WorldWideScience

Sample records for chaperone atpase activity

  1. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. PMID:26991466

  2. The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

    OpenAIRE

    Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K.; Bindics, János; Ślusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L.; Tamaru, Hisashi

    2014-01-01

    Centromeres are the fundamental unit required for segregation of chromosomes during mitosis and meiosis, and they are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). In contrast to the relatively well-known process of de novo assembly of CenH3 at centromeres, little is known of how CenH3 is actively removed, leading to centromere disassembly, an essential biological process during the life of a cell. This study describes the process of centromere d...

  3. Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

    Science.gov (United States)

    Haga, Asami; Okamoto, Tomoya; Yamada, Shintaroh; Kubota, Toshihiko; Sanpei, Ann; Takahashi, Shota; Nakayama, Masahiro; Nagai, Miki; Otaka, Michiro; Miyazaki, Toshio; Nunomura, Wataru; Grave, Ewa; Itoh, Hideaki

    2013-09-01

    In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70. PMID:23687308

  4. c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

    OpenAIRE

    Diana M. Dunn; Mark R. Woodford; Andrew W. Truman; Sandra M. Jensen; Jacqualyn Schulman; Tiffany Caza; Taylor C. Remillard; David Loiselle; Donald Wolfgeher; Brian S.J. Blagg; Lucas Franco; Timothy A. Haystead; Soumya Daturpalli; Matthias P. Mayer; Jane B. Trepel

    2015-01-01

    Summary The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ...

  5. TOWARD UNDERSTANDING ALLOSTERIC SIGNALING MECHANISMS IN THE ATPASE DOMAIN OF MOLECULAR CHAPERONES

    OpenAIRE

    Liu, Ying; Bahar, Ivet

    2010-01-01

    The ATPase cycle of the heat shock protein 70 (HSP70) is largely dependent on the ability of its nucleotide binding domain (NBD), also called ATPase domain, to undergo structural changes between its open and closed conformations. We present here a combined study of the Hsp70 NBD sequence, structure and dynamic features to identify the residues that play a crucial role in mediating the allosteric signaling properties of the ATPase domain. Specifically, we identify the residues involved in the ...

  6. Fingerprinting differential active site constraints of ATPases

    OpenAIRE

    Hacker, Stephan M.; Hardt, Norman; Buntru, Alexander; Pagliarini, Dana; Möckel, Martin; Mayer, Thomas U; Scheffner, Martin; Hauck, Christof R.; Marx, Andreas

    2013-01-01

    The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different A...

  7. Trypsin-induced ATPase activity in potato mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Jung, D.W.; Laties, G.G.

    1976-04-01

    Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg/sup 2 +/, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg/sup 2 +/-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60/sup 0/C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.

  8. Radioprotector modifying influence upon the ion transport ATPase activities

    International Nuclear Information System (INIS)

    The effects of aminothiol and biogenic amine radioprotectors (β-mercaptoethylamine, AET, serotonin, dopamine, histamine) on the basic ion transport enzymes, such as Na, K-ATP ase and Mg, Ca-ATPase activities were investigated in the tissues of numerous organs, with different radiosensitivity in the wistar rats. Experimental results showed that intraperitoneal injection of the used radioprotectors caused preliminary inhibition of the Na, K-ATPase activity in tissues from organs with different radioresistance, but had no influence on the Mg, Ca-ATPase activity in membranes of erythrocytes and rat brain cells. (2 tabs.)

  9. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  10. Measuring In Vitro ATPase Activity for Enzymatic Characterization.

    Science.gov (United States)

    Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria

    2016-01-01

    Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary. PMID:27584824

  11. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  12. Natural products triptolide, celastrol, and withaferin A inhibit the chaperone activity of peroxiredoxin i

    NARCIS (Netherlands)

    Zhao, Qian; Ding, Yu; Deng, Zhangshuang; Lee, On Yi; Gao, Peng; Chen, Pin; Rose, Rebecca J.; Zhao, Hong; Zhang, Zhehao; Tao, Xin Pei; Heck, Albert J R; Kao, Richard; Yang, Dan

    2015-01-01

    Peroxiredoxin I (Prx I) plays an important role in cancer development and inflammation. It is a dual-functional protein which acts as both an antioxidant enzyme and a molecular chaperone. While there have been intensive studies on its peroxidase activity, Prx I's chaperone activity remains elusive,

  13. Influence of activating hormones on human platelet membrane Ca/sup 2 +/-ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Resink, T.J.; Dimitrov, D.; Stucki, S.; Buehler, F.R.

    1986-07-16

    Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca/sup 2 +/-ATPase activity determined. Thrombin decreased the V/sub max/ of Ca/sup 2 +/-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca/sup 2 +/-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, while the simultaneous pretreatment with TPA and Ca/sup 2 +/-ionophore decreased Ca/sup 2 +/-ATPase activity. cAMP elevating agents prostaglandin E/sub 1/ (PGE/sub 1/) and forskolin had no influence per se on Ca/sup 2 +/-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca/sup 2 +/-ATPase system.

  14. The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP*

    OpenAIRE

    Niwa, Hajime; Ewens, Caroline A.; Tsang, Chun; Yeung, Heidi O.; Zhang, Xiaodong; Freemont, Paul S.

    2012-01-01

    p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of m...

  15. The influence of repeated irradiation of rats on activity of Ca2+ - ATPase and Mg2+ -ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Rats were daily irradiated at doses 0.5 Gy in period of two weeks. The activity of Ca2+-ATPase Mg2+-ATPase and the extent of lipid peroxidation in thymus were determined. Thee peculiarity of changing of enzymes activity demonstrates the dependence of Mg2+-ATPase on lipid peroxidation

  16. Principles of Quantitative Estimation of the Chaperone-Like Activity

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Molecular chaperones are able to interact with unfolded states of the protein molecule preventing their aggregation and facilitating folding of the polypeptide chain into the native structure. An understanding of the mechanism of protein aggregation is required to estimate the efficiency of action of chaperones in the test-systems based on the suppression of aggregation of protein substrates. The kinetic regimes of aggregation of proteins are discussed. The analysis of the aggregation kinetics of proteins shows that after passing the lag phase, aggregation follows, as a rule, first order kinetics. The quantitative characterization methods of the ability of chaperones to prevent aggregation of protein substrates have been elaborated.

  17. Brain Na+, K+-ATPase Activity In Aging and Disease

    Science.gov (United States)

    de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López

    2014-01-01

    Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways

  18. Enhancement of Chaperone Activity of Plant-Specific Thioredoxin through γ-Ray Mediated Conformational Change

    Directory of Open Access Journals (Sweden)

    Seung Sik Lee

    2015-11-01

    Full Text Available AtTDX, a thioredoxin-like plant-specific protein present in Arabidospis is a thermo-stable and multi-functional enzyme. This enzyme is known to act as a thioredoxin and as a molecular chaperone depending upon its oligomeric status. The present study examines the effects of γ-irradiation on the structural and functional changes of AtTDX. Holdase chaperone activity of AtTDX was increased and reached a maximum at 10 kGy of γ-irradiation and declined subsequently in a dose-dependent manner, together with no effect on foldase chaperone activity. However, thioredoxin activity decreased gradually with increasing irradiation. Electrophoresis and size exclusion chromatography analysis showed that AtTDX had a tendency to form high molecular weight (HMW complexes after γ-irradiation and γ-ray-induced HMW complexes were tightly associated with a holdase chaperone activity. The hydrophobicity of AtTDX increased with an increase in irradiation dose till 20 kGy and thereafter decreased further. Analysis of the secondary structures of AtTDX using far UV-circular dichroism spectra revealed that the irradiation remarkably increased the exposure of β-sheets and random coils with a dramatic decrease in α-helices and turn elements in a dose-dependent manner. The data of the present study suggest that γ-irradiation may be a useful tool for increasing holdase chaperone activity without adversely affecting foldase chaperone activity of thioredoxin-like proteins.

  19. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    Science.gov (United States)

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  20. Dietary selenium increases the antioxidant levels and ATPase activity in the arteries and veins of poultry.

    Science.gov (United States)

    Cao, Changyu; Zhao, Xia; Fan, Ruifeng; Zhao, Jinxin; Luan, Yilin; Zhang, Ziwei; Xu, Shiwen

    2016-07-01

    Selenium (Se) deficiency is associated with the pathogenesis of vascular diseases. It has been shown that oxidative levels and ATPase activity were involved in Se deficiency diseases in humans and mammals; however, the mechanism by how Se influences the oxidative levels and ATPase activity in the poultry vasculature is unclear. We assessed the effects of dietary Se deficiency on the oxidative stress parameters (superoxide dismutase, catalase, and hydroxyl radical) and ATPase (Na(+)K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, and Ca(++)Mg(++)-ATPase) activity in broiler poultry. A total of 40 broilers (1-day old) were randomly divided into a Se-deficient group (L group, fed a Se-deficient diet containing 0.08 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.20 mg/kg Se). Then, arteries and veins were collected following euthanasia when typical symptoms of Se deficiency appeared. Antioxidant indexes and ATPase activity were evaluated using standard assays in arteries and veins. The results indicated that superoxide dismutase activity in the artery according to dietary Se deficiency was significantly lower (p < 0.05) compared with the C group. The catalase activity in the veins and hydroxyl radical inhibition in the arteries and veins by dietary Se deficiency were significantly higher (p < 0.05) compared with the C group. The Se-deficient group showed a significantly lower (p < 0.05) tendency in Na(+)K(+)-ATPase activity, Ca(++)-ATPase activity, and Ca(++)Mg(++)-ATPase activity. There were strong correlations between antioxidant indexes and Ca(++)-ATPase activity. Thus, these results indicate that antioxidant indexes and ATPases may have special roles in broiler artery and vein injuries under Se deficiency. PMID:26637493

  1. Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

    Science.gov (United States)

    Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

    2016-05-01

    The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival. PMID:26894764

  2. Substrate independent ATPase activity may complicate high throughput screening.

    Science.gov (United States)

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  3. The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by rot dependent and independent pathways

    DEFF Research Database (Denmark)

    Jelsbak, Lotte; Ingmer, Hanne; Valihrach, Lukás; Cohn, Marianne Thorup; Christiansen, Mie H.G.; Kallipolitis, Birgitte H.; Frees, Dorte

    2010-01-01

    The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present...

  4. Effects of phenol on ATPase activities in crude gill homogenates of rainbow trout (Salmo gairdneri Richardson)

    Energy Technology Data Exchange (ETDEWEB)

    Poston, T.M.

    1979-01-01

    The ATPase specific activities from crude gill homogenates of rainbow trout were lower than those from microsomal preparations reported in the literature. Sodium pump activity (ouabain sensitive NaK-ATPase) was demonstrable at 37/sup 0/C. An ouabain insensitive NaK-ATPase was demonstrable at temperatures below 30/sup 0/C and may represent a Na-ATPase activity reported by others. Energy of activation at 25/sup 0/C for total NaK-ATPase ws 10,500 cal.mole/sup -1/. Mg-baseline activity had an energy of activation at 25/sup 0/C of 15,600 cal.mole/sup -1/. Mg-baseline activity was thermally labile at temperatures in excess of 30/sup 0/C. Concentrations of Mg/sup +2/ in excess of 5 mM appeared to inhibit total NaK-ATPase activity. At 37/sup 0/C, Na/sup +/ and K/sup +/ exerted little, if any, stimulatory effect on ATPase activities, in spite of the fact that 37/sup 0/C was the only temperature at which sodium pump activity was demonstrable. MS-222 failed to produce any discernible changes in any of the demonstrable ATPase activities in crude gill homogenates. Total NaK-ATPase activities were more sensitive than Mg-baseline activities to in vitro inhibition by phenol. Concentrations of phenol which produce 50% inhibition in total NaK-ATPase produced only 35% inhibition in Mg-baseline activity. The nature of in vitro inhibition was uncompetitive. Sodium pump activity was unaffected by phenol at concentrations as high as 25 mM. An effort was made to demonstrate an in vivo effects of phenol on rainbow trout gill ATPase activites. An infestation of a parasite (Gyrodactilus) on the experimental fish precludes any definative assessment of in vivo effects.

  5. Thyroid hormone stimulation in vitro of red blood cell Ca2+-ATPase activity: interspecies variation.

    Science.gov (United States)

    Davis, F B; Kite, J H; Davis, P J; Blas, S D

    1982-01-01

    In vitro susceptibility to thyroid hormone stimulation of membrane-associated Ca2+-ATPase activity has been examined in red blood cells from rat, rabbit, dog, monkey, and man. Monkey and human red cell Ca2+-ATPase activities responded comparably to 10(-10)M T4 or T3. Basal and thyroid hormone-stimulated Ca2+-ATPase activity in rabbit erythrocytes was four-fold higher than in primate red cells. Rat and dog red cell Ca2+-ATPase did not respond to iodothyronines in vitro. PMID:6459228

  6. Oxidative stress (Glutathionylation) and Na,K-ATPase activity in rat skeletal muscle

    OpenAIRE

    Juel, Carsten

    2014-01-01

    Background Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation) on the Na,K-ATPase in rat skeletal muscle membranes. Results Immunoprecipitation with an anti-glutathione antibody and subsequent ...

  7. Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

    Science.gov (United States)

    Mack, Korrie L.; Shorter, James

    2016-01-01

    Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

  8. Modulation of P-glycoprotein ATPase activity by some phytoconstituents.

    Science.gov (United States)

    Najar, I A; Sachin, B S; Sharma, S C; Satti, N K; Suri, K A; Johri, R K

    2010-03-01

    In the present investigation 16 phytoconstituents, which are active moieties found in several medicinal herbs, have been evaluated for their P-glycoprotein (P-gp) stimulation/inhibition profiles using a P-gp-dependent ATPase assay in rat jejunal membrane (in vitro). Acteoside, agnuside, catechin, chlorogenic acid, picroside -II and santonin showed an inhibitory effect. Negundoside, picroside -I and oleanolic acid caused a stimulatory effect. Andrographolide, apocyanin, berberine, glycyrrhizin, magniferin and piperine produced a biphasic response (stimulation at low concentration and inhibition at high concentration). The results suggested that a possible interaction of these phytoconstituents at the level of P-gp, could be an important parameter in determining their role in several key pharmacodynamic events. PMID:19653312

  9. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    International Nuclear Information System (INIS)

    Plasma membranes of human oat cell carcinoma possess Mg2+- and Ca2+-dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca2+-ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca2+-ATPase activity is more easily inactivated by Triton X-100, while the Mg2+-ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca2+-ATPase and Mg2+-ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [3H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca2+-ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg2+-ATPase has not been demonstrated, but is presently under investigation

  10. Nitric oxide and Na,K-ATPase activity in rat skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten

    2016-01-01

    Aim: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. Method: The study used...... isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Results: Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles...... activity was depressed by oxidized glutathione. Conclusion: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely...

  11. Antioxidation and ATPase activity in the gill of mud crab Scylla serrata under cold stress

    Institute of Scientific and Technical Information of China (English)

    KONG Xianghui; WANG Guizhong; LI Shaojing

    2007-01-01

    Mud crab (Scylla serrata) is an important commercial crustacean in China. An experiment was designed to study the effect of cold stress on S. serrata. After a one-week adaptation at 28 ℃, the temperature is suddenly reduced to 4 ℃. The crabs were sampled every 2 h for 10 h and dissected immediately to measure the enzyme activity. The crabs at room temperature (28 ℃) were used as the control group. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the content of malondialdehyde (MDA) and the activity of 4 ATPases (Na+, K+-ATPase;Mg2+-ATPase; Ca2+-ATPase; Ca2+, Mg2+-ATPase) were measured biochemically. In contrast to the control group, the SOD activity increased significantly from 2 to 6 h after the cold stress, and then decreased. The CAT and GPX activities increased in 2 h, and then decreased gradually. The content of MDA increased gradually in 4 h. The activity ofNa+, K+-ATPase decreased in 2 h, increased up to the top value at Hour 6,then decreased again. The activities of Mg2+-ATPase, Ca2+-ATPase and Ca2+, Mg2+-ATPase increased significantly in 6 h, insignificantly in any other hours. Under cold stress, the activity of antioxidative enzymes in S. serrata was reduced at first then stabilized, ROS-scavenging weakened, and MDA accumulated gradually in the gill after 6 h. The activity of the 4 ATPases in the crab decreased after 6 h,suggesting that the ability to regulate ion concentration has been paralyzed. Therefore, the maximum period to sustain healthy meat in the crab under cold stress is 6 hours.

  12. Contrasting effects of Na+, K+-ATPase activation on seizure activity in acute versus chronic models.

    Science.gov (United States)

    Funck, V R; Ribeiro, L R; Pereira, L M; de Oliveira, C V; Grigoletto, J; Della-Pace, I D; Fighera, M R; Royes, L F F; Furian, A F; Larrick, J W; Oliveira, M S

    2015-07-01

    Epilepsy is a life-shortening brain disorder affecting approximately 1% of the worldwide population. Most epilepsy patients are refractory to currently available antiepileptic drugs (AEDs). Knowledge about the mechanisms underlying seizure activity and probing for new AEDs is fundamental to the discovery of new therapeutic strategies. Brain Na(+), K(+)-ATPase activity contributes to the maintenance of the electrochemical gradients underlying neuronal resting and action potentials as well as the uptake and release of neurotransmitters. Accordingly, a decrease of Na(+), K(+)-ATPase increases neuronal excitability and may predispose to appearing of seizure activity. In the present study, we tested the hypothesis that activation of Na(+), K(+)-ATPase activity with a specific antibody (DRRSAb) raised against a regulatory site in the α subunit would decrease seizure susceptibility. We found that incubation of hippocampal homogenates with DRRSAb (1 μM) increased total and α1 Na(+), K(+)-ATPase activities. A higher concentration (3 μM) increased total, α1 and α2/α3 Na(+), K(+)-ATPase activities. Intrahippocampal injection of DRRSAb decreased the susceptibility of post status epilepticus animals to pentylenetetrazol (PTZ)-induced myoclonic seizures. In contrast, administration of DRRSAb into the hippocampus of naïve animals facilitated the appearance of PTZ-induced seizures. Quantitative analysis of hippocampal electroencephalography (EEG) recordings revealed that DRRSAb increased the percentage of total power contributed by the delta frequency band (0-3 Hz) to a large irregular amplitude pattern of hippocampal EEG. On the other hand, we found no DRRSAb-induced changes regarding the theta functional state. Further studies are necessary to define the potential of Na(+), K(+)-ATPase activation as a new therapeutic approach for seizure disorders. PMID:25907445

  13. Quantitative measurement of membrane Na+-K+ ATPase activity using thallium-201: comparison with rubidium-86

    International Nuclear Information System (INIS)

    Na+-K+ ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na+-K+ ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na+-K+ ATPase activity. Na+-K+ ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na+-K+ ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na+-K+ ATPase activity in vitro. High level of glucose concentration decreased cellular Na+-K+ ATPase activity, but insulin or PMA increased it

  14. Oxidative stress (glutathionylation and Na,K-ATPase activity in rat skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Carsten Juel

    Full Text Available Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation on the Na,K-ATPase in rat skeletal muscle membranes.Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05 in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0-10 mM increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

  15. Relationship between serum adiponectin level and ATPase activity of erythrocyte membrance in patients with 2-type diabetes

    International Nuclear Information System (INIS)

    Objective: To explore the possible mechanism of development nephrosis as related to changes of serum adiponectin levels and alteration of activities of Na+·K+-ATPase and Ca+2·Mg+2-ATPase of erythrocyte membrance in patients with 2-type diabetes. Methods: Serum adiponectin levels (with RIA) and erythrocyte membrance (prepared with Reilnila method) Na+·K+- ATPase and Ca+2·Mg+2-ATPase activity were determined in 45 DM2 patients without nephropathy, 31 DM2 patients with nephropathy and 30 controls. Results: Serum adiponectin levels in the diabetic patients were significantly lower than those in controls (P+·K+-ATPase and Ca+2·Mg+2-ATPase activities were also significantly lower than those in controls (P+·K+-ATPase and Ca+2·Mg+2-ATPase activities of erythrocyte membrance. (authors)

  16. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    Science.gov (United States)

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  17. Pathways of allosteric regulation in Hsp70 chaperones

    OpenAIRE

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly ...

  18. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

    2016-01-01

    Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  19. Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

    OpenAIRE

    Lawrence, W D; Davis, P J; Blas, S D

    1987-01-01

    The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up ...

  20. Leishmania amazonensis: effects of heat shock on ecto-ATPase activity.

    Science.gov (United States)

    Peres-Sampaio, Carlos Eduardo; de Almeida-Amaral, Elmo Eduardo; Giarola, Naira Ligia Lima; Meyer-Fernandes, José Roberto

    2008-05-01

    In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress. PMID:18295760

  1. Changes of Plasma Membrane H+-ATPase Activities of Glycine max Seeds by PEG Treatment

    Institute of Scientific and Technical Information of China (English)

    Yang Yong-qing; Wang Xiao-feng

    2005-01-01

    The soybean (Glycine max) Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol (PEG)treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H+-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H+-pumping activity increased steadily during PEG treatment.Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H+-ATPase activities in soybean seeds.

  2. Unique Residues Involved in Activation of the Multitasking Protease/Chaperone HtrA from Chlamydia trachomatis

    OpenAIRE

    Huston, Wilhelmina M.; Joel D. A. Tyndall; Lott, William B.; Stansfield, Scott H.; Timms, Peter

    2011-01-01

    DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrat...

  3. A novel protease activity assay using a protease-responsive chaperone protein

    International Nuclear Information System (INIS)

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  4. Pathways of allosteric regulation in Hsp70 chaperones.

    Science.gov (United States)

    Kityk, Roman; Vogel, Markus; Schlecht, Rainer; Bukau, Bernd; Mayer, Matthias P

    2015-01-01

    Central to the protein folding activity of Hsp70 chaperones is their ability to interact with protein substrates in an ATP-controlled manner, which relies on allosteric regulation between their nucleotide-binding (NBD) and substrate-binding domains (SBD). Here we dissect this mechanism by analysing mutant variants of the Escherichia coli Hsp70 DnaK blocked at distinct steps of allosteric communication. We show that the SBD inhibits ATPase activity by interacting with the NBD through a highly conserved hydrogen bond network, and define the signal transduction pathway that allows bound substrates to trigger ATP hydrolysis. We identify variants deficient in only one direction of allosteric control and demonstrate that ATP-induced substrate release is more important for chaperone activity than substrate-stimulated ATP hydrolysis. These findings provide evidence of an unexpected dichotomic allostery mechanism in Hsp70 chaperones and provide the basis for a comprehensive mechanical model of allostery in Hsp70s. PMID:26383706

  5. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    , tyrosinase from Agaricus bisporus and equine cytochrome c. Only for the first target β-casein was acting as a molecular chaperone i.e. its presence resulted in higher residual activity (higher degree of the function preservation). β-Casein did not have any influence on the residual activity of tyrosinase...

  6. Bioactive Metabolites from Chaetomium aureum: Structure Elucidation and Inhibition of the Hsp90 Machine Chaperoning Activity

    Science.gov (United States)

    Kabbaj, Fatima Zahra; Lu, Su; Faouzi, My El Abbés; Meddah, Bouchra; Proksch, Peter; Cherrah, Yahya; Altenbach, Hans-Josef; Aly, Amal H.; Chadli, Ahmed; Debbab, Abdessamad

    2014-01-01

    Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48h. PMID:25482429

  7. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  8. Purinergic effects on Na,K-ATPase activity differ in rat and human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Carsten Juel

    Full Text Available P2Y receptor activation may link the effect of purines to increased maximal in vitro activity of the Na,K-ATPase in rat muscle. The hypothesis that a similar mechanism is present in human skeletal muscle was investigated with membranes from rat and human skeletal muscle.Membranes purified from rat and human muscles were used in the Na,K-ATPase assay. Incubation with ADP, the stable ADP analogue MeS-ADP and UDP increased the Na+ dependent Na,K-ATPase activity in rat muscle membranes, whereas similar treatments of human muscle membranes lowered the Na,K-ATPase activity. UTP incubation resulted in unchanged Na,K-ATPase activity in humans, but pre-incubation with the antagonist suramin resulted in inhibition with UTP, suggesting that P2Y receptors are involved. The Na,K-ATPase in membranes from both rat and human could be stimulated by protein kinase A and C activation. Thus, protein kinase A and C activation can increase Na,K-ATPase activity in human muscle but not via P2Y receptor stimulation.The inhibitory effects of most purines (with the exception of UTP in human muscle membranes are probably due to mass law inhibition of ATP hydrolysis. This inhibition could be blurred in rat due to receptor mediated activation of the Na,K-ATPase. The different effects could be related to a high density of ADP sensitive P2Y1 and P2Y13 receptors in rat, whereas the UTP sensitive P2Y11 could be more abundant in human. Alternatively, rat could possesses a mechanism for protein-protein interaction between P2Y receptors and the Na,K-ATPase, and this mechanism could be absent in human skeletal muscle (perhaps with the exception of the UTP sensitive P2Y11 receptor.Rat muscle is not a reliable model for purinergic effects on Na,K-ATPase in human skeletal muscle.

  9. Modulation of the chaperone-like activity of bovine α-crystallin

    OpenAIRE

    Clark, John I.; Huang, Qing-ling

    1996-01-01

    The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of α-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured βL-crystallin or alcohol dehydrogenase (ADH). The ratios of βL-crystallin/α-crystallin and ADH/α-crystallin were adjusted so that partial inhibition of protein aggregation at 60°C or 37°C, respectively, was observed and modulation of the chaperone ac...

  10. Structural basis for proteasome formation controlled by an assembly chaperone nas2.

    Science.gov (United States)

    Satoh, Tadashi; Saeki, Yasushi; Hiromoto, Takeshi; Wang, Ying-Hui; Uekusa, Yoshinori; Yagi, Hirokazu; Yoshihara, Hidehito; Yagi-Utsumi, Maho; Mizushima, Tsunehiro; Tanaka, Keiji; Kato, Koichi

    2014-05-01

    Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle. PMID:24685148

  11. Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity

    Indian Academy of Sciences (India)

    Swasti Raychaudhuri; Rachana Banerjee; Subhasish Mukhopadhyay; Nitai P Bhattacharyya

    2014-09-01

    Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein’s function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of -helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington’s disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.

