secretion function changes after up-regulation of ATP5b. Sixty female SD rats were divided into three groups randomly and equally. The rat model of PCOS with T2DM was established by free access to the high-carbohydrate/high-fat diet, subcutaneous injections of DHEA, and a single injection of streptozotocin. The pancreas was removed for the detection of the ATPsyn-β expression by immunohistochemical staining, Western blotting and reverse transcription-PCR (RT-PCR). The pancreas islets of the rats were cultured, isolated with collagenase V and purified by gradient centrifugation, and the insulin secretion after treatment with different glucose concentrations was tested. Lentivirus ATP5b was successfully constructed with the vector of GV208 and transfected into the pancreas islets for the over-expression of ATPsyn-β. The insulin secretion and intracellular ATP content were determined after transfection of the PCOS-T2DM pancreas islets with Lenti-ATP5b. The results showed that the expression of ATPsyn-β protein and mRNA was significantly decreased in the pancreas of PCOS-T2DM rats. The ATP content in the pancreas islets was greatly increased and the insulin secretion was improved after the up-regulation of ATPsyn-β in the pancreas islets transfected with lenti-ATP5b. These results indicated that for PCOS, the ATPsyn-β might be one of the key factors for the attack of T2DM.

Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

PubMed

Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

2017-07-01

Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels

The process of lipid storage in insect oocytes: The involvement of β-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas' disease vector Panstrongylus megistus (Hemiptera: Reduviidae).

PubMed

Fruttero, Leonardo L; Leyria, Jimena; Ramos, Fabián O; Stariolo, Raúl; Settembrini, Beatriz P; Canavoso, Lilián E

2017-01-01

Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

PubMed

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

PubMed

Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

2011-11-01

In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

PubMed

Esher, Shannon K; Ost, Kyla S; Kohlbrenner, Maria A; Pianalto, Kaila M; Telzrow, Calla L; Campuzano, Althea; Nichols, Connie B; Munro, Carol; Wormley, Floyd L; Alspaugh, J Andrew

2018-06-01

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

PubMed Central

Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

2015-01-01

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

PubMed Central

1989-01-01

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino

ATP-induced current in isolated outer hair cells of guinea pig cochlea.

PubMed

Nakagawa, T; Akaike, N; Kimitsuki, T; Komune, S; Arima, T

1990-05-01

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

PubMed

Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C

2015-09-01

Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Regulation of necrotic cell death p53, PARP1 and Cyclophilin D -overlapping pathways of regulated necrosis?

PubMed Central

Ying, Yuan; Padanilam, Babu J.

2017-01-01

In contrast to apoptosis and autophagy, necrotic cell death was considered to be a random, passive cell death without definable mediators. However, this dogma has been challenged by recent developments suggesting that necrotic cell death can also be a regulated process. Regulated necrosis includes multiple cell death modalities such as necroptosis, parthanatos, ferroptosis, pyroptosis, and mitochondrial permeability transition pore (MPTP)-mediated necrosis. Several distinctive executive molecules, particularly residing on the mitochondrial inner and outer membrane, amalgamating to form the MPTP have been defined. The c-subunit of the F1F0ATP synthase on the inner membrane and Bax/Bak on the outer membrane are considered to be the long sought components that form the MPTP. Opening of the MPTP results in loss of mitochondrial inner membrane potential, disruption of ATP production, increased ROS production, organelle swelling, mitochondrial dysfunction and consequent necrosis. Cyclophilin D, along with adenine nucleotide translocator (ANT) and the phosphate carrier (PiC) are considered to be important regulators involved in the opening of MPTP. Increased production of ROS can further trigger other necrotic pathways mediated through molecules such as PARP1, leading to irreversible cell damage. This review examines the roles of PARP1 and cyclophilin D in necrotic cell death. The hierarchical role of p53 in regulation and integration of key components of signaling pathway to elicit MPTP-mediated necrosis and ferroptosis is explored. In the context of recent insights, the indistinct role of necroptosis signaling in tubular necrosis after ischemic kidney injury is scrutinized. We conclude by discussing the participation of p53, PARP1 and cyclophilin D and their overlapping pathways to elicit MPTP-mediated necrosis and ferroptosis in acute kidney injury. PMID:27048819

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.

PubMed

Oropesa, Manuel; de la Mata, Mario; Maraver, Juan Garrido; Cordero, Mario D; Cotán, David; Rodríguez-Hernández, Angeles; Domínguez-Moñino, Irene; de Miguel, Manuel; Navas, Plácido; Sánchez-Alcázar, José A

2011-04-01

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase*

PubMed Central

Sunamura, Ei-Ichiro; Konno, Hiroki; Imashimizu, Mari; Mochimaru, Mari; Hisabori, Toru

2012-01-01

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30–40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α3β3γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ϵ inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ϵ inhibition. PMID:23012354

Cellulose Microfibril Formation by Surface-Tethered Cellulose Synthase Enzymes.

PubMed

Basu, Snehasish; Omadjela, Okako; Gaddes, David; Tadigadapa, Srinivas; Zimmer, Jochen; Catchmark, Jeffrey M

2016-02-23

Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

Citrulline protects Streptococcus pyogenes from acid stress using the arginine deiminase pathway and the F1Fo-ATPase.

PubMed

Cusumano, Zachary T; Caparon, Michael G

2015-04-01

A common stress encountered by both pathogenic and environmental bacteria is exposure to a low-pH environment, which can inhibit cell growth and lead to cell death. One major defense mechanism against this stress is the arginine deiminase (ADI) pathway, which catabolizes arginine to generate two ammonia molecules and one molecule of ATP. While this pathway typically relies on the utilization of arginine, citrulline has also been shown to enter into the pathway and contribute to protection against acid stress. In the pathogenic bacterium Streptococcus pyogenes, the utilization of citrulline has been demonstrated to contribute to pathogenesis in a murine model of soft tissue infection, although the mechanism underlying its role in infection is unknown. To gain insight into this question, we analyzed a panel of mutants defective in different steps in the ADI pathway to dissect how arginine and citrulline protect S. pyogenes in a low-pH environment. While protection provided by arginine utilization occurred through the buffering of the extracellular environment, citrulline catabolism protection was pH independent, requiring the generation of ATP via the ADI pathway and a functional F1Fo-ATP synthase. This work demonstrates that arginine and citrulline catabolism protect against acid stress through distinct mechanisms and have unique contributions to virulence during an infection. An important aspect of bacterial pathogenesis is the utilization of host-derived nutrients during an infection for growth and virulence. Previously published work from our lab identified a unique role for citrulline catabolism in Streptococcus pyogenes during a soft tissue infection. The present article probes the role of citrulline utilization during this infection and its contribution to protection against acid stress. This work reveals a unique and concerted action between the catabolism of citrulline and the F1Fo-ATPase that function together to provide protection for bacteria in a low