  12. Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex.

    Science.gov (United States)

    Banerjee, Sreeparna; Flores-Rozas, Hernan

    2005-01-01

    Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2-MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2-MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2-MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2-MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype. PMID:15746000

  13. Influence of a protein hydrolysate from green algae on the activity of some ATPase systems in frog skeletal muscle.

    Science.gov (United States)

    Ivanov, R; Georgieva, B; Naumova, P; Mileva, K; Radicheva, N

    1999-06-01

    The present study investigated the effect of a protein hydrolysate from green algae cultured in the Bulgarian region of Rupy, on the enzyme activity of frog skeletal muscle. The activity of pure Mg(2+)-ATPase, Mg2+,Ca(2+)-ATPase, NaHCO3-stimulated Mg(2+)-ATPase and the latter in the presence of the inhibitors NaSCN and NaN3 in mitochondrial (B-3) and membrane (B-12) fractions were determined before and after treatment with the protein hydrolysate from green algae (30 and 300 micrograms/ml). The differences between ATPase activity of mitochondrial and membrane fractions were described and it was established that in the B-3 fraction, the activity of the NaHCO3-stimulated Mg(2+)-ATPase and Ca(2+)-dependent Mg(2+)-ATPase were accelerated by increasing concentrations of the algae protein hydrolysate. Irrespective of the different (equal or inverse) dose-dependent effects, the protein hydrolysate stimulated Mg(2+)-ATPase and that inhibited by NaSCN an NaN3 bicarbonate-stimulated Mg(2+)-ATPase activity. In most of the probes, the protein hydrolysate produced some increase in enzyme activity of NaHCO3-stimulated Mg(2+)-ATPase and Ca(2+)-dependent Mg(2+)-ATPase in B-12 fractions. The observed properties of the algae protein hydrolysate suggest that it is capable of stimulating enzyme processes in addition to having some antitoxic effect in skeletal muscle. PMID:10420389

  14. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  15. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    International Nuclear Information System (INIS)

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

  16. Peroxynitrite induced decrease in Na+, K+-ATPase activity is restored by taurine

    Institute of Scientific and Technical Information of China (English)

    Necla Kocak-Toker; Murat Giris; Feti Tülübas; Müjdat Uysal; Gülcin Aykac-Toker

    2005-01-01

    AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed toONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured.RESULTS: Different concentrations of ONOO- (100, 200,500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO-to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.

  17. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    Science.gov (United States)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  18. Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2005-01-01

    Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others...... have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase. We tested the hypothesis that curcumin may modulate other members of the P2-type ATPase superfamily by studying the effects of curcumin on the activity...... and kinetic properties of the Na,K-ATPase. Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K0.514.6 M). Curcumin decreased the apparent affinity of Na,K-ATPase for K+ and increased it for Na+ and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction...

  19. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. PMID:27372608

  20. Purinergic effects on Na,K-ATPase activity differ in rat and human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten; Nordsborg, Nikolai Baastrup; Bangsbo, Jens

    2014-01-01

    P2Y receptor activation may link the effect of purines to increased maximal in vitro activity of the Na,K-ATPase in rat muscle. The hypothesis that a similar mechanism is present in human skeletal muscle was investigated with membranes from rat and human skeletal muscle....

  1. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. PMID:26944019

  2. Active plasma membrane p-type H+-ATPase reconstituted into nanodiscs is a monomer

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Hansen, Randi Westh; Martens, Helle Juel; Theorin, Lisa; Palmgren, Michael Broberg; Martinez, Karen Laurence; Günther-Pomorski, Thomas; Fuglsang, Anja Thoe

    2013-01-01

    Background: The plasma membrane H+-ATPase generates electrochemical gradients in plants and fungi. The minimal subunit organization required for activity is not known. Results: We developed a protocol for reconstitution of active H+-ATPase in nanodiscs. Conclusion: The minimal functional unit of...

  3. ALKYLTIN INHIBITION OF ATPASE ACTIVITIES IN TISSUE HOMOGENATES AND SUBCELLULAR FRACTIONS FROM ADULT AND NEONATAL RATS (JOURNAL VERSION)

    Science.gov (United States)

    Inhibition of ATPase activities by triethyltin (TET), diethyltin (DET), monoethyltin (MET) and trimethyltin (TMT) was studied in homogenates of brain and liver from adult rats. MET did not produce significant inhibition. ATPase activities in brain and liver homogenates from TET-t...

  4. Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling

    Directory of Open Access Journals (Sweden)

    Irvine Kenneth D

    2008-01-01

    Full Text Available Abstract Background O-fucosyltransferase1 (OFUT1 is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila. Results We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose. Conclusion Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.

  5. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  6. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  7. Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

    Science.gov (United States)

    Booth, Laurence; Shuch, Brian; Albers, Thomas; Roberts, Jane L; Tavallai, Mehrad; Proniuk, Stefan; Zukiwski, Alexander; Wang, Dasheng; Chen, Ching-Shih; Bottaro, Don; Ecroyd, Heath; Lebedyeva, Iryna O; Dent, Paul

    2016-03-15

    We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types. PMID:26887051

  8. Modulation of the chaperone-like activity of bovine alpha-crystallin.

    Science.gov (United States)

    Clark, J I; Huang, Q L

    1996-12-24

    The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of alpha-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured beta L-crystallin or alcohol dehydrogenase (ADH). The ratios of beta L-crystallin/alpha-crystallin and ADH/alpha-crystallin were adjusted so that partial inhibition of protein aggregation at 60 degrees C or 37 degrees C, respectively, was observed and modulation of the chaperone action of alpha-crystallin could be evaluated easily with selected endogenous metabolites. Enhancement of the anti-aggregation activity in the beta L-crystallin assay was strongest with pantethine, which appeared to interact with alpha-crystallin. Enhancement of the anti-aggregation activity in the ADH assay was strongest with glutathione which appeared to interact with ADH. The results indicated that the products of common metabolic pathways can modulate the chaperone-like effects of alpha-crystallin on protein aggregation. PMID:8986785

  9. Effects of Aluminum on ATPase Activity and Lipid Composition of Plasma Membranes from Wheat Roots

    Institute of Scientific and Technical Information of China (English)

    HE Long-fei; LIU You-liang; SHEN Zhen-guo; WANG Ai-qin

    2002-01-01

    The effects of aluminum on ATPase activity and lipid composition of the plasma membranes isolated from root tips of Al-tolerant (Altas 66) or Al-sensitive (Scout 66) cultivar of Triticum aestivum L.was assayed. The results showed that both cultivars had similar changes in H+ -ATPase and Ca2+ -ATPase activities after aluminum treatment. Exposure of both cultivars to 20 and 100 (mol/L aluminum for 5 d significantly decreased the activities of Ca2+ -ATPase of plasma membranes. The activities of H+-ATPasc in plasma membrane increased under 20 μmol/L aluminum and decreased at 100 μmol/L aluminum. With aluminum treatment, the PL content of plasma membrane decreased, but GL content increased. The ratio of PL to GL decreased more distinctly in Scout 66 than that in Altas 66. Treated with 20 and 100 μmol/L aluminum, linolenic acid content and the index of unsaturated fatty acids decreaced greatly in Scout 66, but the index of unsaturated fatty acids in Altas 66 increased slightly.

  10. Critical findings on the activation cascade of yeast plasma membrane H+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kotyk, Arnošt; Lapathitis, Georgios; Horák, Jaroslav

    2003-01-01

    Roč. 226, č. 1 (2003), s. 175-180. ISSN 0378-1097 R&D Projects: GA ČR GA204/02/1240 Institutional research plan: CEZ:AV0Z5045916; CEZ:AV0Z5011922 Keywords : H+-ATPase * yeast plasma membrane * activation Subject RIV: CE - Biochemistry Impact factor: 1.932, year: 2003

  11. In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP.

    Science.gov (United States)

    Gaume, Xavier; Monier, Karine; Argoul, Françoise; Mongelard, Fabien; Bouvet, Philippe

    2011-01-01

    Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A). We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities. PMID:21403913

  12. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    Science.gov (United States)

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. PMID:22728817

  13. In vivo Study of the Histone Chaperone Activity of Nucleolin by FRAP

    Directory of Open Access Journals (Sweden)

    Xavier Gaume

    2011-01-01

    Full Text Available Nucleolin is a major nucleolar protein involved in various aspects of ribosome biogenesis such as regulation of polymerase I transcription, pre-RNA maturation, and ribosome assembly. Nucleolin is also present in the nucleoplasm suggesting that its functions are not restricted to nucleoli. Nucleolin possesses, in vitro, chromatin co-remodeler and histone chaperone activities which could explain numerous functions of nucleolin related to the regulation of gene expression. The goal of this report was to investigate the consequences of nucleolin depletion on the dynamics of histones in live cells. Changes in histone dynamics occurring in nucleolin silenced cells were measured by FRAP experiments on eGFP-tagged histones (H2B, H4, and macroH2A. We found that nuclear histone dynamics was impacted in nucleolin silenced cells; in particular we measured higher fluorescence recovery kinetics for macroH2A and H2B but not for H4. Interestingly, we showed that nucleolin depletion also impacted the dissociation constant rate of H2B and H4. Thus, in live cells, nucleolin could play a role in chromatin accessibility by its histone chaperone and co-remodeling activities.

  14. Relationship between serum leptin levels, ATPase activity of erythrocyte membrance and development of diabetic nephropathy in patients with DM2

    International Nuclear Information System (INIS)

    Objective: To study the possible mechanism of development of nephrosis affected by changes of serum leptin levels and alteration of activities of Na+K+-ATPase and Ca2+Mg2+-ATPase of erythrocyte membrane in patients with type 2 diabetes(DM2). Methods: Serum leptin levels (with RIA) and erythrocyte membrane Na+K+-ATPase and Ca2+Mg2+-ATPase activitities (with Reinila method) were determined in 40 DM2 patients without nephropathy, 32 DM2 patients with nephropathy and 35 controls. Results Serum leptin levels were significantly higher in the diabetics as a whole than those in controls (P+K+-ATPase and Ca2+Mg2+-ATPase activities were significantly lower (P<0.01). Among the diabetic patients, the serum leptin levels in patients without nephrosis (P<0.05), but the RBC membrance ATPase activities were significantly lower(P<0.05). Conclusion: Development of type 2 diabetes nephrosis might be correlated with the high serum leptin level and decreased ATPase activities of erythrocite membrane. (authors)

  15. Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity.

    Science.gov (United States)

    de Almeida-Amaral, Elmo Eduardo; Caruso-Neves, Celso; Pires, Vanessa Maria Pereira; Meyer-Fernandes, José Roberto

    2008-02-01

    We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite. PMID:17825292

  16. Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis.

    Directory of Open Access Journals (Sweden)

    Wilhelmina M Huston

    Full Text Available DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog. We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

  17. Decreased Erythrocyte NA+,K+-ATPase Activity and Increased Plasma TBARS in Prehypertensive Patients

    Directory of Open Access Journals (Sweden)

    Carlos Ricardo Maneck Malfatti

    2012-01-01

    Full Text Available The essential hypertension has been associated with membrane cell damage. The aim of the present study is investigate the relationship between erythrocyte Na+,K+-ATPase and lipoperoxidation in prehypertensive patients compared to normotensive status. The present study involved the prehypertensive patients (systolic: 136±7 mmHg; diastolic: 86.8±6.3 mmHg; n=8 and healthy men with normal blood pressure (systolic: 110±6.4 mmHg; diastolic: 76.1±4.2 mmHg; n=8 who were matched for age (35±4 years old. The venous blood samples of antecubital vein (5 mL were collected into a tube containing sodium heparin as anticoagulant (1000 UI, and erythrocyte ghosts were prepared for quantifying Na+,K+-ATPase activity. The extent of the thiobarbituric acid reactive substances (TBARS was determined in plasma. The statistical analysis was carried out by Student’s t-test and Pearson’s correlation coefficient. A P<0.05 was considered significant. The Na+,K+-ATPase activity was lower in prehypertensive patients compared with normotensive subjects (4.9 versus 8.0 nmol Pi/mg protein/min; P<0.05. The Na+,K+-ATPase activity correlated negatively with TBARS content (r=-0.6; P<0.05 and diastolic blood pressure (r=-0.84; P<0.05. The present study suggests that Na+,K+-ATPase activity reduction and elevation of the TBARS content may underlie the pathophysiological aspects linked to the prehypertensive status.

  18. FXYD1 negatively regulates Na(+)/K(+)-ATPase activity in lung alveolar epithelial cells.

    Science.gov (United States)

    Wujak, Łukasz A; Blume, Anna; Baloğlu, Emel; Wygrecka, Małgorzata; Wygowski, Jegor; Herold, Susanne; Mayer, Konstantin; Vadász, István; Besuch, Petra; Mairbäurl, Heimo; Seeger, Werner; Morty, Rory E

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na(+)/K(+)-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na(+)/K(+)-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-β, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-β signaling altered Na(+)/K(+)-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na(+)/K(+)-ATPase activity, and contribute to edema persistence. PMID:26410457

  19. Gill Na+, K+-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    International Nuclear Information System (INIS)

    Active transport of Na+ and K+ for osmoregulation in fish involves gill Na+, K+-ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na+, K+-ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na+, K+-ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 microg Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 microg Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P +, K+-ATPase activity was not evident

  20. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Directory of Open Access Journals (Sweden)

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  1. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Science.gov (United States)

    Rocco-Machado, Nathália; Cosentino-Gomes, Daniela; Meyer-Fernandes, José Roberto

    2015-01-01

    Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite. PMID:26070143

  2. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  3. Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

    Directory of Open Access Journals (Sweden)

    Alexander V Chibalin

    Full Text Available Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV while the activity of the α1 isoform decreased (-4.4 mV. Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM, measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63 and Ser(68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

  4. In vitro antioxidant and H + , K + -ATPase inhibition activities of Acalypha wilkesiana foliage extract

    OpenAIRE

    Rajesh Kashi Prakash Gupta; Pradeepa,; Manjunatha Hanumanthappa

    2013-01-01

    Aims: The aim of this study was to evaluate the antioxidant activty and anti-acid property of Acalypha wilkesiana foliage extract. Materials and Methods: Hot and cold aqueous extracts were prepared from healthy leaves of A. wilkesiana. Free radical scavenging activity and H + , K + -ATPase inhibition activities of aqueous foliage extracts was screened by in vitro models. Statistical Analysis Used: All experiments were performed in triplicate and results are expressed as mean ± SEM. Results: A...

  5. Effects of Calcium on ATPase Activity and Lipid Composition of Plasma Membranes from Wheat Roots Under Aluminum Stress

    Institute of Scientific and Technical Information of China (English)

    HE Long-fei; SHEN Zhen-guo; LIU You-liang

    2003-01-01

    Effects of calcium on ATPase activities, lipid contents, and fatty acid compositions of plasma membrane from wheat roots were assayed under aluminum stress. The results showed that the increase of calcium concentration in the nutrient solution increased the activity of H+-ATPase and the phospholipid content, decreased the activity of Ca2+-ATPase and the galactolipid of plasma membrane. Owing to the decrease of linolenic acid content, the index of unsaturated fatty acid (IUFA) and index of double bond (DBI) decreased in Altas66. The IUFA and DBI of plasma membrane from Scout66 roots increased because its linolenic acid content increased obviously and its palmitic acid content decreased apparently.

  6. The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide.

    Science.gov (United States)

    Petrunyaka, V V; Panyushkina, E A; Severina, E P; Orlov, S N

    1990-12-14

    The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes. PMID:2175654

  7. Invariant chain induces B cell maturation in a process that is independent of its chaperonic activity

    Science.gov (United States)

    Matza, Didi; Lantner, Frida; Bogoch, Yoel; Flaishon, Liat; Hershkoviz, Rami; Shachar, Idit

    2002-01-01

    Early stages of B cell development take place in the bone marrow, resulting in formation of immature B cells, which migrate to the spleen for their final differentiation into mature cells. This final maturation step is essential for B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls the differentiation of B cells from the immature to the mature stage. In this study, by generating transgenic mice expressing truncated Ii lacking its luminal domain, we could dissect the chaperonin activity of Ii from its role in B cell maturation. We demonstrate in vivo that Ii N-terminal domain is directly involved in the maturation of B cells and is sufficient to promote B cell differentiation. PMID:11867743

  8. AtDeg2 – a chloroplast protein with dual protease/chaperone activity

    Directory of Open Access Journals (Sweden)

    Przemysław Jagodzik

    2014-07-01

    Full Text Available Chloroplast protease AtDeg2 (an ATP-independent serine endopeptidase is cytosolically synthesized as a precursor, which is imported into the chloroplast stroma and deprived of its transit peptide. Then the mature protein undergoes routing to its functional location at the stromal side of thylakoid membrane. In its linear structure AtDeg2 molecule contains the protease domain with catalytic triad (HDS and two PDZ domains (PDZ1 and PDZ2. In vivo AtDeg2 most probably exists as a supposedly inactive haxamer, which may change its oligomeric stage to form active 12-mer, or 24-mer. AtDeg2 has recently been demonstrated to exhibit dual protease/chaperone function. This review is focused on the current awareness with regard to AtDeg2 structure and functional significance.

  9. Effect of ionizing radiation on regulation of Na,K-ATPase activity in kidneys by univalent cations

    International Nuclear Information System (INIS)

    The effect of ionizing radiation of 0.206 C/kg on the kinetics of activation of rat kidney Na,K-ATPase preparation by Na and K ions was studied as an index of possible qualitative and quantitative changes in the properties of the enzyme. Ionizing radiation was shown not only to increase the enzyme activity but also to change the optimal rate of ATP hydrolysis by Na,K-ATPase and to induce some differences in the shape of the curve for Na,K-ATPase dependence upon Na-sodium//potassium ion ratio in the incubation medium

  10. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  11. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  12. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

    Science.gov (United States)

    Fatehi, Mohammad; Carter, Chris R J; Youssef, Nermeen; Hunter, Beth E; Holt, Andrew; Light, Peter E

    2015-01-01

    ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  13. An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

    OpenAIRE

    Hatori, Yuta; Lutsenko, Svetlana

    2013-01-01

    Significance: Antioxidant protein 1 (Atox1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, Atox1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of Atox1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Recent Advances: Accumulating data suggest that cellular functions of Atox1 are not limited to its c...

  14. Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

    Science.gov (United States)

    Ramos, Inés; Cisint, Susana B; Crespo, Claudia A; Medina, Marcela F; Fernández, Silvia N

    2009-08-01

    The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process. PMID:19397840

  15. Leishmania amazonensis: Increase in ecto-ATPase activity and parasite burden of vinblastine-resistant protozoa.

    Science.gov (United States)

    Giarola, Naira Lígia Lima; Silveira, Thaís Souza; Inacio, Job Domingos Filho; Vieira, Lisvane Paes; Almeida-Amaral, Elmo Eduardo; Meyer-Fernandes, José Roberto

    2014-11-01

    Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of L. amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and

  16. Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

    Institute of Scientific and Technical Information of China (English)

    Derick Shi-Chen Ou; Sung-Bau Lee; Chi-Shuen Chu; Liang-Hao Chang; Bon-chu Chung; Li-Jung Juan

    2011-01-01

    Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown, in this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp 78 gene expression depending on the ATPbinding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of activestate chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.

  17. Stimulation of Na+/K+ ATPase activity and Na+ coupled glucose transport by β-catenin

    International Nuclear Information System (INIS)

    Research highlights: → The oncogenic transcription factor β-catenin stimulates the Na+/K+-ATPase. → β-Catenin stimulates SGLT1 dependent Na+, glucose cotransport. → The effects are independent of transcription. → β-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: β-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. β-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that β-catenin influences membrane transport. To this end, β-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na+/K+-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of β-catenin on the endogenous Na+/K+-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of β-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of β-catenin expression. The stimulating effect of β-catenin on both Na+/K+ ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of transport.

  18. Do nucleic acids moonlight as molecular chaperones?

    Science.gov (United States)

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  19. Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian; Øgendal, Lars Holm

    2014-01-01

    In this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone-like activity of β-casein under elevated temperature. It was shown that β-casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between...

  20. Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities

    Science.gov (United States)

    Nahomi, Rooban B.; DiMauro, Michael A.; Wang, Benlian; Nagaraj, Ram H.

    2015-01-01

    Previous studies have identified peptides in the ‘crystallin-domain’ of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors. PMID:25332102

  1. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  2. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    Science.gov (United States)

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  3. Na+-K+-ATPase in rat skeletal muscle: muscle fiber-specific differences in exercise-induced changes in ion affinity and maximal activity

    DEFF Research Database (Denmark)

    Juel, Carsten

    2008-01-01

    It is unclear whether muscle activity reduces or increases Na(+)-K(+)-ATPase maximal in vitro activity in rat skeletal muscle, and it is not known whether muscle activity changes the Na(+)-K(+)-ATPase ion affinity. The present study uses quantification of ATP hydrolysis to characterize muscle fiber...... type-specific changes in Na(+)-K(+)-ATPase activity in sarcolemmal membranes and in total membranes obtained from control rats and after 30 min of treadmill running. ATPase activity was measured at Na(+) concentrations of 0-80 mM and K(+) concentrations of 0-10 mM. K(m) and V(max) values were obtained...

  4. D-Methionine attenuated cisplatin-induced vestibulotoxicity through altering ATPase activities and oxidative stress in guinea pigs

    International Nuclear Information System (INIS)

    Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatin exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na+, K+-ATPase and Ca2+-ATPase activities in the ampullary tissue with a good dose-response relationship but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na+, K+-ATPase and Ca2+-ATPase activities of the ampullary tissue. D-Methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea pig vestibular labyrinths. D-Methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property

  5. [Changes of sarcolemma Na+/K+ ATPase and sarcoplasmic reticulum membrane Ca2+ ATPase activity after stem cell transplantation in chronic heart failure].

    Science.gov (United States)

    Fan, Zhongcai; Chen, Mao; Deng, Juelin; Liu, Xiaojing; Zhang, Li; Rao, Li; Yang, Qing; Huang, Dejia

    2007-02-01

    To assess the changes of sarcolemma Na+/K+ ATPase (CMNKA) and sarcoplasmic reticulum membrane Ca2+ ATPase (SERCA) activities after stem cells transplantation in heart failure. Rabbit was used as heart failure model by intravenously injecting adriamycin. Autologous bone marrow mononuclear cells (BMCs), bone marrow mesenchymal stem cells (MSCs) or skeletal myoblasts (SMs) were introduced into coronary arteies through the root of aorta when two balloons occluding just above sinus of Valsalva. After 4 weeks, left ventricular ejection fraction (LVEF)was evaluated by echocardiography, and the activities of CMNKA and SERCA were measured by colorimeter. In BMCs (n=8)and MSCs (n=8) group, LVEF were significantly improved (P SMs group (n=6) compared to sham group (n=8). The CMNKA activity in all stem cells groups was significantly increased compared to sham group (P < 0.05). Meanwhile, in comparison with sham group, the incremental tendencies of SERCA activity were seen in stem cells groups. In conclusion, stem cells transplantation could increase the activities of CMNKA and SERCA in heart failure, a possible mechanism to improve heart function. PMID:17333908

  6. Rice Phospholipase Dα is Involved in Salt Tolerance by the Mediation of H+-ATPase Activity and Transcription

    Institute of Scientific and Technical Information of China (English)

    Peng Shen; Rong Wang; Wen Jing; Wenhua Zhang

    2011-01-01

    Phospholipase Dα (PLDα) is involved in plant response to salt stress, but the mechanisms remain unclear.We investigated rice PLDα (OsPLDα) localization and its effect on tonoplast (TP) and plasma membrane (PM) H+-ATPase activity and transcription in response to NaCl. When rice suspension-cultured cells were treated with 100 mM NaCI, PLDα activity in cell extracts showed a transient activation with a threefold increase at 1 h. The amount of OsPLDα protein decreased slightly in the cytosolic fractions, whereas it increased significantly in the TP after NaCI treatment. OsPLDα1 knockdown cells were developed using RNA interference (RNAi) methods. The increase in TP and PM H+-ATPase activity induced by NaCl was significantly inhibited in OsPLDα1-RNAi cells. Knockdown of OsPLDα1 prevented the NaCl-induced increase in the transcript level of OsVHA-A (encodes TP H+-ATPase) and OSA2 (encodes PM H+-ATPase),as well as OsNHX1 (encodes TP Na+/H+ antiporter). The cells died more in OsPLDα1-RNAi mutant than in wild type when they were treated with NaCl. These results suggest that OsPLDα is involved in salt tolerance in rice through the mediation of H+-ATPase activity and transcription.

  7. Difference in 201TlCl accumulation mechanism in brain tumors. A comparison of their Na+-K+ ATPase activities

    International Nuclear Information System (INIS)

    The accumulation levels of 201TlCl and Na+ -K+ ATPase activity in tumor tissue were compared among glioblastoma, benign glioma and meningioma to study the difference in the mechanism of 201TlCl accumulation. The subjects were 19 cases comprised of 6 glioblastoma, 2 oligodendroglioma, 1 fibrillary astrocytoma, 1 pilocytic astrocytoma and 9 meningioma. Preoperative 201TlCl SPECT was performed in all the cases, and Thallium Index (TL index) was calculated by a ratio of 201TlCl in the tumor area and the contralateral area. In addition, cell membrane was extracted from the tumor tissue collected intraoperatively to determine Na+ -K+ ATPase activity. No statistically significant difference in TL index was noted between the glioblastoma group (6.97±2.67) and the meningioma group (5.87±1.99). This fact showed that there was no difference in the accumulation level of 201TlCl between the two groups. On the other hand, the glioblastoma group indicated a higher value of Na+ -K+ ATPase activity (49.13±43.76 μmole/hour/mg protein) than the meningioma group (7.73±13.84 μmol/hour/mg protein) (p+ -K+ ATPase activity in 201TlCl accumulation in glioblastoma and the influences of other accumulation mechanism than Na+ -K+ ATPase activity such as the volume of intratumoral vascular bed in meningioma. (author)

  8. [3H]-ouabain binding sites and (Na+ + K+)ATPase activity in heart of rats fed cholesterol

    International Nuclear Information System (INIS)

    The purpose of this investigation was to determine the effects of cholesterol on the characteristics of ouabain binding sites and (Na+ + K+)ATPase activity in heart. Three groups of male, weanling, Sprague-Dawley rats were fed for 5 weeks diets containing 0, 1 or 2% cholesterol. Membranes were prepared from deoxycholate-treated heart homogenates by differential centrifugation and assayed for ouabain binding and (Na+ + K+)ATPase activity. Membranes were incubated with [3H]-ouabain in the presence of 10 mM Tris-HCl buffer (pH 7.4) and rapidly filtered on glass fiber filters, GF/A. Non-specific binding was measured in the presence of 6 mM non-labeled ouabain. Concentration of [3H]-ouabain binding sites (B/sub max/) was decreased and the binding affinity was increased in the membranes of rats fed 2% cholesterol. The ouabain-sensitive (Na+ + K+)ATPase activity was 50-75% lower in membranes prepared from heart of rats fed cholesterol. The Mg2+-ATPase activity was not changed by dietary cholesterol. The results suggest that cholesterol feeding decreases the number of (Na+ + K+)ATPase units and allosterically modifies the enzyme

  9. Effects of La3+ on ATPase Activities of Plasma Membrane Vesicles Isolated from Casuarina Equisetifolia Seedlings under Acid Rain Stress

    Institute of Scientific and Technical Information of China (English)

    李裕红; 严重玲; 刘景春; 陈英华; 胡俊; 薛博

    2003-01-01

    The effects of La3+ on the growth and the ATPases activities of plasma membrane(PM) vesicles isolated from Casuarina equisetifolia seedlings under artificial acid rain(pH 4.5) stress were studied. The results show that the height, length of roots, fresh weight and PM H+-ATPase activites of Casuarina equisetifolia seedlings increase by the treatments of soaking seeds in LaCl3 solutions with lower concentrations, and those can reach their peak values by treating with 200 mg·L-1 La3+. However, in comparison with the CK, those are inhibited by the higher La3+ concentrations; PM Ca2+-ATPase activity is inhibited with the treatments of La3+. The results also reveal that the H+-ATPase activity and the growth of cell enlarge have a remarkable positive correlation, and La3+ activating H+-ATPase can facilitate plant growth. La3+ also can alleviate cytosolic acidification of plant under acid rain stress and indirectly maintain the stability of intracellular environment. In order to resistant to acid rain and accelerate the growth of Casuarina equisetifolia, the suitable range of La3+ concentrations to soak seeds for 8 h is 50~200 mg*L-1.

  10. Hyperbaric oxygen treatment induces dynamic ATPase activity changes in the rat brain following transient global cerebral ischemia-reperfusion

    Institute of Scientific and Technical Information of China (English)

    Shiming Xu; Hongjuan Wang; Tongnan Gu; Xiuyan Zhou; Rui Chen

    2008-01-01

    BACKGROUND: Energy depletion, induced by ischemia or hypoxia, is one of the first events in neuronal injury. OBJECTIVE: To investigate the dynamic changes of Na+-K+-ATPase and Ca2+-ATPase activity in the rat brain following transient global cerebral ischemia-reperfusion (IR), as well as the effects of hyperbaric oxygen (HBO) treatment. DESIGN, TIME AND SETTING: A randomized and controlled animal study was performed in the Department of Biochemistry and Molecular Biology, Capital Medical University between February and December 2006. MATERIALS: Clean-grade, female, Sprague Dawley rats were provided by the Animal Research Department of Capital Medical University (License number: SYXK11-00-0047). Na+-K+-ATPase and Ca2+-ATPase kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A hyperbaric oxygen chamber (DWC150-300) was supplied by Shanghai 701 Medical Oxygen Chamber Factory (Shanghai, China). METHODS: Sixty-three rats were randomly divided into nine groups: sham operated group (sham-O) as control, groups of IR, and groups treated with hyperbaric oxygen (HBO) after IR. Animal from the IR and HBO groups were sacrificed after four different survival intervals of 6, 24, 48 and 96 hours, respectively. Each group consisted of seven rats. The rats of HBO groups were placed into the hyperbaric chamber. The HBO chamber was flushed with pure oxygen for 5 minutes, followed by a gradual rise in pressure over 5 minutes and stabilization at 0.2 MPa. Then, pure oxygen was supplied for 45 minutes in stabilized pressure, followed by gradually reduced pressure over 15 minutes. The rats of the 6-h HBO group were placed into the HBO chamber following reperfusion for 3 hours on the first day, which was repeated on three consecutive days, always at the same time. Rats in the sham-O group and IR group remained under normal atmospheric pressure. MAIN OUTCOME MEASURES: The Na+-K+-ATPase and Ca2+-ATPase activity in rat brain homogenate was detected by the

  11. Relationship between changes of plasma endothelin (ET) level, ATPase activity of erythrocyte membrane and development of nephropathy in patients with pregnancy induced hypertension

    International Nuclear Information System (INIS)

    Objective: To investigate the possible role played by alteration of plasma ET levels and activities of Na+- K+-APT ase and Ca2+-Mg2+-ATPase of erythrocyte membrane in patients with nephropathy pregnancy induced hypertension. Methods: The concentrations of plasma ET was detected with RIA and erythrocyte membrane ATPase activities were determined with Reilni method in 32 pregnant women with PIH complicated with nephropathy and 70 women with PIH but no nephropathy and 35 normal pregnant women as controls. Results: The plasma ET levels in patients with PHI (both with and without nephropathy) were significantly higher than those in normal preganat women (P+-K+-ATPase and Ca2+-Mg2+-ATPase levels were significantly de- creased (P+-K+-ATPase and Ca2+-Mg2+-ATPase activity of erythrocyte membrane. (authors)

  12. Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

    Science.gov (United States)

    Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

    2015-12-01

    The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. PMID:26589552

  13. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney

    International Nuclear Information System (INIS)

    A partially purified, membrane-bound Na+-K+-ATPase fraction, prepared from the outer medulla of porcine kidney, was incubated in the presence of 0.1-100 mM H2O2 for either 15 or 30 min at 37 degree C. The activity of ouabain-sensitive Na+-K+-ATPase was reduced proportionally to the concentration of H2O2 and the duration of incubation. There were decreases in SH contents and turnover rates of the Na+-K+-ATPase preparation, while malondialdehyde (MDA) and conjugated dienes were generated from the membrane lipids in the course of the incubation. The concentrations of ethanolamine (E) plasmalogen and of arachidonic acid in the E glycerophospholipid molecules were reduced by the free radical reaction. Similarly, a reduction in Na+K+-ATPase activity and the formation of MDA and conjugated dienes, together with a decrease in E glycerophospholipids, were observed when the membrane fraction was exposed to ultraviolet irradiation (254 nm) for 30 min at 4 degree C. Microsomal fractions, prepared from the outer medulla of canine kidney after 1 h of unilateral ischemia and 1 h of reperfusion, showed a decreased Na+-K+-ATPase activity, a reduced amount of SH groups, and an increased MDA. These changes were normalized by the infusion of N-mercaptopropionylglycine. These results support the view (1) that free radical generation affects the enzyme protein as well as membrane lipids, and (2) that free radicals may be formed in the ischemic reperfused kidney

  14. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    Science.gov (United States)

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  15. The influence of transition and heavy metal ions on ATP-ases activity in rat synaptic plasma membranes

    Directory of Open Access Journals (Sweden)

    VESNA VASIC

    2004-07-01

    Full Text Available The influence of transition metal (Cu2+, Zn2+, Fe2+ and Co2+ and heavy metal ions (Hg2+, Pb2+ and Cd2+ on the activities of Na+/K+-ATPase and Mg2+-ATPase isolated from rat synaptic plasma membranes (SPM was investigated. The aim of the study was to elucidate the inhibition of both ATPase activities by exposure to the considered metal ions as a function of their affinity to bind to the –SH containing ligand L-cysteine, as a model system. The half-maximum inhibitory activities (IC50 of the enzymes were determined as parameters of rectangular hyperbolas and correlated with the stability constant (Ks of the respective metal-ion-L-cysteine complex. The linear Dixon plots indicate equilibrium binding of the investigated ions to both enzymes.

  16. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Ahn, Sung-Min, E-mail: smahn@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Department of Translational Medicine, Gachon University Gil Hospital, Incheon (Korea, Republic of); Jang, Ho Hee, E-mail: hhjang@gachon.ac.kr [Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Korea, Republic of); Lee, Sang Yeol [Division of Applied Life Sciences (Brain Korea 21 program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer hPrx1 has RNA-binding properties. Black-Right-Pointing-Pointer hPrx1 exhibits helix-destabilizing activity. Black-Right-Pointing-Pointer Cold stress increases hPrx1 level in the nuclear fraction. Black-Right-Pointing-Pointer hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem-loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  17. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    International Nuclear Information System (INIS)

    Highlights: ► hPrx1 has RNA-binding properties. ► hPrx1 exhibits helix-destabilizing activity. ► Cold stress increases hPrx1 level in the nuclear fraction. ► hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem–loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  18. Direct measurement of gastric H + / K +-ATPase activities in patients with or without Helicobacter pylori-associated chronic gastritis

    Institute of Scientific and Technical Information of China (English)

    Duangporn Thong-Ngam; Pisit Tangkijvanich; Pichet Sampatanukul; Paungpayom Prichakas; Varocha Mahachai; Piyaratana Tosukowong

    2005-01-01

    AIM: The role of Helicobacter pylori (H pylori) infection in gastric acid secretion of patients with chronic gastritisremains controversial. This study was designed to elucidate the effect of H pylori on H+/K+-ATPase activities in gastric biopsy specimens.METHODS: Eighty-two patients with chronic gastritis who had undergone upper endoscopy were included in this study. H pylori infection was confirmed by rapid urease test and histology. Gastric H+/K+-ATPase activities and serum gastrin concentrations were measured by an enzymatic method and radioimmunoassay, respectively. For those patients who received triple therapy for eradicating H pylori, changes in the activity of gastric H+/K+-ATPase and serum gastrin levels were also measured. RESULTS: The mean gastric H+/K+-ATPase activity in H pyloripositive group (42 patients) was slightly higher than thatin H pylori-negative group (29 patients) (169.65±52.9 and eradication of H pylori, the gastric H+/K+-ATPase activities slightly decreased compared to prior therapy (165.03±59.50 The mean basal gastrin concentration was slightly higher in H pylori-positive patients than in H pylori-negative patients (87.92±39.65 pg/mL vs75.04± 42.57 pg/mL, P= 0.228). The gastrin levels fell significantly after the eradication of Hpylori. (Before treatment 87.00±30.78 pg/mL, aftertreatment 64.73±18.96 pg/mL, P = 0.015).CONCLUSION: Gastric H+/K+-ATPase activities are not associated with H pylori status in patients with chronicgastritis.

  19. Spatial distribution and activity of Na(+)/K(+)-ATPase in lipid bilayer membranes with phase boundaries.

    Science.gov (United States)

    Bhatia, Tripta; Cornelius, Flemming; Brewer, Jonathan; Bagatolli, Luis A; Simonsen, Adam C; Ipsen, John H; Mouritsen, Ole G

    2016-06-01

    We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane. PMID:26994932

  20. Effect of green laser light on diabetes mellitus changed ATPase activity in erythrocytes

    International Nuclear Information System (INIS)

    Changes in the membrane bound enzyme activity may report about changes of processes and properties related to the cytoplasmic membrane of cells. Activity of the Na+/K+-ATPase has become objective of our investigation as o tool to evaluate changes of diabetic membranes in comparison to normal membranes of human erythrocytes after laser irradiation with Nd:YAG laser (532 nm) in fluence range 9.5-63.3 J · cm-2. Energies of irradiation 3-20 joules and output power of the laser 30 mW classify this experiment as low-level laser therapy. Bio-stimulation of the enzyme, its activity as well as type-2 diabetes caused disorganisation and alternation of biological membrane and enzyme properties are discussed. (Authors)

  1. ATPase activity of erythrocyte membranes and their permeability for the K-ions as influenced by irradiation and serotonin

    International Nuclear Information System (INIS)

    Na, K-ATPase activity of membranes of erytrocytes after 1 hour of X-ray irradiation of citrate blood of rats (25.8 Kl/kg)-increased, and after irradiation of isolated erytrocytes, placed in the isotonic solution of NaCl did not change. The exflux of K-ions out of irradiated erytrocytes increased equally in both cases. Serotonin (2x10-4 M), added to the probes 10 minutes before irradiation, decreased the exflux of K+ by irradiated erytrocytes, but Na, K-ATPase activity under the influence of amine was without changes

  2. Exercise-induced increase in maximal in vitro Na-K-ATPase activity in human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten; Nordsborg, Nikolai Baastrup; Bangsbo, Jens

    2013-01-01

    The present study investigated whether the maximal in vitro Na,K-ATPase activity in human skeletal muscle is changed with exercise and whether it was altered by acute hypoxia. Needle biopsies from 14 subjects were obtained from vastus lateralis before and after 4 min of intense muscle activity. I...

  3. The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

    Directory of Open Access Journals (Sweden)

    Michael O Daniyan

    Full Text Available Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1 or a human Hsp70 (HSPA1A, indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

  4. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    Science.gov (United States)

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  5. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells

    DEFF Research Database (Denmark)

    Galuska, Dana; Pirkmajer, Sergej; Barres, Romain;

    2011-01-01

    Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in...

  6. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  7. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity.

    Science.gov (United States)

    Schmidt, Bryan H; Osheroff, Neil; Berger, James M

    2012-11-01

    Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. PMID:23022727

  8. Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

    DEFF Research Database (Denmark)

    Rasmussen, M K; Kristensen, M; Juel, C

    2008-01-01

    BACKGROUND: Na(+)/K(+)-ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of alpha-subunits to the outer membrane (translocation). AIM: We investigated the existence of exercise-induced translocation and phosphorylation of...... phospholemman (PLM, FXYD1) protein in rat skeletal muscle and exercise-induced changes in V(max) and K(m) for Na(+) of the Na(+)/K(+)-ATPase. METHODS: Two membrane fractionation methods and immunoprecipitation were used. Results: Both fractionation methods revealed a 200-350% increase in PLM in the sarcolemma...... after 30 min of treadmill running, while the phosphorylation of Ser-68 of PLM appeared to be unchanged. Exercise did not change V(max) or K(m) for Na(+) of the Na(+)/K(+)-ATPase in muscle homogenate, but induced a 67% increase in V(max) in the sarcolemmal giant vesicle preparation; K(m) for Na...

  9. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing.

    OpenAIRE

    Kim, S. H.; Lin, R J

    1996-01-01

    In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases which are involved in several distinct ATP-dependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. Here we report the role of one such DEAH protein, PRP2 of S. ce...

  10. Absence of Malolactic Activity Is a Characteristic of H+-ATPase-Deficient Mutants of the Lactic Acid Bacterium Oenococcus oeni

    OpenAIRE

    Galland, Delphine; Tourdot-Maréchal, Raphaëlle; Abraham, Maud; Chu, Ky Son; Guzzo, Jean

    2003-01-01

    The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR...

  11. Direct measurement of gastric H+/K+-ATPase activities in patients with or without Helicobacter pylori-associated chronic gastritis

    OpenAIRE

    Thong-Ngam, Duangporn; Tangkijvanich, Pisit; Sampatanukul, Pichet; Prichakas, Paungpayom; Mahachai, Varocha; Tosukowong, Piyaratana

    2005-01-01

    AIM: The role of Helicobacter pylori (H pylori ) infection in gastric acid secretion of patients with chronic gastritis remains controversial. This study was designed to elucidate the effect of H pylori on H+/K+-ATPase activities in gastric biopsy specimens.

  12. The Relationship Between Senescence and Ca2+-ATPase Activity of Microsomal Membrane and Lipid Peroxidation in Harvested Peach Fruit

    Institute of Scientific and Technical Information of China (English)

    GUAN Jun-feng; FAN Xiu-cai; DOU Shi-juan; ZHANG Ji-shu; LI Guang-min

    2006-01-01

    Peach fruit easily soften and have a short storage time at normal temperature. In this study, peach fruit (Prunus persica sieb et Zucc cv. Yingqing) were picked and stored at 25 and 4℃ to investigate the senescence in correlation with Ca2+- ATPase activity of microsomal membrane and lipid peroxidation during ripening and senescence. In comparison with that stored at 25℃, the fruit stored at 4℃ exhibited a higher flesh firmness, lower respiration rate, and generated the late bigger peak value of Ca2+-ATPase activity as well as maintained the higher activity of the enzyme. Meanwhile, the lower levels of super oxygen radical (O2-) production and content of malondialdehyde (MDA), a product of membrane lipid peroxidation were observed. Sodium orthovanadate (SO) and erythrosin B (EB), as Ca2+-ATPase inhibitors, could stimulate the respiration rate. The results suggested that the slower senescence rate of peach fruit was closely related to the higher peak value and longer duration of Ca2+-ATPase activity in microsomal membrane, with the slighter membrane lipid peroxidation and lower O2(-) production rate.

  13. Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

    Science.gov (United States)

    Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

    2016-05-01

    Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. PMID:26947936

  14. Evaluation of structure, chaperone-like activity and protective ability of peroxynitrite modified human α-Crystallin subunits against copper-mediated ascorbic acid oxidation.

    Science.gov (United States)

    Ghahramani, Maryam; Yousefi, Reza; Khoshaman, Kazem; Moghadam, Sogand Sasan; Kurganov, Boris I

    2016-06-01

    The copper-catalyzed oxidation of ascorbic acid (ASA) to dehydroascorbate (DHA) and hydrogen peroxide plays a central role in pathology of cataract diseases during ageing and in diabetic patients. In the current study, the structural feature, chaperone-like activity and protective ability of peroxynitrite (PON) modified αA- and αB-Crystallin (Cry) against copper-mediated ASA oxidation were studied using different spectroscopic measurements and gel mobility shift assay. Upon PON modification, additional to protein structural alteration, the contents of nitrotyrosine, nitrotryptophan, dityrosine and carbonyl groups were significantly increased. Moreover, αB-Cry demonstrates significantly larger capacity for PON modification than αA-Cry. Also, based on the extent of PON modification, these proteins may display an improved chaperone-like activity and enhanced protective ability against copper-mediated ASA oxidation. In the presence of copper ions, chaperone-like activity of both native and PON-modified α-Cry subunits were appreciably improved. Additionally, binding of copper ions to native and PON-modified proteins results in the significant reduction of their solvent exposed hydrophobic patches. Overall, the increase in chaperone-like activity/ASA protective ability of PON-modified α-Cry and additional enhancement of its chaperoning action with copper ions appear to be an important defense mechanism offered by this protein. PMID:26896727

  15. AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

    Science.gov (United States)

    Booth, Laurence; Roberts, Jane L; Ecroyd, Heath; Tritsch, Sarah R; Bavari, Sina; Reid, St Patrick; Proniuk, Stefan; Zukiwski, Alexander; Jacob, Abraham; Sepúlveda, Claudia S; Giovannoni, Federico; García, Cybele C; Damonte, Elsa; González-Gallego, Javier; Tuñón, María J; Dent, Paul

    2016-10-01

    We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc. PMID:27187154

  16. Tyrosine 601 of Bacillus subtilis DnaK undergoes phosphorylation and is crucial for chaperone activity and heat shock survival

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2016-04-01

    Full Text Available In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple SILAC-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type, were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the wild type dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

  17. Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna;

    2011-01-01

    A chaperone activity is sufficient for growth at high temperature or oxidative stress, whereas the HtrA protease activity is only essential at conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat-shock response even at...

  18. Heat Treatment of Small Heat Shock Proteins α-Crystallin and Hsp16.3: Structural Changes vs. Chaperone-like Activity

    Institute of Scientific and Technical Information of China (English)

    毛启龙; 柯丹霞; 昌增益

    2001-01-01

    Both α-crystallin from bovine eye lens and Hsp16.3 from Mycobacterium tuberculosis are members of the small heat shock protein family, They were preincubated at 100 C for 15 min and then cooled on ice immediately. The chaperone-like activities of preheated proteins were measured at 37 C using DTT-treated insulin B chains as substrates. Both preheated proteins exhibited greatly enhanced chaperone-like activities, accompanied with almost unchanged secondary structures and surface hydrophobicity but with a minor change in tertiary structures. The dramatically enhanced chaperone-like activities of preheated α-crystallln and Hsp16.3 may have resulted from the irreversible change in the tertiary structure as detected by near-UV CD spectra.

  19. Acid-Denatured Green Fluorescent Protein (GFP as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

    Directory of Open Access Journals (Sweden)

    Marco A. Ramos

    2011-07-01

    Full Text Available Green fluorescent protein (GFP has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for

  20. Age-Dependent Decrease in Chaperone Activity Impairs MANF Expression, Leading to Purkinje Cell Degeneration in Inducible SCA17 Mice

    Science.gov (United States)

    Yang, Su; Huang, Shanshan; Gaertig, Marta A.; Li, Xiao-Jiang; Li, Shihua

    2016-01-01

    SUMMARY Although protein-misfolding-mediated neurodegenerative diseases have been linked to aging, how aging contributes to selective neurodegeneration remains unclear. We established spinocerebellar ataxia 17 (SCA17) knockin mice that inducibly express one copy of mutant TATA box binding protein (TBP) at different ages by tamoxifen-mediated Cre recombination. We find that more mutant TBP accumulates in older mouse and that this accumulation correlates with age-related decreases in Hsc70 and chaperone activity. Consistently, older SCA17 mice experienced earlier neurological symptom onset and more severe Purkinje cell degeneration. Mutant TBP shows decreased association with XBP1s, resulting in the reduced transcription of mesencephalic astrocyte-derived neurotrophic factor (MANF), which is enriched in Purkinje cells. Expression of Hsc70 improves the TBP-XBP1s interaction and MANF transcription, and overexpression of MANF ameliorates mutant TBP-mediated Purkinje cell degeneration via protein kinase C (PKC)-dependent signaling. These findings suggest that the age-related decline in chaperone activity affects polyglutamine protein function that is important for the viability of specific types of neurons. PMID:24462098

  1. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding ▿

    OpenAIRE

    Jones, Christopher P.; Datta, Siddhartha A.K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2010-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid ...

  2. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg;

    2014-01-01

    Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases......) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4...... include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell. © 2013 The Author(s)....

  3. Activity of H(+)-ATPase in ruminal bacteria with special reference to acid tolerance.

    OpenAIRE

    Miwa, T.; Esaki, H; Umemori, J; Hino, T.

    1997-01-01

    Batch culture experiments showed that permeabilized cells and membranes of Ruminococcus albus and Fibrobacter succinogenes, acid-intolerant celluloytic bacteria, have only one-fourth to one-fifth as much H(+)-ATPase as Megasphaera elsdenii and Streptococcus bovis, which are relatively acid tolerant. Even in the cells grown in continuous culture at pH 7.0, the acid-intolerant bacteria contained less than half as much H(+)-ATPase as the acid-tolerant bacteria. The amounts of H(+)-ATPase in the ...

  4. Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

    Science.gov (United States)

    Nakanishi, Kotaro

    2016-09-01

    RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117

  5. Oxidative stress and damage to erythrocytes in patients with chronic obstructive pulmonary disease--changes in ATPase and acetylcholinesterase activity.

    Science.gov (United States)

    Bukowska, Bożena; Sicińska, Paulina; Pająk, Aneta; Koceva-Chyla, Aneta; Pietras, Tadeusz; Pszczółkowska, Anna; Górski, Paweł; Koter-Michalak, Maria

    2015-12-01

    The study indicates, for the first time, the changes in both ATPase and AChE activities in the membrane of red blood cells of patients diagnosed with COPD. Chronic obstructive pulmonary disease (COPD) is one of the most common and severe lung disorders. We examined the impact of COPD on redox balance and properties of the membrane of red blood cells. The study involved 30 patients with COPD and 18 healthy subjects. An increase in lipid peroxidation products and a decrease in the content of -SH groups in the membrane of red blood cells in patients with COPD were observed. Moreover, an increase in the activity of glutathione peroxidase and a decrease in superoxide dismutase, but not in catalase activity, were found as well. Significant changes in activities of erythrocyte membrane enzymes in COPD patients were also evident demonstrated by a considerably lowered ATPase activity and elevated AChE activity. Changes in the structure and function of red blood cells observed in COPD patients, together with changes in the activity of the key membrane enzymes (ATPases and AChE), can result from the imbalance of redox status of these cells due to extensive oxidative stress induced by COPD disease. PMID:26369587

  6. Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1 of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90.

    Directory of Open Access Journals (Sweden)

    Thiago V Seraphim

    Full Text Available The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1 is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1. This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90 with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins' interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90 in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90.

  7. Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90.

    Science.gov (United States)

    Seraphim, Thiago V; Alves, Marina M; Silva, Indjara M; Gomes, Francisco E R; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2013-01-01

    The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins' interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90. PMID:23826147

  8. Effects of Celangulin IV and V From Celastrus angulatus Maxim on Na+/K+-ATPase Activities of the Oriental Armyworm (Lepidoptera: Noctuidae)

    Science.gov (United States)

    Cheng, Dan; Feng, Mingxing; Ji, Yufei; Wu, Wenjun; Hu, Zhaonong

    2016-01-01

    Na+/K+-ATPase (sodium pump) is an important target for the development of botanical pesticide as it is responsible for transforming chemical energy in ATP to osmotic work and maintaining electrochemical Na+ and K+ gradients across the cell membrane of most animal cells. Celangulin IV (C-IV) and V (C-V), which are isolated from the root bark of Celastrus angulatus, are the major active ingredients of this insecticidal plant. The activities of C-IV and C-V on Na+/K+-ATPase were investigated by ultramicro measuring method to evaluate the effects of C-IV and C-V on Na+/K+-ATPase activities of the brain from the fifth Mythimna separata larvae and to discuss the insecticidal mechanism of C-IV and C-V. Results indicate that inhibitory activities of Na+/K+-ATPase by C-IV and C-V possess an obvious concentration-dependent in vitro. Compared with C-IV, the inhibition of C-V on Na+/K+-ATPase was not striking. In vivo, at a concentration of 25 mg/liter, the inhibition ratio of C-IV on Na+/K+-ATPase activity from the brain in narcosis and recovery period was more remarkable than that of C-V. Furthermore, the insects were fed with different mixture ratios of C-IV and C-V. The inhibition extent of Na+/K+-ATPase activity was corresponded with the dose of C-IV. However, C-V had no notable effects. This finding may mean that the mechanism of action of C-IV and C-V on Na+/K+-ATPase were different. Na+/K -ATPase may be an action target of C-IV and C-V. PMID:27324586

  9. Co-factor engineering in lactobacilli: Effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

    DEFF Research Database (Denmark)

    Rud, Ida; Solem, Christian; Jensen, Peter Ruhdal;

    2008-01-01

    The hydrolytic F-1-part of the F1F0-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resul...... have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity. (C) 2008 Elsevier Inc. All rights reserved....

  10. Dimerization and oligomerization of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Ryder, L Rebekka; Steinø, Anne; Højrup, Peter; Hansen, Jesper; Beyer, N Helena; Heegaard, Niels H H; Houen, Gunnar

    2003-01-01

    The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disulfide-bridged dimers under physiological conditions, indicating that the SH group may not be fully accessible in the native...... calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity....

  11. Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats

    OpenAIRE

    Yi-zong ZHAI; Chang-lin HUANG; Chang, Qi; Wang, Jiu-Qing; Zhang, Jia; Guo, Yan-Ling

    2015-01-01

    Objective To explore the effects of percutaneous impulsive current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats, in order to investigate the effect of exercise-induced fatigue. Methods Seventy-two 8-week old male Wistar rats were randomly divided into 4 groups (18 each): control group (group A), fatigue group (group B), stimulation before fatigue group (group C) and stimulation after fatigue group (...

  12. Rapid modulation of Na+/K+-ATPase activity in osmoregulatory tissues of a salmonid fish

    DEFF Research Database (Denmark)

    Tipsmark, Christian Kølbæk; Madsen, Steffen

    2001-01-01

    The effects of cyclic AMP on Na+/K+-ATPase activity were studied in the gill and kidney of the euryhaline brown trout Salmo trutta using two different experimental approaches. In the first series of experiments, in situ Na+/K+-ATPase activity was analyzed by measuring the ouabain-sensitive uptake...... of non-radioactive rubidium (Rb+) into gill cells and blocks of gill and kidney tissue. Rubidium uptake was linear for at least 30 min and was significantly inhibited by 1 mmol x l(-1) ouabain. Several agents presumed to increase the intracellular cyclic AMP concentration inhibited ouabain......-sensitive Rb+ uptake in both gill (0.5 and 2 mmol x l(-1) dibutyryl-cyclic AMP, 1 mmol x l(-1) theophylline, 10 micromol x l(-1) forskolin and 10 micromol x l(-1 )isoproterenol) and kidney (10 micromol x l(-1) forskolin) tissue from freshwater-acclimated fish. In a separate series of experiments, ATP hydrolase...

  13. Effect of Rejection on Electrophysiologic Function of Canine Intestinal Grafts: Correlation with Histopathology and Na–K-ATPase Activity

    OpenAIRE

    Takeyoshi, Izumi; Zhang, Shimin; KOKUDO, YASUTAKA; Kenjiro NAKAMURA; Ikoma, Akira; Zhu, Yue; Starzl, Thomas E.; Todo, Satoru

    1995-01-01

    To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na–K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18–24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were switched between a pair of dogs (allograft). Animals receivi...

  14. The effects of low-intensity electromagnetic irradiation at the frequencies of 51.8 and 53 GHz and antibiotic ceftazidime on Lactobacillus acidophilus F0F1 ATP-ase activity

    International Nuclear Information System (INIS)

    The effects of low intensity electromagnetic irradiation (EMI) at the frequencies 51.8 and 53 GHz and antibiotic ceftazidime on N,N'-dicyclohexylcarbodiimide (DCCD), inhibited ATP-ase activity of Lactobacillus acidophilus membrane vesicles were investigated. It was shown that both frequencies decreased the ATP-ase activity, moreover, ceftazidime increase the sensitivity of cells to DCCD, inhibitor of the F0F1-ATP-ase. EMI combined with ceftazidime and DCCD markedly decreased the ATPase activity. The F0F1-ATP-ase is suggested can be a target for the effects observed

  15. Changes in the F0F1-ATPase activity of irradiated Lactobacillus acidophilus in the presence of ceftazidime at low pH

    International Nuclear Information System (INIS)

    The aim of this study was the investigation of the effects of low intensity electromagnetic irradiation (EMI) at the frequencies of 51.8 and 53 GHz and of antibiotic ceftazidime on the N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity of membrane vesicles of lactic acid bacteria Lactobacillus acidophilus grown at low pH (pH 4.0 or 6.5) and assayed at the same pH. It was shown that both frequencies EMI stimulated ATPase activity of L. acidophilus grown at pH 4.0, but EMI combined with ceftazidime and DCCD decreased ATPase activity at pH 4.0 and pH 6.5. It was suggested that the F0F1-ATPase might be a target for EMI even at low pH

  16. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

    OpenAIRE

    Hallows, Kenneth R.; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M.

    2009-01-01

    Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3−, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) reg...

  17. Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+, K+-ATPase activity

    International Nuclear Information System (INIS)

    Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (370C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[3H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[3H]amphetamine binding, suggesting the involvement of Na+, K+-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na+,K+-ATPase activity and the number of specific high-affinity binding sites for [3H]ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. These data suggest that the (+)-[3H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na+,K+-ATPase activity, and the latter may be involved in the glucostatic regulation of appetite

  18. Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly.

    Science.gov (United States)

    Wu, Guowei; Adachi, Hironori; Ge, Junhui; Stephenson, David; Query, Charles C; Yu, Yi-Tao

    2016-03-15

    Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre-mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre-mRNA splicing, leading to growth-deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA-dependent ATPase involved in monitoring the U2 BSRR-branch site base-pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre-mRNA splicing by directly altering the binding/ATPase activity of Prp5. PMID:26873591

  19. An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.

    Science.gov (United States)

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro. PMID:27473959

  20. Pentylenetetrazol-induced seizures are associated with Na⁺,K⁺-ATPase activity decrease and alpha subunit phosphorylation state in the mice cerebral cortex.

    Science.gov (United States)

    Marquezan, Bárbara P; Funck, Vinícius R; Oliveira, Clarissa V; Pereira, Letícia M; Araújo, Stífani M; Zarzecki, Micheli S; Royes, Luiz Fernando F; Furian, Ana Flávia; Oliveira, Mauro S

    2013-08-01

    The present study aimed to investigate whether Na(+),K(+)-ATPase activity and phosphorylation state of the catalytic α subunit are altered by pentylenetetrazol (PTZ)-induced seizures. PTZ (30, 45 or 60 g/kg, i.p.) was administered to adult male Swiss mice, and Na(+),K(+)-ATPase activity and phosphorylation state were measured in the cerebral cortex 15 min after PTZ administration. Na(+),K(+)-ATPase activity significantly decreased after PTZ-induced seizures (60 mg/kg). Immunoreactivity of phosphorylated Ser943 at α subunit was increased after PTZ-induced seizures. A significant positive correlation between Na(+),K(+)-ATPase activity and latency to myoclonic jerks and generalized seizures was found. Conversely, a strong negative correlation between Ser943 phosphorylation and latency to generalized seizures was detected. Given the role of Na(+),K(+)-ATPase as a major regulator of brain excitability, Ser943 at Na(+),K(+)-ATPase α subunit may represent a potentially valuable new target for drug development for seizure disorders. PMID:23602551

  1. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

    Directory of Open Access Journals (Sweden)

    Dana Galuska

    Full Text Available BACKGROUND: Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC in control and hyperglycemic conditions. METHODOLOGY/PRINCIPAL FINDINGS: HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86Rb(+ uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM. DNA binding activity was determined by electrical mobility shift assay (EMSA. Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1-subunit protein expression, accompanied with increase in (86Rb(+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6, concomitant with Na,K-ATPase α(1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing. CONCLUSIONS/SIGNIFICANCE: Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription

  2. A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

    Science.gov (United States)

    Kumar, C Sudheer; Swamy, Musti J

    2016-07-01

    HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch. PMID:27292547

  3. Effects of percutaneous midband pulse current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats

    Directory of Open Access Journals (Sweden)

    Yi-zong ZHAI

    2015-06-01

    Full Text Available Objective To explore the effects of percutaneous impulsive current stimulation in hepatic region on the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase in exercise-induced fatigued rats, in order to investigate the effect of exercise-induced fatigue. Methods Seventy-two 8-week old male Wistar rats were randomly divided into 4 groups (18 each: control group (group A, fatigue group (group B, stimulation before fatigue group (group C and stimulation after fatigue group (group D. Exhaustion of animals in B, C and D groups were reproduced by prolonged swimming. Current stimulation (1024Hz, 10mA, current cycle 1sec for 20 minutes was given to the rats of group C before swimming, and to those in group D after exhaustion. At the weekend of 1st, 3rd and 5th week after modeling, the rats were sacrificed in batches from each group (6 each. The activities of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase were determined by spectrophotometry, and Bradfood protein quantification was employed to quantitate the protein in rats' hepatic mitochondria. Results No significant difference was found in swimming-exhaustion time among 3 groups at the first weekend (P>0.05, while the swimming-exhaustion time was significantly prolonged at the 3rd and 5th weekends in group D than in group B and C (P0.05, while the enzyme activities were obviously lower at the 3rd and 5th weekend in group B than that in groups A, C and D (P<0.05, and they were also lower in group C than that in group D (P<0.05. Conclusions Exercise-induced fatigue can lower the activity of hepatic mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase. Percutaneous pulsive current stimulating hepatic region of exercise-induced fatigued rats may improve the enzyme activity, reduce the concentration of free calcium and calcium overload in mitochondria, stimulate the oxidative phosphorylation, accelerate the rate of respiratory chain, promote exercise endurance and score, and

  4. Potentiating activity of luteolin on membrane permeabilizing agent and ATPase inhibitor against methicillin-resistant Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    Dae-Ki Joung; Dong-Won Shin; Dong-Yeul Kwon; Young-Seob Lee; Sin-Hee Han; Sang-Won Lee; Seon-Woo Cha; Su-Hyun Mun; Ryong Kong; Ok-Hwa Kang; Ho-Jun Song

    2016-01-01

    Objective: To investigate the mechanism of antibacterial activity of luteolin (LUT) against methicillin-resistant Staphylococcus aureus (MRSA). Methods: The mechanism of anti-MRSA activity of LUT was analyzed by the viability assay in membrane permeabilizing agent, ATPase inhibitors, and peptidoglycan (PGN) derived from Staphylococcus aureus (S. aureus). Also, transmission electron microscopy was used to monitor survival characteristics and changes in S. aureus morphology. Results: Compared to the LUT alone, the optical density of suspensions treated with the combination of 125 μg/mL Tris and 250 μg/mL DCCD were reduced to 60%and 46%, respectively. PGN (15.6 μg/mL) gradually impeded the activity of LUT, and PGN (62.5 μg/mL) completely blocked the activity of LUT on S. aureus. Conclusions: Increased susceptibility to LUT with the Tris and DCCD combinations is evident in all tested MRSA isolates. The results indicate LUT synergy in increasing cytoplasmic membrane permeability and inhibiting ATPase. S. aureus PGN directly blocks the antibacterial activity of LUT, suggesting the direct binding of LUT with PGN. These findings may be validated for the development of antibacterial agent for low MRSA resistance.

  5. Optimisation of recombinant production of active human cardiac SERCA2a ATPase

    OpenAIRE

    Antaloae, Ana V.; Cédric Montigny; Marc le Maire; Watson, Kimberly A.; Thomas L-M Sørensen

    2013-01-01

    Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain...

  6. Synchronous In Situ ATPase Activity, Mechanics, and Ca2+ Sensitivity of Human and Porcine Myocardium

    Czech Academy of Sciences Publication Activity Database

    Griffiths, P. J.; Isackson, H.; Pelc, Radek; Redwood, C.S.; Funari, S.S.; Watkins, H.; Ashley, C. C.

    2009-01-01

    Roč. 97, č. 9 (2009), s. 2503-2512. ISSN 0006-3495 R&D Projects: GA MŠk(CZ) LC06063 Grant ostatní: EC(XE) RII3-CT-2004-506008 Institutional research plan: CEZ:AV0Z50110509 Keywords : myocardium * actomyosin-ATPase * synchrotron-radiation Subject RIV: ED - Physiology Impact factor: 4.390, year: 2009

  7. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    OpenAIRE

    Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

    2002-01-01

    Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarizat...

  8. Quantification of Anti-Aggregation Activity of Chaperones: A Test-System Based on Dithiothreitol-Induced Aggregation of Bovine Serum Albumin

    OpenAIRE

    Vera A Borzova; Markossian, Kira A.; Dmitriy A. Kara; Natalia A Chebotareva; Makeeva, Valentina F.; Poliansky, Nikolay B.; Muranov, Konstantin O.; Kurganov, Boris I.

    2013-01-01

    The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin ...

  9. Effect of organic solvents on nervous cell membrane as measured by changes in the (Ca2+/Mg2+) ATPase activity and fluidity of synaptosomal membrane.

    Science.gov (United States)

    Edelfors, S; Ravn-Jonsen, A

    1992-03-01

    The effect of various solvents on the central nervous system was studied by using rat brain synaptosomal membranes as an in vitro model. The activity of (Ca2+/Mg2+) ATPase and the membrane fluidity was determined. The alteration of the ATPase activity depended on the physio-chemical characteristics of the solvent in question. Incubation with aliphatic alkanes caused a stimulation of the ATPase activity whereas mixed hydrocarbons as kerosene, white spirit and gasoline inhibited the enzyme. Incubation with chlorinated hydrocarbons caused a biphasic response dependent on the concentration. Oxygen-containing hydrocarbons exhibited various effects as found after incubation with hydrocarbons. The different effects of the solvents on the ATPase activity suggest that the lipophilicity of the solvents is one of more parameters affecting the membrane. Furthermore, the biphasic response following the incubation with chlorinated hydrocarbons indicates that more mechanisms are involved in the enzyme effect. The membrane fluidity is increased with higher concentrations of the solvents. From the results it is concluded that the ATPase activity depends not only on the membrane fluidity and volume, but also on the hydrophilic vicinity of the enzyme molecule. PMID:1533717

  10. Long-term decrease in Na+,K+-ATPase activity after pilocarpine-induced status epilepticus is associated with nitration of its alpha subunit.

    Science.gov (United States)

    Funck, Vinícius Rafael; Ribeiro, Leandro Rodrigo; Pereira, Letícia Meier; de Oliveira, Clarissa Vasconcelos; Grigoletto, Jéssica; Fighera, Michele Rechia; Royes, Luiz Fernando Freire; Furian, Ana Flávia; Oliveira, Mauro Schneider

    2014-12-01

    Temporal lobe epilepsy (TLE) is the most common type of epilepsy with about one third of TLE patients being refractory to antiepileptic drugs. Knowledge about the mechanisms underlying seizure activity is fundamental to the discovery of new drug targets. Brain Na(+),K(+)-ATPase activity contributes to the maintenance of the electrochemical gradients underlying neuronal resting and action potentials as well as the uptake and release of neurotransmitters. In the present study we tested the hypothesis that decreased Na(+),K(+)-ATPase activity is associated with changes in the alpha subunit phosphorylation and/or redox state. Activity of Na(+),K(+)-ATPase decreased in the hippocampus of C57BL/6 mice 60 days after pilocarpine-induced status epilepticus (SE). In addition, the Michaelis-Menten constant for ATP of α2/3 isoforms increased at the same time point. Nitration of the α subunit may underlie decreased Na(+),K(+)-ATPase activity, however no changes in expression or phosphorylation state at Ser(943) were found. Further studies are necessary define the potential of nitrated Na(+),K(+)-ATPase as a new therapeutic target for seizure disorders. PMID:25311690

  11. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.

    Science.gov (United States)

    Hallows, Kenneth R; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M

    2009-04-01

    Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. PMID:19211918

  12. Effects of fructose-1,6-diphosphate on concentration of calcium and activities of sarcoplosnic Ca2+-ATPase in cardiomyocytes of Adriamycin-treated rats

    Institute of Scientific and Technical Information of China (English)

    CAI Wei; CHEN Jun-zhu; RUAN Li-ming; WANG Yi-na

    2005-01-01

    Objective: To observe the effects of fructose-1,6-diphosphate (FDP) on serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), as well as the concentration of calcium in cardiomyocytes (Myo[Ca2+]) and activity of sarcoplosnic Ca2+-ATPase (SRCa2+-ATPase) in Adriamycin (ADR)-treated rats. Methods: Rats were intraperitoneally injected with ADR (2.5mg/kg every other day for 6 times) and then with different dosages of FDP (every other day for twenty-one times). Bi-antibodies sandwich Enzyme linked immune absorption assay (ELISA) was performed to detect serum level of cTnI. CK-MB was detected by monoclonal antibody, Myo[Ca2+] was detected by fluorescent spectrophotometry and the activity of SRCa2+-ATPase was detected by inorganic phosphate method. Results: FDP (300, 600, 1200 mg/kg) significantly reduced the serum levels of cTnI and CK-MB, while at the same time decreased calcium concentration and increased SRCa2+-ATPase activity in cardiomyocytes of ADR-treated rats (P<0.01). Conclusions: FDP might alleviate the cardiotoxic effects induced by ADR through decreasing calcium level as well as increasing SRCa2+-ATPase activity in cardiomyocytes.

  13. Kinase-Mediated Regulation of P4-ATPases

    DEFF Research Database (Denmark)

    Frøsig, Merethe Mørch

    Abstract Kinase-Mediated Regulation of P4-ATPases Understanding kinase-mediated regulation and designing novel tools to study regulatory proteins of P4-ATPases P4-ATPases play a critical role in the biogenesis of transport vesicles in the secretory and endocytic pathways, and P4-ATPase activity...

  14. Structural differences between bovine A(1) and A(2) β-casein alter micelle self-assembly and influence molecular chaperone activity.

    Science.gov (United States)

    Raynes, J K; Day, L; Augustin, M A; Carver, J A

    2015-04-01

    Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) β-CN variant formed smaller micelles than A(1) β-CN, with the monomer-micelle equilibrium of A(2) β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A(2) β-CN and most likely led to enhanced chaperone activity of A(2) β-CN compared with A(1) β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk. PMID:25648798

  15. Inhibition of nitrate reductase and ATPase activities in Zea mays roots by tungsten and N, N'-dicyclohexylcarbodiimide

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-02-01

    Full Text Available The activity of soluble and membrane-bound ATPase obtained from corn roots was in vivo markedly inhibited by N,N' -dicyclohexylcanbodiimide (DCCD and W042- ions. DCCD (2.5 X 10-5 M added to the nutrient solution strongly decreased in vivo nitrate reductase (NR activity after 12-h growth of plants while it had no effect in experiments in vitro on NR activity. Tungsten in a concentration of 10-4 M completely blocked NR activity after 24 h. In the above used concentrations neither DCCD nor W042- inhibited completely N03- absorption by corn roots. The results suggest that there must exist in corn roots another or an additional mechanism of N03- assimilation apart from of that proposed by Butz and Jackson (1977.

  16. Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

    Science.gov (United States)

    Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey

    2014-06-01

    Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. PMID:24723394

  17. Changes in soluble interleukin-2 receptor level in serumand Na+ -K+- exchanging ATPase activity in semen of infertile men caused by antisperm antibody

    Institute of Scientific and Technical Information of China (English)

    JiangNI; Qing-LeiLI; WeiZHANG; Jian-SongXIE; Shu-LingBIAN

    2000-01-01

    Aim: To explore the possible mechanisms of male infertility caused by antisperm antibody (AsAb). Methods: The soluble interleukin-2 receptor (sIL-2R) level in serum was analyzed by ELISA and Na+ -K+ -exchanging ATPase activity in semen by phosphorus (Pi) assay. Results: The sIL-2R level in serum was significantly higher and the Na+-K+- exchanging ATPase activity in semen significantly lower in AsAb positive infertile men when compared with the controls. Conclusion: The AsAb titer varies with the sIL-2R level in serum. A decrease in Na+-K+-exchanging ATPase activity in semen may play a role in male infertility caused by AsAb.

  18. Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

    OpenAIRE

    Friedrich, T.; Bamberg, E; Nagel, G

    1996-01-01

    The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were mark...

  19. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Directory of Open Access Journals (Sweden)

    Elisa Zorzi

    2011-10-01

    Full Text Available Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs. However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70, and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

  20. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    Energy Technology Data Exchange (ETDEWEB)

    Zorzi, Elisa [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Bonvini, Paolo, E-mail: paolo.bonvini@unipd.it [OncoHematology Clinic of Pediatrics, University-Hospital of Padova, 35100 Padova (Italy); Fondazione Città della Speranza, 36030 Monte di Malo, Vicenza (Italy)

    2011-10-21

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

  1. Inducible Hsp70 in the Regulation of Cancer Cell Survival: Analysis of Chaperone Induction, Expression and Activity

    International Nuclear Information System (INIS)

    Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more “addicted” to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells

  2. The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

    Science.gov (United States)

    Silva, Lilian N D; Ribeiro-Neto, José A; Valadares, Jéssica M M; Costa, Mariana M; Lima, Luciana A R S; Grillo, Luciano A M; Cortes, Vanessa F; Santos, Herica L; Alves, Stênio N; Barbosa, Leandro A

    2016-08-01

    Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae. PMID:26993642

  3. Δ²,³-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases.

    Science.gov (United States)

    Noël, François; Pimenta, Paulo Henrique Cotrim; Dos Santos, Anderson Rouge; Tomaz, Erick Carlos Loureiro; Quintas, Luis Eduardo Menezes; Kaiser, Carlos Roland; Silva, Claudia Lucia Martins; Férézou, Jean-Pierre

    2011-01-01

    Looking at a new putative target for the large spectrum antiparasitic drug ivermectin, we recently showed that avermectin-derived drugs are active against promastigote and amastigote forms of Leishmania amazonensis at low micromolar concentrations. However, we then reported that at this concentration range ivermectin is also able to inhibit three important mammalian P-type ATPases so that unacceptable adverse effects could occur if this drug were used at such high doses therapeutically. The present work aimed to test the activity of ten ivermectin analogs on these rat ATPases in search of a compound with similar leishmanicidal activity but with no effect on the mammalian (host) ATPases at effective concentrations. We synthesized three new ivermectin analogs for testing on rat SERCA (1a and 1b), Na+, K+-ATPase (α₁ and α₂/α₃ isoforms) and H+/K+-ATPase activity, along with seven analogs already characterized for their leishmanicidal activity. Our main finding is that one of the prepared derivatives, Δ²,³-ivermectin ethyl secoester 8, is equipotent to ivermectin 1 for the in vitro leishmanicidal effects but is nearly without effect on the rat ATPases, indicating that it could have a better therapeutic index in vivo and could serve as a candidate for hit-to-lead progression. This conclusion is further supported by the fact that compound 8 produced only 6% (vs 77% for ivermectin) inhibition of the human kidney enzyme at 5 μM, a concentration corresponding to the IC₅₀ for the activity against L. amazonensis amastigotes. PMID:21088826

  4. Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

    DEFF Research Database (Denmark)

    Axelsen, K.B.; Venema, K.; Jah, T.;

    1999-01-01

    The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme....... Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP...

  5. Polypeptide binding properties of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C S; Heegaard, N H; Holm, A; Højrup, P; Houen, G

    2000-01-01

    Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be...

  6. Evaluation of molecular chaperons Hsp72 and neuropeptide Y as characteristic markers of adaptogenic activity of plant extracts.

    Science.gov (United States)

    Asea, Alexzander; Kaur, Punit; Panossian, Alexander; Wikman, Karl Georg

    2013-11-15

    We have previously demonstrated that ADAPT-232, a fixed combination of adaptogenic substances derived from Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract stimulated the expression and release of neuropeptide Y (NPY) and molecular chaperone Hsp72 from isolated human neurolgia cells. Both of these mediators of stress response are known to play an important role in regulation of neuroendocrine system and immune response. We further demonstrated that ADAPT-232 induced release of Hsp70 is mediated by NPY, suggesting an existence of NPY-mediated pathway of activation of Hsp72 release into the blood circulation system. The objective of this study was to determine whether this pathway is common for adaptogens and whether NPY and/or Hsp72 can be considered as necessary specific biomarkers for adaptogenic activity. The release of NPY and Hsp72 from neuroglia cells in response to treatment with various plant extracts (n=23) including selected validated adaptogens, partly validated adaptogens, claimed but negligibly validated adaptogens and some other plant extracts affecting neuroendocrine and immune systems but never considered as adaptogens was measured using high throughput ELISA techniques. We demonstrated that adaptogens, e.g. R. rosea, S. chinensis and E. senticosus stimulate both NPY and Hsp70 release from neuroblastoma cells, while tonics and stimulants have no significant effect on NPY in this in vitro test. In the groups of partly validated adaptogens the effect of Panax ginseng and Withania somnifera was not statistically significant both on NPY and Hsp70 release, while the activating effect of Bryonia alba and Rhaponticum cartamoides was significant only on Hsp70. In contrast, all tested non-adaptogens, such as antiinflammatoty plant extracts Matricaria recutita, Pelargonium sidoides, Hedera helix and Vitis vinifera significantly inhibit Hsp70 release and have no influence on NPY release from neuroblastoma

  7. Gymnastics of molecular chaperones.

    Science.gov (United States)

    Mayer, Matthias P

    2010-08-13

    Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families. PMID:20705236

  8. A kinetic characterization of (Na+, K+)-ATPase activity in the gills of the pelagic seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae).

    Science.gov (United States)

    Leone, Francisco Assis; Lucena, Malson Neilson; Rezende, Luciana Augusto; Garçon, Daniela Pereira; Pinto, Marcelo Rodrigues; Mantelatto, Fernando Luis; McNamara, John Campbell

    2015-04-01

    We characterize the kinetic properties of a gill (Na(+), K(+))-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na(+), K(+))-ATPase activity, but also containing mitochondrial F0F1- and Na(+)- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈ 110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na(+), K(+))-ATPase hydrolyzed ATP obeying Michaelis-Menten kinetics with VM = 109.5 ± 3.2 nmol Pi min(-1) mg(-1) and KM = 0.03 ± 0.003 mmol L(-1). Mg(2+) (VM = 109.8 ± 2.1 nmol Pi min(-1 )mg(-1), K0.5 = 0.60 ± 0.03 mmol L(-1)), Na(+) (VM = 117.6 ± 3.5 nmol Pi min(-1 ) mg(-1), K0.5 = 5.36 ± 0.14 mmol L(-1)), K(+) (VM = 112.9 ± 1.4 nmol Pi min(-1 )mg(-1), K0.5 = 1.32 ± 0.08 mmol L(-1)), and NH4 (+) (VM = 200.8 ± 7.1 nmol Pi min(-1 )mg(-1), K0.5 = 2.70 ± 0.04 mmol L(-1)) stimulated (Na(+), K(+))-ATPase activity following site-site interactions. K(+) plus NH4 (+) does not synergistically stimulate (Na(+), K(+))-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K(+) binding that can be occupied by NH4 (+), stimulating the enzyme. Ouabain (KI = 84.0 ± 2.1 µmol L(-1)) and orthovanadate (KI = 0.157 ± 0.001 µmol L(-1)) inhibited total ATPase activity by ≈ 50 and ≈ 44 %, respectively. Ouabain inhibition increases ≈ 80 % in the presence of NH4 (+) with a threefold lower KI, suggesting that NH4 (+) is likely transported as a K(+) congener. PMID:25534346

  9. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    International Nuclear Information System (INIS)

    Highlights: ► Pepper vein banding potyvirus VPg harbors Walker motifs. ► VPg exhibits ATPase activity in the presence of NIa-Pro. ► Plausible structural and functional interplay between VPg and NIa-Pro. ► Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  10. Leishmania amazonensis: heme stimulates (Na(+)+K(+))ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways.

    Science.gov (United States)

    Almeida-Amaral, Elmo Eduardo; Cardoso, Viviane Carrozino; Francioli, Fernanda Gomes; Meyer-Fernandes, José Roberto

    2010-04-01

    In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na(+)+K(+))ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH(3) and U73122, specific inhibitors of PI-PLC. (Na(+)+K(+))ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50nM. This effect was completely reversed by 10nM calphostin C, an inhibitor of PKC. Thus, the effect of 50nM heme on (Na(+)+K(+))ATPase activity is completely abolished by ET-18-OCH(3) and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na(+)+K(+))ATPase activity through a PI-PLC/PKC signaling pathway. PMID:20045694

  11. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  12. The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity.

    Directory of Open Access Journals (Sweden)

    Amit P Bhavsar

    Full Text Available To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

  13. Ca2+- and Mg2+-ATPase activities in winter wheat root plasma membranes as affected by NaCl stress during growth

    NARCIS (Netherlands)

    Mansour, MMF; van Hasselt, PR; Kuiper, PJC

    1998-01-01

    Winter wheat seedlings were grown in Hoagland nutrient solution with or without 100 mmol/L NaCl added. Plasma membranes from root cells were prepared by aqueous polymer two phase partitioning and the stimulation of plasma membrane ATPase activity by Mg2+ and Ca2+ was investigated. The enzyme was act

  14. Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

    Science.gov (United States)

    Vasconcelos, Andrea Rodrigues; Kinoshita, Paula Fernanda; Yshii, Lidia Mitiko; Marques Orellana, Ana Maria; Böhmer, Ana Elisa; de Sá Lima, Larissa; Alves, Rosana; Andreotti, Diana Zukas; Marcourakis, Tania; Scavone, Cristoforo; Kawamoto, Elisa Mitiko

    2015-05-01

    Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS. PMID:25818175

  15. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology

    Institute of Scientific and Technical Information of China (English)

    范平

    2012-01-01

    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  16. Páginas de Na-K-ATPase activity in the guinea pig stria vascularis in experimentally-induced endolymphatic hydrops

    OpenAIRE

    Nishiyama, S.; T. Okada; Kobayahsi, T.; García del Saz, E.; Seguchi, H.

    1994-01-01

    The effect of endolymphatic hydrops on the Na-K-ATPase activity in the guinea pig stria vascularis was electron microscopically and enzyme cytochemically investigated one year after experimental induction. The morphological observations revealed intercellular dropsy in the basal infoldings of the marginal cells, and shrinkage and disappearance of intermediate cells. Moreover, shrinkage of the marginal cells, especially of the basal infoldings, was occasiona...

  17. Effect of Salinity on Hemolymph Osmotic Pressure, Sodium Concentration and Na+-K+-ATPase Activity of Gill of Chinese Crab, Eriocheir sinensis

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The effects of salinity on hemolymph osmotic pressure, Na+ concentration and Na+-K+-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na+ concentration increased significantly (P<0.05), and the Na+-K+-ATPase activity of gills decreased significantly (P<0.05) when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na+ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na+-K+-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na+ concentration and lower Na+-K+-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct (P>0.05); However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1 d, and then tended to be stable from day 1 to day 15.

  18. Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

    International Nuclear Information System (INIS)

    The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells (Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H+-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H+-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H+-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2mM dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H+-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH. This rapid irreversible inhibition of the plasma membrane H+-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material. (author)

  19. Inhibitors of the AAA+ Chaperone p97

    Directory of Open Access Journals (Sweden)

    Eli Chapman

    2015-02-01

    Full Text Available It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®, which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+ chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and

  20. Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Thomassen, Martin; Lundby, Carsten; Pilegaard, Henriette; Bangsbo, Jens

    2005-01-01

    The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na+-K+-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na+-K+-ATPase subunit a1, a2, a3,...

  1. Reduced activity of SKC a and Na-K ATPase underlies the accelerated impairment of EDH-type relaxations in mesenteric arteries of aging spontaneously hypertensive rats.

    Science.gov (United States)

    Kong, Billy W C; Man, Ricky Y K; Gao, Yuansheng; Vanhoutte, Paul M; Leung, Susan W S

    2015-06-01

    Aging is accompanied by endothelial dysfunction due to reduced bioavailability of nitric oxide (NO) and/or reduced endothelium-dependent hyperpolarizations (EDH). This study examines the hypothesis that hypertension aggravates the impairment of EDH-type relaxation due to aging. EDH-type relaxations were studied in superior mesenteric arteries isolated from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats of 12, 36, 60, and 72 weeks of age. EDH-type relaxations in WKY were reduced with aging, and this was associated with an impairment of the function of small-conductance calcium-activated potassium channels (SKC a) and sodium-potassium ATPase (Na-K ATPase). EDH-type relaxation in SHR was smaller than that in WKY arteries, and further reduction occurred with aging. Pharmacological experiments suggested a reduced involvement of SKC a and Na-K ATPase and activation of adenosine monophosphate-activated protein kinase and silent information regulator T1 (sirtuin-1; SIRT1) in mesenteric arteries of 12-week-old SHR. These pharmacological findings suggest that in superior mesenteric arteries of the rat, the reduction in EDH-type relaxation occurs with aging and that such a reduction is exacerbated in hypertension. The latter exacerbation appears to involve proteins associated with the process of cellular senescence and is related to impaired function of SKC a and Na-K ATPase, a phenomenon that is also observed in mesenteric arteries of older normotensive rats. PMID:26171229

  2. Fungal plasma membrane H⁺-ATPase inhibitory activity of o-hydroxybenzylated flavanones and chalcones from Uvaria chamae P. Beauv.

    Science.gov (United States)

    Kongstad, Kenneth T; Wubshet, Sileshi G; Kjellerup, Lasse; Winther, Anne-Marie Lund; Staerk, Dan

    2015-09-01

    In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H(+)-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H(+)-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H(+)-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays. PMID:26102180

  3. Changes of plasma membrane ATPase activity,membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities

    Institute of Scientific and Technical Information of China (English)

    ZHAO Zhong-qiu; ZHENG Hai-lei; ZHU Yong-guan

    2004-01-01

    The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0 ‰, 10 ‰, 20 ‰, 30 ‰, 40 ‰) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A.marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity(A. marina and roots of K. candel: 0-30‰;leaves of K.candel: 0-20‰), the activity of ATPase increased with increasing salinity, while high salinity(above 30‰ or 20‰) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0-10‰(K. candel) or 0-20‰(A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

  4. Revisiting the mechanisms of copper toxicity to rainbow trout: Time course, influence of calcium, unidirectional Na(+) fluxes, and branchial Na(+), K(+) ATPase and V-type H(+) ATPase activities.

    Science.gov (United States)

    Chowdhury, M Jasim; Girgis, Mina; Wood, Chris M

    2016-08-01

    In order to resolve uncertainties as to the mechanisms of toxic action of Cu and the protective effects of water [Ca], juvenile rainbow trout were acclimated to baseline soft water (SW, [Na(+)]=0.07, [Ca(2+)]=0.15, [Mg(2+)]=0.05mmolL(-1)) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27mmolL(-1)). The 96-h LC50 was 7-fold higher (63.8 versus 9.2μgCuL(-1); 1.00 versus 0.14μmolCuL(-1)) at [Ca]=3.0 versus 0.15mmolL(-1). Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24h, the gill Cu load (LA50≈0.58μgCug(-1); 9.13nmolCug(-1)) predictive of 50% mortality by 96h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na(+) losses (J(Na)net) by increasing unidirectional Na(+) efflux rates (J(Na)out) and inhibiting unidirectional Na(+) uptake rates (J(Na)in). The effect on J(Na)out was virtually immediate, whereas the effect on J(Na)in developed progressively over 24h and was associated with an inhibition of branchial Na(+), K(+) ATPase activity. The J(Na)in inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL(-1)) than the J(Na)out stimulation (100μg Cu L(-1)). Elevated Ca protected against both effects, as well as against the inhibition of Na(+), K(+) ATPase activity. Branchial V-type H(+) ATPase activity was also inhibited by Cu exposure (100μgCuL(-1)), but only after 24h at high [Ca] (3.0mmolL(-1)). These novel results therefore reinforce the applicability of BLM theory to Cu, clarify that whether Na(+) influx or efflux is more sensitive depends on the duration of Cu exposure, show that elevated water [Ca], independent of alkalinity, is protective against both mechanisms of Cu toxicity, and identify V-type H(+)ATPase as a new Cu target for future investigation. PMID:27262060

  5. Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in rat cardiac pressure-overload hypertrophy

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Aim: To observe effects of angiotensin (Ang) Ⅱ receptor antagonist (AT1) irbesartan and angiotensin-converting enzyme (ACE) inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in the model of pressure-overload cardiac hypertrophy. Methods: Forty male adult Sprague Dawley rats were divided into 5 groups.One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1: sham group, rats (n=8) were gavaged with normal saline 2 ml/(kg·d)(ig); Group 2: control group, rats (n=8) were treated with normal saline 2 ml/(kg·d) (ig); Group 3: rats (n=8) were given perindopril 2 mg/(kg·d) (ig); Group 4: rats (n=8) were treated with irbesartan 20 mg/(kg·d) (ig); Group 5: rats (n=8) were given irbesartan 20 mg/(kg·d) plus perindopril 2 mg/(kg·d) (ig). Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Results: Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca2+-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca2+-ATPase. Conclusion:These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca2+-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of

  6. Difference in {sup 201}TlCl accumulation mechanism in brain tumors. A comparison of their Na{sup +}-K{sup +} ATPase activities

    Energy Technology Data Exchange (ETDEWEB)

    Sugo, Nobuo; Kuroki, Takao; Nemoto, Masaaki; Mito, Toshiaki; Seiki, Yoshikatsu; Shibata, Iekado [Toho Univ., Tokyo (Japan). Omori Hospital

    2000-07-01

    The accumulation levels of {sup 201}TlCl and Na{sup +} -K{sup +} ATPase activity in tumor tissue were compared among glioblastoma, benign glioma and meningioma to study the difference in the mechanism of {sup 201}TlCl accumulation. The subjects were 19 cases comprised of 6 glioblastoma, 2 oligodendroglioma, 1 fibrillary astrocytoma, 1 pilocytic astrocytoma and 9 meningioma. Preoperative {sup 201}TlCl SPECT was performed in all the cases, and Thallium Index (TL index) was calculated by a ratio of {sup 201}TlCl in the tumor area and the contralateral area. In addition, cell membrane was extracted from the tumor tissue collected intraoperatively to determine Na{sup +} -K{sup +} ATPase activity. No statistically significant difference in TL index was noted between the glioblastoma group (6.97{+-}2.67) and the meningioma group (5.87{+-}1.99). This fact showed that there was no difference in the accumulation level of {sup 201}TlCl between the two groups. On the other hand, the glioblastoma group indicated a higher value of Na{sup +} -K{sup +} ATPase activity (49.13{+-}43.76 {mu}mole/hour/mg protein) than the meningioma group (7.73{+-}13.84 {mu}mol/hour/mg protein) (p<0.05, t test). These results suggested the involvement of Na{sup +} -K{sup +} ATPase activity in {sup 201}TlCl accumulation in glioblastoma and the influences of other accumulation mechanism than Na{sup +} -K{sup +} ATPase activity such as the volume of intratumoral vascular bed in meningioma. (author)

  7. MODULATION OF Na + /K + , Mg 2 + and Ca 2+ ATPase ACTIVITY IN DIFFERENT REGIONS OF RAT BRAIN DURING ROTENONE INDUCED PARKINSON'S DISEASE AND PROTECTIVE ROLE OF BACOPA MONNIERI

    Directory of Open Access Journals (Sweden)

    Gunduluru Swathi

    2013-02-01

    Full Text Available Bacopa monnieri(BM; Family: Scrophulariaceae, also referred as Brahmi or Jalbrahmi has been used for centuries in Ayurvedic system of medicine as a brain tonic, memory enhancer, revitaliser of sensory organs, anti-anxiety, cardio-tonic, diuretic, antidepressant and anticonvulsant agent, and the pharmacological actions are mainly attributed to the saponin compounds present in the alcoholic extract of the plant. The present study was carried out with a specific aim to examine the neuroprotective effect of Bacopa monnieriduring Rotenone (RT induced Parkinson’s disease (PD with particular reference to Na+/K+, Mg2+and Ca2+-ATPase activities in different regions of rat brain. In the experiment conducted rats were divided into four groups of six in each group, group 1 received Salinewater (1 ml/kg, group 2 received RT (2.5 mg/kg through i.p. route administration for 60 days to induce PD. The third group received BM extract (180 mg/kg/day for 20 days orally before induction of PD and group 4 received Levodopa (LD (10 mg/kg/day orally which is referred as drug control. The levels of Na+/K+, Mg2+and Ca2+-ATPase activities were measured. Na+/K+, Mg2+and Ca2+-ATPase activities were significantly depleted in different brain regions of rat during RT induced PD when compared to control rats. Treatment with BM and LD caused significant elevation in the activity levels of Na+/K+, Mg2+and Ca2+-ATPase in different brain regions of rats when compared to induced PD rats. Our results suggest the ability of BM extract to modulate Na+/K+, Mg2+and Ca2+- ATPase activities in different brain regions of RT induced rodent model of PD and thus offers effective management in the treatment of PD.

  8. Comparison of Plasma Membrane H+-ATPase Activity in Two Ecotypes of Reed (Phragmites communis) Leaves from Different Habitats

    Institute of Scientific and Technical Information of China (English)

    JINGYan; GONGHai-Jun; ZHAOZhi-Guang; CHENGuo-Cang; WANGSuo-Min; ZHANGCheng-Lie

    2004-01-01

    Plasma membrane (PM) vesicles of the leaves of two ecotypes of reed (Phragrnites communis Trin.), swamp reed (SR) and heavy salt meadow reed (HSMR) growing in the desert region of Northwest China, were purified by two-phase partitioning and the properties of their PM H+-ATPases (EC 3.6.1.35) were compared. The specific activity of this enzyme was greater in HSMR than in SR and the Km lower (1.27mmol/L in SR and 0.30mmol/L in HSMR), and the Vmax of ATP hydrolysis activity showed no significant difference between the two ecotypes. The PM H+-ATPase was more sensitive to denaturing temperatures in HSMR than in SR, and the pH profile also showed a slight difference, suggesting that the catalytic mechanism of this enzyme was different in HSMR compared with that in SR. The p-nitrophenyl phosphate (PNPP) hydrolysis activity of H+-ATPase was higher in HSMR than in SR at low concentrations of PNPP, but showed no difference at high PNPP concentration. The Km for PNPP hydrolysis was 3.61mmol/L and 1.92mmol/L in SR and HSMR, respectively. And the Vmax of PNPP hydrolysis showed no significant difference in the two reed ecotypes. An experiment with the inhibitor vanadate showed that the catalytic mechanisms of the phosphatase domain of the ATPase were different in the two ecotypes. The data obtained following trypsin treatment showed a difference in the enzyme activity pattern, suggesting that there existed a possible change in the C-terminus of the ATPase, either in the structure or in the property or both of them. In addition, compared with SR, the ATP-dependent H+ pumping activity of ATPase and the coupling between proton transport and ATP hydrolysis in HSMR were increased. These results indicated that the properties of PM H+-ATPase were changed in HSMR with compared to those in SR, which might include enzyme modifications and different isoforms expressed. The alterations of the properties of this enzyme might be an adaptive response to the habitat.

  9. Light induces changes in activities of Na(+)/K(+)-ATPase, H(+)/K(+)-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam, Tridacna squamosa.

    Science.gov (United States)

    Ip, Yuen K; Ching, Biyun; Hiong, Kum C; Choo, Celine Y L; Boo, Mel V; Wong, Wai P; Chew, Shit F

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH(+) 4 or H(+), and activities of enzymes involved in converting NH(+) 4 to glutamate/glutamine in inner mantle, outer mantle, and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH(+) 4 in substitution for K(+) to activate Na(+)/K(+)-ATPase (NKA), manifested as a significant increase in the Na(+)/NH(+) 4-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H(+) released from CaCO3 deposition could react with NH3 to form NH(+) 4 in the extrapallial fluid, and NH(+) 4 could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH(+) 4 and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM)-sensitive-V-H(+)-ATPase (VATPase) in the inner mantle, and significant increases in the Na(+)/K(+)-activated-NKA, H(+)/NH(+) 4-activated-H(+)/K(+)-ATPase, and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na(+) homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH(+) 4 from the environment. This is the first report on light having direct

  10. Light induces changes in activities of Na+/K+(NH4+-ATPase, H+/K+(NH4+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam Tridacna squamosa

    Directory of Open Access Journals (Sweden)

    Alex Y K Ip

    2015-03-01

    Full Text Available The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control, on enzymatic activities of transporters involved in the transport of NH4+ or H+, and activities of enzymes involved in converting NH4+ to glutamate/glutamine in inner mantle, outer mantle and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH4+ in substitution for K+ to activate Na+/K+-ATPase (NKA, manifested as a significant increase in the Na+/NH4+-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H+ released from CaCO3 deposition could react with NH3 to form NH4+ in the extrapallial fluid, and NH4+ could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH4+ and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM-sensitive-V-H+-ATPase (VATPase in the inner mantle, and significant increases in the Na+/K+-activated-NKA, H+/NH4+-activated-H+/K+-ATPase and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na+ homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH4+ from the environment. This is the first report on light having direct enhancing effects on activities of certain

  11. Crystal Structures of Cisplatin Bound to a Human Copper Chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Boal, Amie K.; Rosenzweig, Amy C.; (NWU)

    2010-08-16

    Copper trafficking proteins, including the chaperone Atox1 and the P{sub 1B}-type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.

  12. X-ray effects on the activity of a Mg2+-dependent, Na+- and K+-activable microsomal membrane ATP-ase system

    International Nuclear Information System (INIS)

    The bahviour of a Mg2+-dependent, Na+- and K+-activable ATP-ase sytem on irradiation was investigated using a microsome fraction of guinea pig myocardial cells prepared by fractionated centrifugation. The Na+- and K+-activable component, transport-ATPase, was particularly radiation-sensitive. Three stages of development were observed for a 1,500 R radiation damage until 24 h p.r.. In the first stage, until 30 minutes p.r., the activity of transport-ATP-ase was inhibited. This was followed by repair processes which had reached a peak value clearly higher than the control values at 4 hours p.r.. In the third stage, the activity was reduced again; 15 and 24 hours after termination of exposure, values again were nearly the same as after 30 minutes where a maximum was observed for this radiation dose. Radiation-induced electrolyte displacements, active transport, and radiation-induced inhibition of transport-ATP-ase were correlated and discussed; the assumption was that changes in, the electrolyte conditions in the membranes on irradiation are at least partly due to the described inhibition of transport-ATP-ase. (orig./AJ)

  13. Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

    International Nuclear Information System (INIS)

    Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K+ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K+ channels and Na+/K+-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O2− production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K+-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K+-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K+ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress. -- Highlights: ► Increased free radicals production ► Increased Na+/K+ ATPase activity ► Promotes activation of the K+ channels and reduced vascular reactivity ► These effects preserve endothelial function against oxidative stress. ► Low concentrations constitute environmental cardiovascular

  14. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Science.gov (United States)

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression. PMID:21531199

  15. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    Science.gov (United States)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  16. Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

    OpenAIRE

    Mahmmoud, Yasser A.

    2005-01-01

    Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase.We tested the hypothesis that curcumin may modulat...

  17. The investigation for the relationship among serum leptin, erythrocyte membrane Ca2+-ATPase activity and hypertensive disorder complicating pregnancy

    Institute of Scientific and Technical Information of China (English)

    Chunfang Li; Wenli Gou; Xuelian Chen; Shuping Zhang

    2007-01-01

    Objective: To study the significance of Leptin and the activity of erythrocyte membrane Ca2+-ATPase (EMCA) in the development of hypertensive disorder complicating pregnancy. Methods: Radioimmunoassay was used to test the level of serum Leptin,and the activity of EMCA was determined chemically in 38 pregnant women with hypertensive disorder complicating pregnancy and 36 normotensive pregnant women. Results: The level of serum Leptin in hypertensive disorder complicating pregnancy(gestational hypertension: 13.76 ± 3.46 ng/ml; preeclampsia:15.76 ± 5.47 ng/ml; eclampsia: 18.32 ± 6.38 ng/ml)was significantly higher than that in normotensive pregnant women (11.33 ± 2.93 ng/ml), respectively. The average EMCA activity of patients with hypertensive disorder complicating pregnancy (gestational hypertension: 1.65 ± 0.24 μmol· pi/mg·h; preeclampsia: 1.37 ± 0.19 μmol·pi/mg·h; eclampsia:1.12 ± 0.14 μ mol·pi/mg·h) was significantly lower than that of normotensive pregnant women(1.83 ±0.38 μ mol·pi/mg·h),respectively. There was a negative correlation between the level of serum Leptin and the activity of RMCA in hypertensive disorder complicating pregnancy (r = -0.63). Conclusion: Inhibition of EMCA activity of erythrocyte in hypertensive disorder complicating pregnancy may increase cytoplasmic free calcium, which contributes to the development of hypertensive disorder complicating pregnancy. The negative correlation between the level of serum Leptin and the activity of EMCA, also suggested that serum Leptin and the activity of EMCA may play a role in the development of hypertensive disorder complicating pregnancy.

  18. Discovery of a novel target for the dysglycemic chromogranin A fragment pancreastatin: interaction with the chaperone GRP78 to influence metabolism.

    Directory of Open Access Journals (Sweden)

    Nilima Biswas

    Full Text Available RATIONALE: The chromogranin A-derived peptide pancreastatin (PST is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced. METHODS AND RESULTS: We used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273-301-amide as "bait" and mouse liver homogenate as "prey", and identified GRP78 (a.k.a. "78 kDa Glucose Regulated Protein", HSPA5, BIP as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones, involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the "adaptive UPR" in CHGA(-/- mice compared to wild-type (+/+. By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(-/- mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM. PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis. In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP. CONCLUSIONS: Our results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion, but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic

  19. Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

    Energy Technology Data Exchange (ETDEWEB)

    Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.; Yu, Zhanqian; Powers, Evan T.; Kelly, Jeffery W.; Lieberman, Raquel L. (Scripps); (GIT)

    2013-03-07

    Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, such as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.

  20. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    Science.gov (United States)

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators. PMID:24316836

  1. Effects of synthetic and naturally occurring flavonoids on Na+, K+-ATPase: Aspects of the structure-activity relationship and action mechanism

    International Nuclear Information System (INIS)

    A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na+, K+ -adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na+, K+ -ATPase from dog kidney cortex when present at concentrations from 40-1000 μM. Flavones possessing cyclohexyl instead of the phenyl group were the most potent with IC50 at 257-320 μM. Structure-activity relationships were observed among the following mono-substituted flavones as: (i) 2-cyclohexyl-benzopyran-4-one much-gt 2-phenyl-benzopyran-4-one; (ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one > 2-cyclohexyl-6-hydroxy-benzopyran-4-one > 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly and of the flavonoids were able to block [3H] ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na+, K+ -ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme

  2. Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences

    DEFF Research Database (Denmark)

    Jacobs, M; Kontinen, V; Sarvas, M;

    1993-01-01

    In prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including alpha-amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted by...... the N-terminal pro-sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin-alkaline phosphatase fusion genes in the prsA3 mutant. We found massive...... degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro-sequence (like subtilisin...

  3. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ54-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP–BeFx

    International Nuclear Information System (INIS)

    This study reports the crystallization of a new nucleotide state of the ATPase domain of a bacterial transcription activator NtrC1 from the hyperthermophilic bacterium Aquifex aeolicus. Wild-type NtrC1 ATPase domain was crystallized in the presence of the ATP analog ADP–BeFx–Mg and the crystals diffracted anisotropically to at best 3.2, 5.2 and 3.2 Å resolution in the a*, b* and c* directions, respectively. One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ70 RNA polymerase (RNAP), σ54 RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP–BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators

  4. Overproduction of the Escherichia coli Chaperones GroEL-GroES in Rhodococcus ruber Improves the Activity and Stability of Cell Catalysts Harboring a Nitrile Hydratase.

    Science.gov (United States)

    Tian, Yuxuan; Chen, Jie; Yu, Huimin; Shen, Zhongyao

    2016-02-01

    Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpEGroEL- GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases. PMID:26562693

  5. N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

    Science.gov (United States)

    Cha, Joon-Yung; Lee, Sang-Hoon; Seo, Kyung Hye; Choi, Young Jin; Cheong, Mi Sun; Son, Daeyoung

    2016-02-01

    Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance. PMID:26724757

  6. Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content.

    Science.gov (United States)

    Sjögren, Lars L E; MacDonald, Tara M; Sutinen, Sirkka; Clarke, Adrian K

    2004-12-01

    ClpC is a molecular chaperone of the Hsp100 family. In higher plants there are two chloroplast-localized paralogs (ClpC1 and ClpC2) that are approximately 93% similar in primary sequence. In this study, we have characterized two independent Arabidopsis (Arabidopsis thaliana) clpC1 T-DNA insertion mutants lacking on average 65% of total ClpC content. Both mutants display a retarded-growth phenotype, leaves with a homogenous chlorotic appearance throughout all developmental stages, and more perpendicular secondary influorescences. Photosynthetic performance was also impaired in both knockout lines, with relatively fewer photosystem I and photosystem II complexes, but no changes in ATPase and Rubisco content. However, despite the specific drop in photosystem I and photosystem II content, no changes in leaf cell anatomy or chloroplast ultrastructure were observed in the mutants compared to the wild type. Previously proposed functions for envelope-associated ClpC in chloroplast protein import and degradation of mistargeted precursors were examined and shown not to be significantly impaired in the clpC1 mutants. In the stroma, where the majority of ClpC protein is localized, marked increases of all ClpP paralogs were observed in the clpC1 mutants but less variation for the ClpR paralogs and a corresponding decrease in the other chloroplast-localized Hsp100 protein, ClpD. Increased amounts of other stromal molecular chaperones (Cpn60, Hsp70, and Hsp90) and several RNA-binding proteins were also observed. Our data suggest that overall ClpC as a stromal molecular chaperone plays a vital role in chloroplast function and leaf development and is likely involved in photosystem biogenesis. PMID:15563614

  7. Effect of salinity on hemolymph osmotic pressure, sodium concentration and Na+-K+-ATPase activity of gill of Chinese crab, Eriocheir sinensis

    Science.gov (United States)

    Liu, Hongyu; Pan, Luqing; Fu, Lü

    2008-02-01

    The effects of salinity on hemolymph osmotic pressure, Na+ concentration and Na+-K+-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na+ concentration increased significantly ( Phemolymph osmotic pressure and Na+ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na+-K+-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na+ concentration and lower Na+-K+-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct ( P>0.05); However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1 d, and then tended to be stable from day 1 to day 15.

  8. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed

    International Nuclear Information System (INIS)

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed 45Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of 45Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis

  9. Chaperoning ribosome assembly

    OpenAIRE

    Karbstein, Katrin

    2010-01-01

    Chaperones help proteins fold in all cellular compartments, and many associate directly with ribosomes, capturing nascent chains to assist their folding and prevent aggregation. In this issue, new data from Koplin et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200910074) and Albanèse et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201001054) suggest that in addition to promoting protein folding, the chaperones ribosome-associated complex (RAC), nascent chain–associated complex (NAC), and Jjj1 also...

  10. Vanadate inhibits the ATPase activity and DNA binding capability of bacterial MutS. A structural model for the vanadate–MutS interaction at the Walker A motif

    OpenAIRE

    Pezza, Roberto J.; Villarreal, Marcos A.; Montich, Guillermo G.; Argaraña, Carlos E.

    2002-01-01

    MutS, a member of the ABC ATPases superfamily, is a mismatch DNA-binding protein constituent of the DNA post-replicative mismatch repair system (MMRS). In this work, it is shown that the ATPase activity of Pseudomonas aeruginosa and Escherichia coli MutS is inhibited by ortho- and decavanadate. Structural comparison of the region involved in the ATP binding of E.coli MutS with the corresponding region of other ABC ATPases inhibited by vanadate, including the myosin– orthovanadate–Mg complex, ...

  11. Chaperoning the Chaperone: A Role for the Co-chaperone Cpr7 in Modulating Hsp90 Function in Saccharomyces cerevisiae

    OpenAIRE

    Zuehlke, Abbey D.; Johnson, Jill L.

    2012-01-01

    Heat-shock protein 90 (Hsp90) of Saccharomyces cerevisiae is an abundant essential eukaryotic molecular chaperone involved in the activation and stabilization of client proteins, including several transcription factors and oncogenic kinases. Hsp90 undergoes a complex series of conformational changes and interacts with partner co-chaperones such as Sba1, Cpr6, Cpr7, and Cns1 as it binds and hydrolyzes ATP. In the absence of nucleotide, Hsp90 is dimerized only at the carboxy-terminus. In the pr...

  12. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  13. Functional roles of Na+/K+-ATPase in active ammonia excretion and seawater acclimation in the giant mudskipper, Periophthalmodon schlosseri

    Directory of Open Access Journals (Sweden)

    Shit F Chew

    2014-04-01

    Full Text Available The giant mudskipper, Periophthalmodon schlosseri, is an amphibious fish that builds burrows in the mudflats. It can actively excrete ammonia through its gills, and tolerate high environmental ammonia. This study aimed to examine the effects of seawater (salinity 30; SW acclimation and/or environmental ammonia exposure on the kinetic properties of Na+/K+-ATPase (Nka from, and mRNA expression and protein abundance of nka/Nka α–subunit isoforms in, the gills of P. schlosseri pre-acclimated to slightly brackish water (salinity 3; SBW. Our results revealed that the Nka from the gills of P. schlosseri pre-acclimated to SBW for 2 wk had substantially higher affinity to (or lower Km for K+ than NH4+, and its affinity to NH4+ decreased significantly after 6-d exposure to 75 mmol l-1 NH4Cl in SBW. Hence, Nka transported K+ selectively to maintain intracellular K+ homeostasis, instead of transporting NH4+ from the blood into ionocytes during active NH4+ excretion as previously suggested. Two nkaα isoforms, nkaα1 and nkaα3, were cloned and sequenced from the gills of P. schlosseri. Their deduced amino acid sequences had K+ binding sites identical to that of Nkaα1c from Anabas testudineus, indicating that they could effectively differentiate K+ from NH4+. Six days of exposure to 75 mmol l-1 NH4Cl in SBW, or to SW with or without 50 mmol l-1 NH4Cl led to significant increases in Nka activities in the gills of P. schlosseri. However, a significant increase in the comprehensive Nkaα protein abundance was observed only in the gills of fish exposed to 50 mmol l-1 NH4Cl in SW. Hence, post-translational modification could be an important activity modulator of branchial Nka in P. schlosseri. The fast modulation of Nka activity and concurrent expressions of two branchial nkaα isoforms could in part contribute to the ability of P. schlosseri to survive abrupt transfer between SBW and SW or abrupt exposure to ammonia.

  14. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ54-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP–BeFx

    OpenAIRE

    Sysoeva, Tatyana A.; Yennawar, Neela; Allaire, Marc; Nixon, B. Tracy

    2013-01-01

    This study reports the crystallization of a new nucleotide state of the ATPase domain of a bacterial transcription activator NtrC1 from the hyperthermophilic bacterium Aquifex aeolicus. Wild-type NtrC1 ATPase domain was crystallized in the presence of the ATP analog ADP–BeFx–Mg and the crystals diffracted anisotropically to at best 3.2, 5.2 and 3.2 Å resolution in the a*, b* and c* directions, respectively.

  15. Influence of phosphorylation of lymphocyte's plasma-membrane proteins and calmodulin on Ca2+, Mg2+ -ATPase activity under irradiation

    International Nuclear Information System (INIS)

    We establish that the regulation of Ca2+, Mg2+ - ATPase from plasma membranes of rat spleen lymphocytes is controlled by calmodulin and the Ca2+, calmodulin-dependent phosphorylation system. The mechanisms of regulation of this process are sensitive to the total X-ray irradiation in doses of 0.5 and 1 Gy

  16. Assessment of D-methionine protecting cisplatin-induced otolith toxicity by vestibular-evoked myogenic potential tests, ATPase activities and oxidative state in guinea pigs.

    Science.gov (United States)

    Lo, Wu-Chia; Chang, Chih-Ming; Liao, Li-Jen; Wang, Chi-Te; Young, Yi-Ho; Chang, Yih-Leong; Cheng, Po-Wen

    2015-01-01

    To date, inadequate study has been devoted to the toxic vestibular effects caused by cisplatin. In addition, no electrophysiological examination has been conducted to assess cisplatin-induced otolith toxicity. The purposes of this study are thus two-fold: 1) to determine whether cervical vestibular-evoked myogenic potentials (VEMPs) and ocular VEMPs are practical electrophysiological methods of testing for cisplatin-induced otolith toxicity and 2) to examine if D-methionine (D-met) pre-injection would protect the otolith organs against cisplatin-induced changes in enzyme activities and/or oxidative status. Guinea pigs were intraperitoneally treated once daily with the following injections for seven consecutive days: sterile 0.9% saline control, cisplatin (5 mg/kg) only, D-met (300 mg/kg) only, or a combination of d-met (300 mg/kg) and cisplatin (5 mg/kg), respectively, with a 30 minute window in between. Each animal underwent the oVEMP and cVEMP tests before and after treatment. The changes in the biochemistry of the otolith organs, including membranous Na(+), K(+)-ATPase and Ca(2+)-ATPase, lipid peroxidation (LPO) levels and nitric oxide (NO) levels, were also evaluated. In the cisplatin-only treated guinea pigs, the mean amplitudes of the oVEMP tests were significantly (ppigs receiving both D-met and cisplatin, the amplitudes of their oVEMP tests were significantly larger (p<0.05) than those of the cisplatin-only group, but smaller (p<0.05) than those of the saline control or D-met-only group. However, no significant difference of the amplitudes of cVEMP tests was noted among the four groups. In comparison with the other three groups, the cisplatin-only group had the lowest (ps<0.05) mean Na(+), K(+)-ATPase and Ca(2+)-ATPase, and the highest (ps<0.05) LPO and NO levels. The oVEMP tests were feasible for the evaluation of cisplatin-related otolith dysfunction. D-Met attenuated the reduced ATPase activities and increased oxidative stress induced by cisplatin

  17. Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

    Science.gov (United States)

    Balsamo, R A; Uribe, E G

    1988-02-01

    Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces. PMID:24226399

  18. Stimulating effect of low doses of ionizing radiation on the activity of chicken liver and spleen plasma membrane Ca+2 ATPase during different periods of development

    International Nuclear Information System (INIS)

    Effect of pre incubative irradiation of chickens on the activity of chicken liver and spleen plasma membrane Ca+2-ATPase in 13, 15, 17 day embryos and 1, 5, 10, 20, 30, 40, 50, 60 day chickens has been studied. Low doses of radiation are discovered to stimulate liver and spleen enzyme activity. On the basis of data obtained it is suggested that in the cells of radiosensitive and radio resistive organs molecular mechanisms of stimulating effect of low doses are similar. (author). 10 refs.; 1 fig.; 2 tabs

  19. Obstacle Effects on One-Dimensional Translocation of ATPase

    Institute of Scientific and Technical Information of China (English)

    WANG Xian-Ju; AI Bao-Quan; LIU Liang-Gang

    2002-01-01

    We apply a general random walk model to the study of the ATPase's one-dimensional translocation along obstacle biological environment, and show the effects of random obstacles on the ATPase translocation along single stranded DNA. We find that the obstacle environment can reduce the lifetime of ATPase lattice-bound state which results in the inhibition of ATPase activity. We also carry out the ranges of rate constant of ATPase unidirectonal translocation and bidirectional translocation. Our results are consistent with the experiments and relevant theoretical consideration, and can be used to explain some physiological phenomena.

  20. Saccharomyces cerevisiae vacuolar H+-ATPase regulation by disassembly and reassembly: one structure and multiple signals.

    Science.gov (United States)

    Parra, Karlett J; Chan, Chun-Yuan; Chen, Jun

    2014-06-01

    Vacuolar H(+)-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved. PMID:24706019

  1. sup 86 Rb(K) influx and ( sup 3 H)ouabain binding by human platelets: Evidence for beta-adrenergic stimulation of Na-K ATPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Turaihi, K.; Khokher, M.A.; Barradas, M.A.; Mikhailidis, D.P.; Dandona, P. (Royal Free Hospital and School of Medicine, London (England))

    1989-08-01

    Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), {sup 86}Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific ({sup 3}H)ouabain binding by the human platelet. This {sup 86}Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active {sup 86}Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active {sup 86}Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

  2. Attenuation by methyl mercury and mercuric sulfide of pentobarbital induced hypnotic tolerance in mice through inhibition of ATPase activities and nitric oxide production in cerebral cortex

    Energy Technology Data Exchange (ETDEWEB)

    Chuu, Jiunn-Jye; Huang, Zih-Ning; Yu, Hsun-Hsin; Chang, Liang-Hao [College of Engineering, Southern Taiwan University, Institute of Biotechnology, Tainan (China); Lin-Shiau, Shoei-Yn [College of Medicine, National Taiwan University, Institute of Pharmacology, Taipei (China)

    2008-06-15

    This study is aimed at exploring the possible mechanism of hypnosis-enhancing effect of HgS or cinnabar (a traditional Chinese medicine containing more than 95% HgS) in mice treated with pentobarbital. We also examined whether the effect of HgS is different from that of the well-known methyl mercury (MeHg). After a short period (7 days) of oral administration to mice, a nontoxic dose (0.1 g/kg) of HgS not only significantly enhanced pentobarbital-induced hypnosis but also attenuated tolerance induction; while a higher dose (1 g/kg) of HgS or cinnabar exerted an almost irreversible enhancing effect on pentobarbital-hypnosis similar to that of MeHg (2 mg/kg) tested, which was still effective even after 10 or 35 days cessation of administration. To study comparatively the effects of different mercury forms from oral administration of MeHg and HgS on membrane ATPase activities of experimental mice, analysis of the Hg content in the cerebral cortex revealed that correlated with the decrease of Na{sup +}/K{sup +}-ATPase and Ca{sup 2+}-ATPase activities. Furthermore, NO levels of blood but not that of cerebral cortex were also decreased by mercuric compounds. Although pentobarbital alone enhanced cytochrome p450-2C9 in time dependent manner, all of mercurial compounds tested had no such effect. All of these findings indicated that the mercurial compounds including cinnabar, HgS and MeHg exert a long-lasting enhancing hypnotic activity without affecting pentobarbital metabolism, which provides evidence-based sedative effect of cinnabar used in Chinese traditional medicine for more than 2,000 years. The nontoxic HgS dosing (0.1 g/kg/day) for consecutive 7 days is perhaps useful for delaying or preventing pentobarbital-tolerance. (orig.)

  3. Inhibition of vacuolar ATPase attenuates the TRAIL-induced activation of caspase-8 and modulates the trafficking of TRAIL receptosomes

    Czech Academy of Sciences Publication Activity Database

    Horová, Vladimíra; Hradilová, Naďa; Jelínková, Iva; Koc, Michal; Švadlenka, Jan; Bražina, Jan; Klíma, Martin; Slavík, J.; Vaculová, Alena; Anděra, Ladislav

    2013-01-01

    Roč. 280, č. 14 (2013), s. 3436-3450. ISSN 1742-464X R&D Projects: GA ČR GAP301/10/1971; GA ČR(CZ) GAP301/11/1730; GA MŠk 1M0506 Institutional support: RVO:68378050 ; RVO:68081707 Keywords : acidification * apoptosis * caspase-8 * TRAIL * V-ATPase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.986, year: 2013

  4. In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli.

    Science.gov (United States)

    Sugimoto, Shinya; Saruwatari, Kozue; Higashi, Chihana; Tsuruno, Keigo; Matsumoto, Shunsuke; Nakayama, Jiro; Sonomoto, Kenji

    2008-03-01

    In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli. PMID:18323638

  5. The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair

    OpenAIRE

    Jacobs-Palmer, Emily; Hingorani, Manju M.

    2006-01-01

    MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the T. aquaticus MutS dimer binds a mismatch rapidly (kON = 3 x 106 M−1s−1) and forms a stable c...

  6. AB185. Semen pH effects sperm motility and capacitation by influencing Na/K-ATPase activity and Ca concentration in spermoplasm

    OpenAIRE

    Li CHEN; Ge, Yifeng; Liang, Yuanjiao; Yao, Bing

    2014-01-01

    Background The pH of semen is one of the important elements for maintaining spermatic normal functions. Variation of semen pH could lead to male infertility, but the mechanism of which is still unknown. Methods The healthy human spermatozoa cultured in sperm nutrition solution with pH 5.2, 6.2, 7.2, 8.2, 9.2 and 10.2 respectively were analyzed for sperm motility, vitality, hypoosmotic swelling rate, Na/K-ATPase activity and the intracellular Caconcentration. Results The results showed that sp...

  7. Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

    Science.gov (United States)

    Batista, Fernanda A H; Almeida, Glessler S; Seraphim, Thiago V; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2015-01-01

    The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. PMID:25369258

  8. Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory.

    Science.gov (United States)

    Zhang, Xiao-min; Yan, Xun-yi; Zhang, Bin; Yang, Qian; Ye, Mao; Cao, Wei; Qiang, Wen-bin; Zhu, Li-jun; Du, Yong-lan; Xu, Xing-xing; Wang, Jia-sheng; Xu, Fei; Lu, Wei; Qiu, Shuang; Yang, Wei; Luo, Jian-hong

    2015-07-01

    The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca(2+)-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation. PMID:26088419

  9. P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na(+),K(+)-ATPase.

    Science.gov (United States)

    Geibel, S; Barth, A; Amslinger, S; Jung, A H; Burzik, C; Clarke, R J; Givens, R S; Fendler, K

    2000-09-01

    P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems. PMID:10968997

  10. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    Science.gov (United States)

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  11. Characterization and Structure of a Zn[superscript 2+] and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus fulgidus

    Energy Technology Data Exchange (ETDEWEB)

    Sazinsky, Matthew H.; LeMoine, Benjamin; Orofino, Maria; Davydov, Roman; Bencze, Krisztina Z.; Stemmler, Timothy L.; Hoffman, Brian M.; Arguello, Jose M.; Rosenzweig, Amy C. (Worcester); (WSU-MED); (NWU)

    2010-03-08

    Bacterial CopZ proteins deliver copper to P{sub 1B}-type Cu{sup +}-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 {angstrom} resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu{sup +} to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu{sup 2+} to Cu{sup +}. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.

  12. Lipid Chaperones and Metabolic Inflammation

    Directory of Open Access Journals (Sweden)

    Masato Furuhashi

    2011-01-01

    Full Text Available Over the past decade, a large body of evidence has emerged demonstrating an integration of metabolic and immune response pathways. It is now clear that obesity and associated disorders such as insulin resistance and type 2 diabetes are associated with a metabolically driven, low-grade, chronic inflammatory state, referred to as “metaflammation.” Several inflammatory cytokines as well as lipids and metabolic stress pathways can activate metaflammation, which targets metabolically critical organs and tissues including adipocytes and macrophages to adversely affect systemic homeostasis. On the other hand, inside the cell, fatty acid-binding proteins (FABPs, a family of lipid chaperones, as well as endoplasmic reticulum (ER stress, and reactive oxygen species derived from mitochondria play significant roles in promotion of metabolically triggered inflammation. Here, we discuss the molecular and cellular basis of the roles of FABPs, especially FABP4 and FABP5, in metaflammation and related diseases including obesity, diabetes, and atherosclerosis.

  13. The THERMOSENSITIVE MALE STERILE 1 Interacts with the BiPs via DnaJ Domain and Stimulates Their ATPase Enzyme Activities in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Zhao-Xia Ma

    Full Text Available The Arabidopsis TMS1 encodes a heat shock protein identical to the Hsp40 protein AtERdj3A and plays important roles in the thermotolerance of pollen tubes and other plant tissues. Despite its importance to plant growth and reproduction, little has been known about its mechanisms underlying thermotolerance of plants. In this study, the relationship between TMS1 and the Hsp70 proteins, Binding Immunoglobulin Proteins (BiPs was explored to understand the molecular mechanisms of TMS1 in thermotolerance of plants. The expression of TMS1 was induced not only by heat shock, but also by dithiothreitol (DTT and L-azetidine-2-carboxylic acid (AZC, similarly to the three BiP genes, indicating that TMS1 may be involved in unfolded protein response (UPR. The firefly luciferase complementary imaging (LCI, GST pull-down and ATPase enzyme activity assays demonstrated that the DnaJ domain of TMS1 could interact with BiP1 and BiP3, and could stimulate their ATPase enzyme activities. In addition, the expression level of TMS1 was reduced in the bzip28 bzip60 double mutant. These results suggest that TMS1 may function at the downstream of bZIP28 and bZIP60 and be involved in termotolerance of plants, possibly by participating in refolding or degradation of unfolded and misfolded proteins through interaction with the BiPs.

  14. Iron oxide nanoparticles to an Indian major carp, Labeo rohita: Impacts on hematology, iono regulation and gill Na+/K+ ATPase activity

    Directory of Open Access Journals (Sweden)

    Anand Sadanandan Remya

    2015-04-01

    Full Text Available In this study, the chronic toxicity effects of iron oxide (Fe2O3 nanoparticles (NPs (500 mg l−l on certain hematological, ionoregulatory and gill Na+/K+ ATPase activity of an Indian major carp, Labeo rohita were estimated for a period of 25 days under static bioassay. A significant increase in hemoglobin (Hb content, red blood cell (RBC count and hematocrit (Ht value was noticed throughout the study period when compared to control groups. In contrast, mean cellular volume (MCV, mean cellular hemoglobin (MCH (except on 5th day and mean cellular hemoglobin concentration (MCHC levels and white blood cell (WBC counts were found to be decreased during the above study period. Fe2O3 NPs also caused alterations in iono regulation resulting in hyponatremia (Na+, hypochloremia (Cl− (except on 5th day and hypokalemia (K+ (except up to 15th day. A biphasic trend in gill Na+/K+-ATPase activity was noticed during the above treatment period. Our results demonstrate that high Fe2O3 NP concentrations in the aquatic environment may have adverse physiological effects on fish. These data may be useful to assess the environmental risk posed by NPs. However the toxicity of various sizes of the nanoparticle could be evaluated using different aquatic organisms.

  15. Development of Classification Models for Identifying “True” P-glycoprotein (P-gp Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

    Directory of Open Access Journals (Sweden)

    Anna Maria Bianucci

    2012-06-01

    Full Text Available P-glycoprotein (P-gp is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

  16. Engineering of recombinant crystallization chaperones

    OpenAIRE

    Koide, Shohei

    2009-01-01

    The preparation of diffraction quality crystals remains the major bottleneck in macromolecular x-ray crystallography. A crystallization chaperone is an auxiliary protein, such as fragments of monoclonal antibodies, that binds to and increases the crystallization probability of a target molecule of interest. Such chaperones reduce conformational heterogeneity, mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and provide phasing information. Crys...

  17. Molecular chaperones and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Neurodegenerative diseases are characterized by the accumulation of intracellular or extracellular protein aggregates that result from conformational changes in proteins. These diseases may result from an imbalance between the production of misfolded proteins and normal chaperone capacity. Molecular chaperones provide a first line of defence against misfolded, aggregation-prone proteins and are, therefore, promising therapeutic targets for neurodegenerative diseases.

  18. Effect of Pyrethrins and Rosemary on the ATPase Activities of Rat Cerebral Synaptosome%除虫菊酯和迷迭香对大鼠大脑突触体ATP酶的活性影响

    Institute of Scientific and Technical Information of China (English)

    刘子超; 梁醒财

    2008-01-01

    Pyrethrins are the widely used insecticides in agriculture and households, and they act on integral membrane proteins, causing neurotoxic effects on animals. Rosemary, as an antioxidant, is developed to improve the stability of pyrethrins and applied for many commercially pyrethrinrelated products. This paper aimed to study the effects of pyrethrins and rosemary, as well as their combination on the cerebral synaptosome ATPase of rat. The Percoll gradient method was used to isolate synaptosomes. Total ATPase and Mg2+-ATPase activities were analyzed by determining the amount of inorganic phosphate. The results indicated that the activities of total ATPase and Mg2+-ATPase in 10 μmol/L pyrethrins were reduced to 80.3% and 46.9%, respectively, compared with CK. However, rosemary at concentration of 0.3 to 30 μmol/L showed little effect on ATPase, while at the concentration of 3 000 μmol/L, the total ATPase and the Mg2+-ATPase activities were decreased to 66.8% and 54.5%, respectively. The mixture of 10 μmol/L pyrethrin and 30 μmol/L rosemary decreased the activities of the total ATPase and Mg2+-ATPase to 72.9% and 33.4%, respectively. It is concluded that pyrethrins can inhibit ATPase activity of rat cerebral synaptosome dramatically; rosemary inhibits ATPase only at high concentration; and rosemary slightly enhances inhibition of pyrethrins on ATPase activity. Fig 3, Ref 19%除虫菊酯越来越广泛地被应用于农业和家庭昆虫防治,主要通过作用于膜结合蛋白而对动物起神经毒性.迷迭香因其抗氧化功能而被应用于很多商业化的除虫菊酯产品中.本实验以大鼠大脑突触体ATP酶为研究对象,对除虫菊酯和迷迭香的药理学进行了研究.用Percoll梯度离心法分离突触体,通过检测无机磷的量来测定总ATP酶和Mg2+-ATP酶活性.结果表明,除虫菊酯在浓度为10 μmol/L时总ATP酶和Mg2+-ATP酶分别降低到对照的80.3%和46.9%.迷迭香在浓度为0.3~30 μmol/L时几乎不

  19. Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness.

    Science.gov (United States)

    McGuire, Christina; Cotter, Kristina; Stransky, Laura; Forgac, Michael

    2016-08-01

    V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics

  20. P-type ATPases.

    Science.gov (United States)

    Palmgren, Michael G; Nissen, Poul

    2011-01-01

    P-type ATPases form a large superfamily of cation and lipid pumps. They are remarkably simple with only a single catalytic subunit and carry out large domain motions during transport. The atomic structure of P-type ATPases in different conformations, together with ample mutagenesis evidence, has provided detailed insights into the pumping mechanism by these biological nanomachines. Phylogenetically, P-type ATPases are divided into five subfamilies, P1-P5. These subfamilies differ with respect to transported ligands and the way they are regulated. PMID:21351879

  1. Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Sørensen, P

    1991-01-01

    The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium al...

  2. The roles of co-chaperone CCRP/DNAJC7 in Cyp2b10 gene activation and steatosis development in mouse livers.

    Directory of Open Access Journals (Sweden)

    Marumi Ohno

    Full Text Available Cytoplasmic constitutive active/androstane receptor (CAR retention protein (CCRP and also known as DNAJC7 is a co-chaperone previously characterized to retain nuclear receptor CAR in the cytoplasm of HepG2 cells. Here we have produced CCRP knockout (KO mice and demonstrated that CCRP regulates CAR at multiple steps in activation of the cytochrome (Cyp 2b10 gene in liver: nuclear accumulation, RNA polymerase II recruitment and epigenetic modifications. Phenobarbital treatment greatly increased nuclear CAR accumulation in the livers of KO males as compared to those of wild type (WT males. Despite this accumulation, phenobarbital-induced activation of the Cyp2b10 gene was significantly attenuated. In ChIP assays, a CAR/retinoid X receptor-α (RXRα heterodimer binding to the Cyp2b10 promoter was already increased before phenobarbital treatment and further pronounced after treatment. However, RNA polymerase II was barely recruited to the promoter even after phenobarbital treatment. Histone H3K27 on the Cyp2b10 promoter was de-methylated only after phenobarbital treatment in WT but was fully de-methylated before treatment in KO males. Thus, CCRP confers phenobarbital-induced de-methylation capability to the promoter as well as the phenobarbital responsiveness of recruiting RNA polymerase II, but is not responsible for the binding between CAR and its cognate sequence, phenobarbital responsive element module. In addition, KO males developed steatotic livers and increased serum levels of total cholesterol and high density lipoprotein in response to fasting. CCRP appears to be involved in various hepatic regulations far beyond CAR-mediated drug metabolism.

  3. Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases with antiviral activity against human respiratory syncytial virus.

    Science.gov (United States)

    Cui, Rui; Wang, Yizhuo; Wang, Liu; Li, Guiming; Lan, Ke; Altmeyer, Ralf; Zou, Gang

    2016-08-01

    Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants and young children worldwide, yet no vaccine or effective antiviral treatment is available. To search for new anti-RSV agents, we developed a cell-based assay that measures inhibition of RSV-induced cytopathic effect (CPE) and identified cyclopiazonic acid (CPA), an intracellular calcium ATPase inhibitor as a RSV inhibitor (EC50 values 4.13 μM) by screening of natural product library. CPA inhibited the replication of RSV strains belonging to both A and B subgroups and human parainfluenza virus type 3, but not Enterovirus 71. Mechanism of action study by time-of-addition assay and minigenome assay revealed that CPA acts at the step of virus genome replication and/or transcription. Moreover, two other calcium ATPase inhibitors (Thapsigargin and BHQ) and calcium ionophores (A23187 and ionomycin), but not calcium channel blockers (nifedipine, nimodipine, and tetrandrine), also had similar effect. These results indicate that an increase in intracellular calcium concentration is detrimental to RSV replication. Thus, our findings provide a new strategy for anti-RSV therapy via increasing intracellular calcium concentration. PMID:27210812

  4. Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

    Science.gov (United States)

    Kristjansson, H.; Hochstein, L. I.

    1986-01-01

    Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

  5. New crystal structure of the proteasome-dedicated chaperone Rpn14 at 1.6 Å resolution

    International Nuclear Information System (INIS)

    A new crystal structure of yeast Rpn14 with an E384A mutation was determined at 1.6 Å resolution. The improved high-resolution structure provides a framework for understanding proteasome assembly. The 26S proteasome is an ATP-dependent protease responsible for selective degradation of polyubiquitylated proteins. Recent studies have suggested that proteasome assembly is a highly ordered multi-step process assisted by specific chaperones. Rpn14, an assembly chaperone for ATPase-ring formation, specifically recognizes the ATPase subunit Rpt6. The structure of Rpn14 at 2.0 Å resolution in space group P64 has previously been reported, but the detailed mechanism of Rpn14 function remains unclear. Here, a new crystal structure of Rpn14 with an E384A mutation is presented in space group P21 at 1.6 Å resolution. This high-resolution structure provides a framework for understanding proteasome assembly

  6. Discovery, production and purification of the Na+, K+ activated ATPase inhibitor, L-681,110 from the fermentation broth of Streptomyces sp. MA-5038.

    Science.gov (United States)

    Huang, L; Albers-Schonberg, G; Monaghan, R L; Jakubas, K; Pong, S S; Hensens, O D; Burg, R W; Ostlind, D A; Conroy, J; Stapley, E O

    1984-09-01

    The maximum yield for the production of L-681,110 by Streptomyces sp. MA-5038 (ATCC 31587) was observed after 5 days' incubation at 28 degrees C and pH about 8.3. L-681,110 was isolated from the fermentation broth by acetone extraction of the mycelia, absorption to Amberlite XAD-2 resin and two separations by thin-layer chromatography. The structure of L-681,110 was found to consist of a sixteen-membered lactone with a new type of substitution. The inhibition of ATPase, activity against Caenorhabditis elegans and stimulation of gamma-aminobutyric acid release indicate that L-681,110 possesses some characteristics of both oligomycin and avermectin. L-681,110 was also active against tapeworm and ticks in an in vivo assay. PMID:6094416

  7. Ultracytochemical Localization and Functional Analysis of ATPase During the Endosperm Development in Oryza sativa L.

    Institute of Scientific and Technical Information of China (English)

    WEI Cun-xu; LAN Sheng-yin; XU Zhen-xiu

    2003-01-01

    Ultracytochemical localization of ATPase during development of rice endosperm was performed using a lead phosphate precipitation technique. The results indicated that, at the coenocyte and ceilularization stages, active ATPase was mainly distributed in an embryo sac wall, nucleus, and plasma membrane. At the early stage of development and differentiation, active ATPase was observed in the plasma membrane. At the grain filling stage, ATPase was highly active in the plasma membrane, intercellular space, and plasmodesmata in aleurone, moderately active on the plasma membrane in subaleurone. In starchy endosperm, ATPase was localized in the plasma membrane and degenerated nucleus. ATPase activity also appeared around vacuole and protein body in endosperm cell. The relationships between the ultracytochemical localization of ATPase and its function during the development of rice endosperm were discussed. Overall, ATPase was involved in the process of nutrition absorption and protein synthesis.

  8. Nitrate transport in cucumber leaves is an inducible process involving an increase in plasma membrane H+-ATPase activity and abundance

    Directory of Open Access Journals (Sweden)

    Nikolic Miroslav

    2012-05-01

    Full Text Available Abstract Background The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level. Results The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction. However, it did not exhibit typical biphasic kinetics and was characterized by a higher Km with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1 and NRT2 transporter (CsNRT2 in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process. Conclusions Obtained results provide for the first time evidence that a saturable

  9. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein: Importance of the C-terminal unstructured tail

    OpenAIRE

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interac...

  10. Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism.

    OpenAIRE

    Robben, J.H.; Sze, M.; Knoers, N.V.A.M.; Deen, P. M. T.

    2006-01-01

    Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogeni...

  11. Active glucose transport and proton pumping in tonoplast membrane of Zea mays L. coleoptiles are inhibited by anti-H+-ATPase antibodies

    International Nuclear Information System (INIS)

    A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg2+-ATP dependent accumulation of 3-O-methyl-D-glucose (OMG) as determined by a membrane filtration technique using 14C-labeled substrate. OMG transport showed an apparently saturable component with a K/sub m/ of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H+-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes

  12. Future migratory behaviour predicted from premigratory levels of gill Na+/K(+-)ATPase activity in individual wild brown trout ( Salmo trutta )

    DEFF Research Database (Denmark)

    Nielsen, C.; Aarestrup, Kim; Norum, U.;

    2004-01-01

    The relationship between premigratory gill Na+/K(+-)ATPase activity, determined at two dates during spring, and future migratory behaviour was investigated using non-lethal gill biopsies and PIT-tagging in wild brown trout (Salmo trutta) from two tributaries. No significant relationship between......(-1)), with an average of 91 % of the predictions being correct. The present study shows that a non-lethal premigratory biochemical measurement can successfully select individual brown trout with high probability of migration...... obtained. The ability of this regression model from the tributaries to predict future migratory behaviour in an independent group of trout caught in early April in the mainstream was evaluated. A threshold probability of migration was used to predict the behaviour of the mainstream individuals as either...

  13. [Electrogenic activity of Na-K-ATPase and calcium ions in m. soleus fibers of rats and Mongolian gerbil during simulation of gravitational unloading].

    Science.gov (United States)

    Kravtsova, V V; Ogneva, I V; Altaeva, E G; Razgovorova, I A; Tiapkina, O V; Nikol'skiĭ, E E; Shenkman, B S; Krivoĭ, I I

    2010-01-01

    Some of the electrophysiological parameters of m. soleus of rat and Mongolian gerbil, and Ca ions content in fiber myoplasm were compared in different periods of gravitational unloading simulated by tail-suspension. No difference was found between the control animals as for membrane potential at rest, electrogenic activities of Na-K-ATPase and its isoforms, and input resistance of m. soleus fibers. At the same time, unlike rats, gerbils exhibited a substantial Ca decrease in myoplasm. From day one to 14 of gravitational unloading the pace of electrophysiological changes in gerbil's m. soleus was noticeably slower than of rat's, whereas Ca ions depositing in myoplasm was observed in both species already at the beginning ofsuspension. Analysis of the results suggests that adaptive changes in m. soleus of Mongolian gerbil and rat during simulated gravitational unloading are fundamentally different due to, probably, peculiar water-electrolyte metabolism, type of locomotion, and other factors which are still unclear. PMID:20799658

  14. Chaperoning prions: the story unfolds

    OpenAIRE

    O'Connor, David; Jones, Gary

    2006-01-01

    Prions are infectious proteins that are responsible for a number of mammalian degenerative diseases. The discovery of prions in yeast has allowed detailed genetic analysis to be carried out to identify cellular factors involved in prion propagation. It is now clear that a complex relationship exists between molecular chaperones and prion propagation. Prions may actually have evolved to exploit the cell's chaperone machinery to ensure their own propaga...

  15. The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity

    DEFF Research Database (Denmark)

    Martuseviciene, Giedre; Hofman-Bang, Jacob; Clausen, Torben; Olgaard, Klaus; Lewin, Ewa

    2011-01-01

    The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be...

  16. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome.

    Science.gov (United States)

    Förster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners. PMID:19653995

  17. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    International Nuclear Information System (INIS)

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  18. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    Energy Technology Data Exchange (ETDEWEB)

    Foerster, Friedrich [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany); Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Lasker, Keren [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv (Israel); Beck, Florian; Nickell, Stephan [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany); Sali, Andrej [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Baumeister, Wolfgang, E-mail: baumeist@biochem.mpg.de [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany)

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  19. Chaperones in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    The hepatitis C virus (HCV) infects approximately 3% ofthe world population or more than 185 million peopleworldwide. Each year, an estimated 350000-500000deaths occur worldwide due to HCV-associated diseasesincluding cirrhosis and hepatocellular carcinoma. HCV isthe most common indication for liver transplantation inpatients with cirrhosis worldwide. HCV is an envelopedRNA virus classified in the genus Hepacivirus in theFlaviviridae family. The HCV viral life cycle in a cellcan be divided into six phases (1) binding and internalization;(2) cytoplasmic release and uncoating; (3)viral polyprotein translation and processing; (4) RNAgenome replication; (5) encapsidation (packaging) andassembly; and (6) virus morphogenesis (maturation)and secretion. Many host factors are involved in theHCV life cycle. Chaperones are an important group ofhost cytoprotective molecules that coordinate numerouscellular processes including protein folding, multimericprotein assembly, protein trafficking, and proteindegradation. All phases of the viral life cycle requirechaperone activity and the interaction of viral proteinswith chaperones. This review will present our currentknowledge and understanding of the role of chaperonesin the HCV life cycle. Analysis of chaperones in HCVinfection will provide further insights into viral/hostinteractions and potential therapeutic targets for bothHCV and other viruses.

  20. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase......Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na...

  1. High-Level Formation of Active Pseudomonas cepacia Lipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

    OpenAIRE

    Quyen, Dinh Thi; Schmidt-Dannert, Claudia; Schmid, Rolf D.

    1999-01-01

    The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chape...

  2. Engineering a prototypic P-type ATPase Listeria Monocytogenes Ca(2+)-ATPase 1 for single-molecule FRET studies

    DEFF Research Database (Denmark)

    Dyla, Mateusz; Andersen, Jacob; Kjaergaard, Magnus;

    2016-01-01

    Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrol...

  3. Effects of aqueous extract of Hibiscus sabdariffa on renal Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in Wistar rats%玫瑰茄提取物对Wistar大鼠肾Na+-K+-ATP酶及Ca2+-Mg2+-ATP酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    Lawrence A.Olatunji; Taofeek O.Usman; Joseph O.Adebayo; Victoria A.Olatunji

    2012-01-01

    OBJECTIVE:To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in rats.METHODS:The 25 and 50 mg/(kg · d) of aqueous extracts of H.sabdariffa were respectively given to rats in the experimental groups for 28 d,and rats in the control group received an appropriate volume of distilled water as vehicle.Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in the kidney were assayed by spectrophotometric method. RESULTS:Administrations of 25 and 50 mg/(kg · d) of aqueous extract of H.sabdariffa significantly decreased the Ca2+-Mg2+-ATPase activity in the kidney of rats (P <0.05).However,the renal Na+-K+-ATpase activity of the experimental rats was not affected by either dose of the extract.And the plasma Na+,K+ and Ca2+ levels of the experimental rats had no significant changes.Administration of either dose of the extract did not result in any significant changes in body and kidney weights,the concentrations of plasma albumin and total protein,and alkaline phosphatase,aspartate aminotransferase and alanine aminotransferase activities.However,concentrations of creatinine and urea were significantly reduced by 50 mg/kg of the extract (P<0.05). CONCLUSION:The present study indicates that oral administration of aqueous extract of H.sabdariffa may preserve the renal function despite a decreased renal Ca2+-Mg2+-ATPase activity.%目的:研究口服玫瑰茄水提取物对大鼠肾Na+-K+-ATP酶和Ca2+- Mg2+-ATP酶活性的影响.方法:连续28 d分别给予实验大鼠口服25和50 mg/kg的玫瑰茄水提物,同时给予对照组大鼠灌胃适当剂量的蒸馏水.用光谱测定法分析大鼠肾脏中Na+-K+-ATP酶和Ca2+ -Mg2+-ATP酶的活性.结果:口服25和50 mg/kg的玫瑰茄提取物后,实验组大鼠肾Ca2+-Mg2+-ATP酶活性显著降低(P<0.05),然而肾Na+ -K+ -ATP酶的活性却未受到任何影响.实验组大鼠体质量、肾脏质量,血浆白蛋白和总蛋白浓

  4. Activation of K{sup +} channels and Na{sup +}/K{sup +} ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

    Energy Technology Data Exchange (ETDEWEB)

    Fiorim, Jonaina, E-mail: nanafiorim@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Ribeiro Júnior, Rogério Faustino, E-mail: faustino43@oi.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Azevedo, Bruna Fernades, E-mail: brunafernandes.azevedo@gmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Simões, Maylla Ronacher, E-mail: yllars@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Padilha, Alessandra Simão, E-mail: ale_spadilha@yahoo.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Stefanon, Ivanita, E-mail: ivanita@pq.cnpq.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Alonso, Maria Jesus, E-mail: mariajesus.alonso@urjc.es [Departamento de Ciencias de la Salud III, Universidad Rey Juan Carlos, Alcorcón (Spain); Salaices, Mercedes, E-mail: mercedes.salaices@uam.es [Departamento de Farmacología, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPaz) (Spain); Vassallo, Dalton Valentim, E-mail: daltonv2@terra.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil)

    2012-07-01

    Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K{sup +} channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K{sup +} channels and Na{sup +}/K{sup +}-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O{sub 2}{sup −} production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K{sup +}-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K{sup +}-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K{sup +} channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress. -- Highlights: ► Increased free radicals production ► Increased Na{sup +}/K{sup +} ATPase activity ► Promotes activation of the K{sup +} channels and reduced vascular reactivity ► These effects preserve endothelial function against oxidative

  5. Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori.

    Science.gov (United States)

    Ti, Xiaonan; Tuzuki, Nobuhiko; Tani, Naoki; Morigami, Etsuko; Isobe, Minoru; Kai, Hidenori

    2004-11-01

    We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments. PMID:15607508

  6. Adaptive alterations on gill Na(+), K(+)-ATPase activity and mitochondrion-rich cells of juvenile Acipenser sinensis acclimated to brackish water.

    Science.gov (United States)

    Zhao, Feng; Wu, Beibei; Yang, Gang; Zhang, Tao; Zhuang, Ping

    2016-04-01

    Understanding the physiological changes and osmoregulatory strategy is critical for anadromous species to adapt to large changes between freshwater and marine environments. In this study, juvenile Chinese sturgeon (Acipenser sinensis) were acclimated for 2 months to freshwater (FW, c. 0 ‰) and brackish water (BW, 15 ‰). Blood was assessed for changes in osmolality and ions. Gill tissue was assayed for Na(+), K(+)-ATPase (NKA) activity and immunohistochemical analysis on mitochondria-rich cells (MRCs). Serum osmolality and ions concentrations (Na(+), Cl(-) and K(+)) examined, except K(+), increased significantly in those specimens adapted to BW. However, the variations were within the range of effective hyperosmotic adaptation. The specific activity of gill NKA of juveniles adapted to BW was significantly higher (c. 1.6 times) than that of fish adapted to FW. MRCs were mainly presented in the interlamellar region of the filament and at the base of the lamella in either FW- or BW-acclimated individuals. In BW, the number and size of MRCs on filaments greatly increased. However, there was no significant difference in the number and size of the MRCs at the lamella region. Results show that juvenile Chinese sturgeon keep osmotic homeostasis in hyperosmotic environments by increasing gill NKA activity and MRCs' size and number, which is similar to other sturgeons and euryhaline teleosts. PMID:26614501

  7. Effects of salinity on activities of H+-ATPase, H+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean (Glycine max L.) seedlings

    Institute of Scientific and Technical Information of China (English)

    YU Bing-jun; LAM Hon-ming; SHAO Gui-hua; LIU You-ling

    2005-01-01

    The effects of NaCl stress on the H+ -ATPase, H+ -PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars( Glycine max L. ) differing in salt tolerance(Wenfeng7,salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3% (W/V) NaCl for 3 d, the H+ -ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H+ -PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios ofphospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.

  8. The mammalian homologue of yeast Afg1 ATPase (lactation elevated 1) mediates degradation of nuclear-encoded complex IV subunits.

    Science.gov (United States)

    Cesnekova, Jana; Rodinova, Marie; Hansikova, Hana; Houstek, Josef; Zeman, Jiri; Stiburek, Lukas

    2016-03-15

    Mitochondrial protein homeostasis is crucial for cellular function and integrity and is therefore maintained by several classes of proteins possessing chaperone and/or proteolytic activities. In the present study, we focused on characterization of LACE1 (lactation elevated 1) function in mitochondrial protein homeostasis. LACE1 is the human homologue of yeast mitochondrial Afg1 (ATPase family gene 1) ATPase, a member of the SEC18-NSF, PAS1, CDC48-VCP, TBP family. Yeast Afg1 was shown to mediate degradation of mitochondrially encoded complex IV subunits, and, on the basis of its similarity to CDC48 (p97/VCP), it was suggested to facilitate extraction of polytopic membrane proteins. We show that LACE1, which is a mitochondrial integral membrane protein, exists as part of three complexes of approximately 140, 400 and 500 kDa and is essential for maintenance of fused mitochondrial reticulum and lamellar cristae morphology. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4 (cytochrome c oxidase 4), COX5A and COX6A, and is required for normal activity of complexes III and IV of the respiratory chain. Using affinity purification of LACE1-FLAG expressed in a LACE1-knockdown background, we show that the protein interacts physically with COX4 and COX5A subunits of complex IV and with mitochondrial inner-membrane protease YME1L. Finally, we demonstrate by ectopic expression of both K142A Walker A and E214Q Walker B mutants, that an intact ATPase domain is essential for LACE1-mediated degradation of nuclear-encoded complex IV subunits. Thus the present study establishes LACE1 as a novel factor with a crucial role in mitochondrial protein homeostasis. PMID:26759378

  9. Screening for Pharmacological Chaperones in Fabry Disease

    OpenAIRE

    Shin, Sang-Hoon; Murray, Gary J.; Kluepfel-Stahl, Stefanie; Cooney, Adele M.; Quirk, Jane M.; Schiffmann, Raphael; Brady, Roscoe O.; Kaneski, Christine R.

    2007-01-01

    As a prerequisite for full clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease we developed a rapid screening assay for enhancement of endogenous α-galactosidase A (α-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1- deoxygalactonojirimycin (DGJ). When patient derived T-cells were grown in the presence of DGJ α-Gal A activity increased to more than 50% of normal in several mutations ...

  10. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

    Science.gov (United States)

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-04-18

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  11. Effects of anthropogenic sound on digging behavior, metabolism, Ca2+/Mg2+ ATPase activity, and metabolism-related gene expression of the bivalve Sinonovacula constricta

    Science.gov (United States)

    Peng, Chao; Zhao, Xinguo; Liu, Saixi; Shi, Wei; Han, Yu; Guo, Cheng; Jiang, Jingang; Wan, Haibo; Shen, Tiedong; Liu, Guangxu

    2016-04-01

    Anthropogenic sound has increased significantly in the past decade. However, only a few studies to date have investigated its effects on marine bivalves, with little known about the underlying physiological and molecular mechanisms. In the present study, the effects of different types, frequencies, and intensities of anthropogenic sounds on the digging behavior of razor clams (Sinonovacula constricta) were investigated. The results showed that variations in sound intensity induced deeper digging. Furthermore, anthropogenic sound exposure led to an alteration in the O:N ratios and the expression of ten metabolism-related genes from the glycolysis, fatty acid biosynthesis, tryptophan metabolism, and Tricarboxylic Acid Cycle (TCA cycle) pathways. Expression of all genes under investigation was induced upon exposure to anthropogenic sound at ~80 dB re 1 μPa and repressed at ~100 dB re 1 μPa sound. In addition, the activity of Ca2+/Mg2+-ATPase in the feet tissues, which is directly related to muscular contraction and subsequently to digging behavior, was also found to be affected by anthropogenic sound intensity. The findings suggest that sound may be perceived by bivalves as changes in the water particle motion and lead to the subsequent reactions detected in razor clams.

  12. 女贞子提取物对大鼠不同组织Ca2+-ATPase活性的影响%Effects of Ligustrum lucidum Extracts on Ca2+-ATPase Activity in Different Tissues of Rat

    Institute of Scientific and Technical Information of China (English)

    马云慧; 熊正英

    2012-01-01

    研究了女贞子提取物对大强度耐力训练大鼠不同组织Ca2+ -ATPase活性的影响,探讨了女贞子提取物对大鼠运动能力的作用机制.结果表明:运动组和运动+女贞子组大鼠各组织Ca2+-ATPasee活性均显著低于安静组,运动+女贞子组大鼠不同组织Ca2+-ATPase活性显著高于运动组;运动+女贞子组大鼠力竭运动时间比运动组延长23.09%.女贞子提取物可以调节大鼠不同组织Ca2+ -ATPase活性,延长运动至疲劳的时间.%To study the mechanism of Ligustrum lucidum extract on the exercise performance of rat through examining the effects of Ligustrum lucidum extracts on Ca2+ -ATPase activity in different tissue of rats in endurance training. The results showed that the Ca2+ -ATPase activity in the rat tissue of exercise control group and exercise+ extract feeding group was significantly lower than that of the sedentary control group (/> increase Ca2+-ATPase activity, and extend the time from exercise to fatigue.

  13. Focal cerebral ischaemia induces a decrease in activity and a shift in ouabain affinity of Na+, K+-ATPase isoforms without modifications in mRNA and protein expression.

    Science.gov (United States)

    Jamme, I; Barbey, O; Trouvé, P; Charlemagne, D; Maixent, J M; MacKenzie, E T; Pellerin, L; Nouvelot, A

    1999-02-20

    In a mouse model of focal cerebral ischaemia, we observed after 1 h of ischaemia, that the total Na+, K+-ATPase activity was decreased by 39.4%, and then did not vary significantly up to 6 h post-occlusion. In the sham group, the dose-response curves for ouabain disclosed three inhibitory sites of low (LA), high (HA) and very high (VHA) affinity. In ischaemic animals, we detected the presence of only two inhibitory sites for ouabain. After 1 h of permanent occlusion, the first site exhibited a low affinity while the second site presented an affinity intermediate between those of HA and VHA sites, which evolved after 3 h and 6 h of occlusion towards that of the VHA site. The presence of only two ouabain sites for Na+, K+-ATPase after ischaemia could result from a change in ouabain affinity of both HA and VHA sites (alpha2 and alpha3 isoforms, respectively) to form a unique component. Irrespective of the duration of ischaemia, the smaller activity of this second site accounted entirely for the loss in total activity. Surprisingly, no modifications in protein and mRNA expression of any alpha or beta isoforms of the enzyme were observed, thus suggesting that ischaemia could induce intrinsic modifications of the Na+, K+-ATPase. PMID:10082868

  14. Review: P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas

    DEFF Research Database (Denmark)

    Andersen, Jens P; Vestergaard, Anna L; Mikkelsen, Stine A;

    2016-01-01

    P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood...... focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms....

  15. Cerebral Oedema, Blood-Brain Barrier Breakdown and the Decrease in Na(+),K(+)-ATPase Activity in the Cerebral Cortex and Hippocampus are Prevented by Dexamethasone in an Animal Model of Maple Syrup Urine Disease.

    Science.gov (United States)

    Rosa, Luciana; Galant, Leticia S; Dall'Igna, Dhébora M; Kolling, Janaina; Siebert, Cassiana; Schuck, Patrícia F; Ferreira, Gustavo C; Wyse, Angela T S; Dal-Pizzol, Felipe; Scaini, Giselli; Streck, Emilio L

    2016-08-01

    Maple syrup urine disease (MSUD) is a rare metabolic disorder associated with acute and chronic brain dysfunction. This condition has been shown to lead to macroscopic cerebral alterations that are visible on imaging studies. Cerebral oedema is widely considered to be detrimental for MSUD patients; however, the mechanisms involved are still poorly understood. Therefore, we investigated whether acute administration of branched-chain amino acids (BCAA) causes cerebral oedema, modifies the Na(+),K(+)-ATPase activity, affects the permeability of the blood-brain barrier (BBB) and alters the levels of cytokines in the hippocampus and cerebral cortex of 10-day-old rats. Additionally, we investigated the influence of concomitant administration of dexamethasone on the alterations caused by BCAA. Our results showed that the animals submitted to the model of MSUD exhibited an increase in the brain water content, both in the cerebral cortex and in the hippocampus. By investigating the mechanism of cerebral oedema, we discovered an association between H-BCAA and the Na(+),K(+)-ATPase activity and the permeability of the BBB to small molecules. Moreover, the H-BCAA administration increases Il-1β, IL-6 and TNF-α levels in the hippocampus and cerebral cortex, whereas IL-10 levels were decreased in the hippocampus. Interestingly, we showed that the administration of dexamethasone successfully reduced cerebral oedema, preventing the inhibition of Na(+),K(+)-ATPase activity, BBB breakdown and the increase in the cytokines levels. In conclusion, these findings suggest that dexamethasone can improve the acute cerebral oedema and brain injury associated with high levels of BCAA, either through a direct effect on brain capillary Na(+),K(+)-ATPase or through a generalized effect on the permeability of the BBB to all compounds. PMID:26133302

  16. Genomic organization of ATOX1, a human copper chaperone

    Directory of Open Access Journals (Sweden)

    Kaler Stephen G

    2003-02-01

    Full Text Available Abstract Background Copper is an essential trace element that plays a critical role in the survival of all living organisms. Menkes disease and occipital horn syndrome (OHS are allelic disorders of copper transport caused by defects in a X-linked gene (ATP7A that encodes a P-type ATPase that transports copper across cellular membranes, including the trans-Golgi network. Genetic studies in yeast recently revealed a new family of cytoplasmic proteins called copper chaperones which bind copper ions and deliver them to specific cellular pathways. Biochemical studies of the human homolog of one copper chaperone, ATOX1, indicate direct interaction with the Menkes/OHS protein. Although no disease-associated mutations have been reported in ATOX1, mice with disruption of the ATOX1 locus demonstrate perinatal mortality similar to that observed in the brindled mice (Mobr, a mouse model of Menkes disease. The cDNA sequence for ATOX1 is known, and the genomic organization has not been reported. Results We determined the genomic structure of ATOX1. The gene contains 4 exons spanning a genomic distance of approximately 16 kb. The translation start codon is located in the 3' end of exon 1 and the termination codon in exon 3. We developed a PCR-based assay to amplify the coding regions and splice junctions from genomic DNA. We screened for ATOX1 mutations in two patients with classical Menkes disease phenotypes and one individual with occipital horn syndrome who had no alterations detected in ATP7A, as well as an adult female with chronic anemia, low serum copper and evidence of mild dopamine-beta-hydroxylase deficiency and no alterations in the ATOX1 coding or splice junction sequences were found. Conclusions In this study, we characterized the genomic structure of the human copper chaperone ATOX1 to facilitate screening of this gene from genomic DNA in patients whose clinical or biochemical phenotypes suggest impaired copper transport.

  17. Heterologous Expression of MeLEA3: A 10 kDa Late Embryogenesis Abundant Protein of Cassava, Confers Tolerance to Abiotic Stress in Escherichia coli with Recombinant Protein Showing In Vitro Chaperone Activity.

    Science.gov (United States)

    Barros, Nicolle L F; da Silva, Diehgo T; Marques, Deyvid N; de Brito, Fabiano M; dos Reis, Savio P; de Souza, Claudia R B

    2015-01-01

    Late embryogenesis abundant (LEA) proteins are small molecular weight proteins involved in acquisition of tolerance to drought, salinity, high temperature, cold, and freezing stress in many plants. Previous studies revealed a cDNA sequence coding for a 10 kDa atypical LEA protein, named MeLEA3, predicted to be located into mitochondria with potential role in salt stress response of cassava (Manihot esculenta Crantz). Here we aimed to produce the recombinant MeLEA3 protein by heterologous expression in Escherichia coli and evaluate the tolerance of bacteria expressing this protein under abiotic stress. Our result revealed that the recombinant MeLEA3 protein conferred a protective function against heat and salt stress in bacterial cells. Also, the recombinant MeLEA3 protein showed in vitro chaperone activity by protection of NdeI restriction enzyme activity under heat stress. PMID:25990084

  18. Regulation of vacuolar H+-ATPase in microglia by RANKL

    International Nuclear Information System (INIS)

    Vacuolar H+-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  19. Regulation of Na+/K+-ATPase activity by nitric oxide in the kidney and gill of the brown trout (Salmo trutta)

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Madsen, Steffen S

    2003-01-01

    In teleost fish, successful osmoregulation involves controlled ion transport mechanisms in kidney and gill epithelia. In this study, the effect of nitric oxide (NO) on Na(+)/K(+)-ATPase was investigated in vitro in these two tissues in brown trout (Salmo trutta) acclimated to freshwater. The NO...... donor sodium nitroprusside (SNP) inhibited in situ Na(+)/K(+)-ATPase activity, measured as ouabain-sensitive Rb(+) uptake, in both samples of kidney and gill tissue and in isolated gill cells. The effect was dose-dependent in both tissues, with a maximal observed inhibition of approximately 40-50% (1...... mmol l(-1)). To further investigate the mechanism of the NO effect, whole-tissue Na(+) and K(+) levels were analysed. In kidney, SNP (1 mmol l(-1)) led to an increase in tissue Na(+) levels and a decrease in K(+) levels in a 3:2 ratio. In gill tissue, no change in either ion was observed. These...

  20. Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

    Science.gov (United States)

    Chan, Sze Wah Samuel; Yau, Jason; Ing, Christopher; Liu, Kaiyin; Farber, Patrick; Won, Amy; Bhandari, Vaibhav; Kara-Yacoubian, Nareg; Seraphim, Thiago V; Chakrabarti, Nilmadhab; Kay, Lewis E; Yip, Christopher M; Pomès, Régis; Sharpe, Simon; Houry, Walid A

    2016-07-01

    Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation. PMID:27265850

  1. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

    Directory of Open Access Journals (Sweden)

    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

  2. Fungal plasma membrane H+-ATPase inhibitory activity of o-hydroxybenzylated flavanones and chalcones from Uvaria chamae P. Beauv

    DEFF Research Database (Denmark)

    Kongstad, Kenneth Thermann; Wubshet, Sileshi Gizachew; Kjellerup, Lasse;

    2015-01-01

    In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H+-ATPase. Ethyl acetate...... extract of U. chamae was subjected to high-resolution fungal PM H+-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC–HRMS–SPE–NMR). This led to...

  3. Interactions between curcumin and Hsp90 and effects of curcumin on Hsp90 ATPase activity%姜黄素与Hsp90相互作用以及对Hsp90ATPase 活性的影响

    Institute of Scientific and Technical Information of China (English)

    范莹娟; 许建华

    2013-01-01

    目的 研究姜黄素(curcumin)与Hsp90的结合作用,及其对Hsp90-ATPase 活性抑制作用.方法 采用荧光光谱实验,选取280 nm为激发波长,290~510 nm的波长范围内进行荧光光谱扫描,研究不同浓度curcumin与Hsp90的相互作用.采用孔雀绿磷钼酸铵-无机磷检测法,研究curcumin对Hsp90-ATPase活性抑制.结果 curcumin解离常数为(21.608±1.752) μmol·L-1,格尔德霉素(GA)为(17.372±1.200) μmol·L-1.当ATP为1 mmol·L-1时,GA作用于Hsp90的IC50值为0.38 μmol·L-1,curcumin的IC50值为3.75 μmol·L-1也有较强的Hsp90-ATPase抑制活性.结论 经过荧光光谱分析,可以确定curcumin与Hsp90的结合机制,且curcumin能抑制Hsp90-ATPase活性.

  4. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    Directory of Open Access Journals (Sweden)

    Gennady Verkhivker

    2013-11-01

    Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

  5. Inhibition Effect of Eugenol on Na+-K+-ATPase Activity in Brain and Spinal Cord of Cyprinus carpio%丁香酚对鲤鱼脑和脊髓中钠钾泵活性的抑制效果

    Institute of Scientific and Technical Information of China (English)

    孙文渊; 吕世明; 谭艾娟; 林艳红; 安苗; 华夏; 李博岩; 焦亚琴

    2015-01-01

    为探明钠钾泵(Na+-K+-ATP 酶)是否为丁香酚作用的靶位之一,以了解丁香酚的作用机制和进一步开发利用提供参考,将体重900~1000 g、体长40~45 cm 的鲤鱼随机分为对照组(10尾,不含丁香酚乳剂的水溶液)和丁香酚处理组(30尾),测定其不同麻醉时期(诱导期、麻醉期和恢复期)鲤鱼大脑、中脑、间脑、小脑、延脑和脊髓中 Na+-K+-ATP 酶的活性。结果表明:经丁香酚麻醉后不同麻醉时期鲤鱼各脑和脊髓中 Na+-K+-ATP 酶活性均不同程度下降,丁香酚的抑制作用随麻醉程度加深而加强,在麻醉期时抑制作用最强,与对照组比,丁香酚处理组各脑和脊髓中酶活性在麻醉期性均极显著下降(P <0.01);与麻醉期比,恢复期除间脑和延脑外,其余各脑组织和脊髓中酶活性均呈极显著升高(P <0.01)。在麻醉的各时期丁香酚对鲤鱼各脑和脊髓中的 Na+-K+-ATP 酶有明显的抑制作用,对鲤鱼的麻醉作用可能与其抑制脑 Na+-K+-ATP 酶的活性有关。%The Na+-K+-ATPase activity in brain, midbrain, interbrain, cerebellum, medulla oblongata and spinal cord of Cyprinus carpio treated with eugenol at different anesthesia stages (induction stage,narcosis stage and restoration stage)was detected to probe the effect of eugenol on Na+-K+-ATPase activity and provide a reference for further development and utilization of eugenol.The results showed that the Na+-K+-ATPase activity in brain,midbrain,interbrain,cerebellum,medulla oblongata and spinal cord of C.carpio treated with eugenol reduces at different anesthesia stages to varying degrees, and the inhibition effect of eugenol increases with increase of anesthesia degree.That is,the inhibition effect of eugenol is the maximum at narcosis stage.The Na+-K+-ATPase activity in brain,midbrain, interbrain,cerebellum,medulla oblongata and spinal cord of Cyprinus carpio Linnaeus

  6. Positive Cooperativity of the p97 AAA ATPase Is Critical for Essential Functions*

    OpenAIRE

    Nishikori, Shingo; Esaki, Masatoshi; Yamanaka, Kunitoshi; Sugimoto, Shinya; Ogura, Teru

    2011-01-01

    p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by d...

  7. Hsp100/ClpB Chaperone Function and Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Vierling, Elizabeth [University of Massachusetts

    2015-01-27

    The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpB proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.

  8. Sodium ions as substitutes for protons in the gastric H,K-ATPase

    International Nuclear Information System (INIS)

    In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes

  9. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Chingakham R. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Lovell, Scott; Mehzabeen, Nurjahan [University of Kansas, Del Shankel Structural Biology Center, Lawrence, KS 66047 (United States); Chowdhury, Wasimul Q.; Geanes, Eric S. [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States); Battaile, Kevin P. [IMCA-CAT Hauptman–Woodward Medical Research Institute, 9700 South Cass Avenue, Building 435A, Argonne, IL 60439 (United States); Roelofs, Jeroen, E-mail: jroelofs@ksu.edu [Kansas State University, 338 Ackert Hall, Manhattan, KS 66506 (United States)

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  10. Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function*

    OpenAIRE

    Hayes, David; Napoli, Vanessa; Mazurkie, Andrew; Stafford, Walter F.; Graceffa, Philip

    2009-01-01

    The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical u...

  11. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar

    2014-10-01

    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  12. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

    Directory of Open Access Journals (Sweden)

    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  13. In vitro effect of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+, K+-ATPase

    OpenAIRE

    Anne Caroline Candido Gomes; Luzia da Silva Sampaio; Paulo André da Silva; Marcelo Einicker Lamas; Cassia Mônica Sakuragui; Cleber Bomfim Barreto Junior; Naomi Kato Simas; Ricardo Machado Kuster

    2014-01-01

    The present study aimed to investigate the in vitro effects of isoschaftoside isolated from Syngonium podophyllum on pig kidney Na+,K+-ATPase. The Na+, K+-ATPase activity was determined by colorimetric measurement of inorganic phosphate (Pi), resulting from ATP hydrolysis. Isoschaftoside significantly decreased the renal Na+, K+-ATPase activity at the highest concentration as well as at a lower concentration. Our work suggests that isoschaftoside is a promising compound for the treatment of h...

  14. A sulfur-based transport pathway in Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Zhang, Limei; Sitsel, Oleg; Pedersen, Lotte Thue; Moncelli, Maria Rosa; Tadini-Buoninsegni, Francesco; Gourdon, Pontus Emanuel; Rees, Douglas C; Nissen, Poul; Meloni, Gabriele

    Legionella pneumophila Cu(+)-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu(+) is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382......Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB-type Cu(+)-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu(+) across cellular membranes. Crystal...... structures of a copper-free Cu(+)-ATPase are available, but the mechanism of Cu(+) recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the...

  15. Change Characteristics of Short Track Speed Skaters on Na +, K + -ATPase Activity%短道速滑运动员Na+,K+-ATP酶活性变化特征

    Institute of Scientific and Technical Information of China (English)

    李兆鹏

    2013-01-01

    Based on the Na+,K+ -ATPase activity with high sensitivity to the training loads, using the Na+,K+ -ATPase activity to measure the training effect and to evaluate different training methods will have important value of improving the short track speed skaters’ performance and promoting the scientific training of short track speed skating.The paper investigates the Na+, K+ -ATPase activity change of the profession-al skaters from province and city teams after 4 weeks interval training and lasting training.The results show that the Na+,K+ -ATPase activity of training group is significantly higher than the untrained group, and the longer training the higher Na+, K+ -ATPase activity.The interval training can more easily increase the ac-tivity than the lasting training, the better a skater’ motion coordination and the higher his Na+, K+ -AT-Pase.The results indicated that the Na+,K+ -ATPase activity can be used as a sensitive index to evaluate the skaters’ training load and training effect.The interval training method has better effect than the lasting training method for the short track speed skaters.%  Na+,K+-ATP 酶活性对训练负荷具有较高的敏感性,利用 Na+,K+-ATP 酶活性衡量训练效果,评定不同训练方法对提高短道速滑运动员能力的效能,对促进短道速滑项目科学化训练具有重要价值。通过对省、市队短道速滑和未经训练受试对象采用间歇训练法和持续训练法运动4周后观察 Na+,K+-ATP 酶活性变化,研究结果表明:经过短道速滑训练的受试对象 Na+,K+-ATP 酶活性要高于非训练组,并且训练年限越长,Na+,K+-ATP 酶活性就越高;采用间歇训练法要比持续训练法更容易提升 Na+,K+-ATP 酶活性;动作协调性好的短道速滑运动员 Na+,K+-ATP 酶活性高于协调性差的短道速滑运动员。实验提示:Na+,K+-ATP 酶活性也可作为短道速滑运动员评定训练负荷和衡量训练效果的敏感指标,短道速滑运

  16. 低温对钝顶螺旋藻质膜H+-ATP酶活性的影响%EFFECT OF LOW TEMPERATURE STRESS ON THE ACTIVITIES OF PM H+-ATPASE IN SPIRULINA(ARTHROSPIRA) PLATENSIS

    Institute of Scientific and Technical Information of China (English)

    张三润; 杨茜

    2015-01-01

    Objective:To explore the variation of PM H+-ATPase of the Spirulina platensis ( S1 ) from alkaline lake in Erdos Plateau and its relationship with low temperature resistance under low tem-peratures stress,and comparing them with that of S. platensis( S2 ) from Chad Lake in Africa at the same time. Methods:After the purification of the plasma membranes of both Spirulina platensis ( S1 ) from alkaline lake in Erdos Plateau and S. platensis( S2 ) from Chad Lake in Africa with aqueous polymer two-phase partitioning system, the activities of PM H+-ATPase are determined by Mo-Blue-color method. Results:It is discovered that they being exposed right to low temperature or after exercised under low temperature,as temperature dropping and days lasting,the activities of PM H+-ATPase of both S1 and S2 rise first,and drop afterward. The stability of PM H+-ATPase activity of S1 is better than that of S2 at the same conditions above. The activity of PM H+-ATPase can be improved through low temperature exercise. The altering range of PM H+-ATPase activities is S1activity of S1 is 0℃,S210℃,while it is 0℃ for S1 ,5℃for S2 after exercised under low temperatures. Conclusion:S1 has better adaptability to low temperature.%目的::探讨低温下鄂尔多斯高原碱湖钝顶螺旋藻( S1) PM H+-ATPase活性的变化及其与抗低温的关系,并与非洲Chad 湖钝顶螺旋藻( S2)进行比较。方法:以鄂尔多斯钝顶螺旋藻( S1)和非洲Chad 湖钝顶螺旋藻( S2)为材料,采用两相法提纯质膜( plasms membrae PM),钼蓝法测定PM H+-ATPase活性。结果:结果显示:低温直接处理和锻炼后低温处理,S1和S2 PM H+-ATPase活性都是先升后降。 S1 PM H+-ATPase活性比S2 PM H+-ATPase活性稳定。低温锻炼可提高PM H+-ATPase活性。 PM H+-ATPase活性变化范围S1

  17. A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B12 utilization

    OpenAIRE

    Tal, Nir; Ovcharenko, Elena; Lewinson, Oded

    2013-01-01

    In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. In this large superfamily of proteins, two catalytic sites hydrolyze ATP to power uphill substrate translocation. A central question in the field concerns the relationship between the two ATPase catalytic sites: Are the sites independent of one another? Are both needed for function? Do they function cooperatively? These issues have been resolved for type I ABC transporters but never for ...

  18. The Role of the Plasma Membrane H+-ATPase in Plant-Microbe Interactions

    Institute of Scientific and Technical Information of China (English)

    James Mitch Elmore; Gitta Coaker

    2011-01-01

    T Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plantpathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

  19. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.;

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.......(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was...

  20. Insight into the assembly of chaperones

    Energy Technology Data Exchange (ETDEWEB)

    May, R.P. [Institut Max von Laue - Paul Langevin (ILL), 38 - Grenoble (France); Stegmann, R.; Manakova, E.; Roessle, M.; Hermann, T.; Heumann, H. [Max-Planck-Institut fuer Biochemie, Martinsried (Germany); Axmann, S.; Plueckthun, A. [Zurich Univ. (Switzerland); Wiedenmann, A. [HMI, Berlin (Germany)

    1997-04-01

    Chaperones are proteins that help other proteins (substrate proteins) to acquire a `good` conformation. The folding is a dynamic process and involves repetitive binding and release of the chaperone components and of the substrate protein. Small-angle neutron scattering is used to investigate the structural changes that appear to happen during the folding process. (author). 2 refs.

  1. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

    NARCIS (Netherlands)

    Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The exten

  2. Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    Full Text Available Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected

  3. K+ congeners that do not compromise Na+ activation of the Na+,K+-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser A; Kopec, Wojciech; Khandelia, Himanshu

    2015-01-01

    water molecules, and 3) the rotamer transition is mediated by water traffic into the ion binding cavity. Accordingly, dehydration induced by osmotic stress enhanced the interaction of the congeners with the outward facing sites and profoundly modified the organization of membrane domains of the α......-subunit. These results assign a catalytic role for water in pump function, and shed light on a backbone-independent but a conformation-dependent switch between H-bond and dispersion contact as part of the catalytic mechanism of the Na(+),K(+)-ATPase....

  4. 女贞子提取物对大鼠不同组织Na+,K+-ATPase活性的影响%Effect of Fructus Ligustri Lucidi Extracts on Na+,K+-ATPase Activities in Different Rat Tissues

    Institute of Scientific and Technical Information of China (English)

    马云慧

    2011-01-01

    The mechanism of the influence of the extracts of Ligustrum lucidum Ait.Fruit (Fructus Ligustri Lucidi) on the activities of Na+ ,K+-ATPases in different tissues of rats under strong tolerant exercises was investigated.Twenty four SD rats were randomly divided into sedentary control group, exercise control group and exercise + Fructus Ligustri Lucidi group (n = 8).Exercise control group received high-intensity treadmill training for 6 weeks, exercise + Fructus Ligustri Lucidi group was treated with 400 mg/kg of Ligustrum lucidum extract (2 mL) other than high-intensity treadmill training daily.Sedentary control group and exercise control group was treated with 0.5% Tween-80 solution (2 mL).After 6 weeks,sedentary control group, exercise control and exercise + Fructus Ligustri Lucidi group were given an exhaustive exercise, then samples were taken, and Na+ and K+-ATPase activities in different rat tissues of each group were measured.The Na+ and K+-ATPase activities in the rat tissue of exercise control group and exercise+ Fructus Ligustri Lucidi group was significantly lower than those of the sedentary control group (p<0.01) ; the Na+ and K+-ATPase activities of the exercise + Fructus Ligustri Lucidi group were significantly higher than those of the control group (p<0.01, p<0.05); the exhaustive exercise time of exercise + Fructus Ligustri Lucidi rats was 23.09% longer than that of the control group.The results indicated that supplemented Fructus Ligustri Lucidi extract could regulate and increase the activities of Na+ and K+-ATPase of rats, and extend the time from exercise to fatigue.%通过女贞子提取物对大强度耐力训练大鼠不同组织Na+,K+-ATPase活性的影响,探讨女贞子提取物对大鼠运动能力的作用机制.以24只SD大鼠随机分为安静对照组、运动对照组和运动+女贞子组,每组8只.运动对照组进行6周大强度跑台训练,运动+女贞子组除大强度跑台训练外,每天灌服2 mL浓度为400 mg

  5. Light induces changes in activities of Na+/K+-ATPase, H+/K+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam, Tridacna squamosa

    OpenAIRE

    Ip, Yuen K.; Ching, Biyun; Kum C Hiong; Choo, Celine Y. L.; Boo, Mel V.; Wong, Wai P.; Chew, Shit F.

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH+ 4 or H+, and activities of enzymes involved in converting NH+ 4 to glutamate/glutamine in inner mantle, outer mantle, and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH+ 4 in substitution for K+ to ac...

  6. Light induces changes in activities of Na+/K+(NH4+)-ATPase, H+/K+(NH4+)-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam Tridacna squamosa

    OpenAIRE

    Alex Y K Ip; Biyun eChing; Kum C Hiong; Yen Ling eChoo; Mel Veen Boo; WaiP eWong; S F Chew

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH4+ or H+, and activities of enzymes involved in converting NH4+ to glutamate/glutamine in inner mantle, outer mantle and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH4+ in substitution for K+ to activa...

  7. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  8. Sub-chronic effect of neem based pesticide (Vepacide) on acetylcholinesterase and ATPases in rat.

    Science.gov (United States)

    Rahman, M F; Siddiqui, M K; Jamil, K

    1999-09-01

    Acetylcholinesterases (AChE), Na(+)-K+, Mg2+ and Ca(2+)-ATPases were monitored in rat brain when treated orally with 80, 160 and 320 mg/kg of Vepacide, an active ingredient from neem seed oil, daily for 90 days. Brain AChE, Na(+)-K+ and Ca(2+)-ATPases were inhibited whereas Mg(2+)-ATPase levels were enhanced in both the sexes after 45 and 90 days of treatment. The relative sensitivities of these ATPases to Vepacide indicated that Ca(2+)-ATPase being more sensitive than Na(+)-K(+)-ATPase in both the sexes. The magnitude of Ca(2+)-ATPase inhibited by this compound was higher than that of brain AChE. It appears to be sexual dimorphism in the alterations of brain AChE, Na(+)-K+ and Mg(2+)-ATPases by Vepacide with females being significant when compared with males. After 28 days of post treatment the alterations observed were approached to those of controls both in male and female rats showing reversal of the toxicity. These results indicated that the ATPases were potently inhibited by Vepacide and seemed to be its precise target among the enzyme studied. This can be used as biochemical marker of exposure to this neem derived product. PMID:10466107

  9. VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

    Science.gov (United States)

    Ramachandran, Nivetha; Munteanu, Iulia; Wang, Peixiang; Ruggieri, Alessandra; Rilstone, Jennifer J; Israelian, Nyrie; Naranian, Taline; Paroutis, Paul; Guo, Ray; Ren, Zhi-Ping; Nishino, Ichizo; Chabrol, Brigitte; Pellissier, Jean-Francois; Minetti, Carlo; Udd, Bjarne; Fardeau, Michel; Tailor, Chetankumar S; Mahuran, Don J; Kissel, John T; Kalimo, Hannu; Levy, Nicolas; Manolson, Morris F; Ackerley, Cameron A; Minassian, Berge A

    2013-03-01

    X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy. PMID:23315026

  10. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  11. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  12. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae

    DEFF Research Database (Denmark)

    L. Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard;

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H+-ATPases are...

  13. Aging cellular networks: chaperones as major participants

    OpenAIRE

    Soti, Csaba; Csermely, Peter

    2006-01-01

    We increasingly rely on the network approach to understand the complexity of cellular functions. Chaperones (heat shock proteins) are key "networkers", which have among their functions to sequester and repair damaged protein. In order to link the network approach and chaperones with the aging process, we first summarize the properties of aging networks suggesting a "weak link theory of aging". This theory suggests that age-related random damage primarily affects the overwhelming majority of t...

  14. Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.

    Science.gov (United States)

    Esmann, M; Fedosova, N U; Maunsbach, A B

    2000-09-29

    Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range. PMID:11018676

  15. Water and molecular chaperones act as weak links of protein folding networks: energy landscape and punctuated equilibrium changes point towards a game theory of proteins

    OpenAIRE

    Kovacs, Istvan A.; Szalay, Mate S.; Csermely, Peter

    2004-01-01

    Water molecules and molecular chaperones efficiently help the protein folding process. Here we describe their action in the context of the energy and topological networks of proteins. In energy terms water and chaperones were suggested to decrease the activation energy between various local energy minima smoothing the energy landscape, rescuing misfolded proteins from conformational traps and stabilizing their native structure. In kinetic terms water and chaperones may make the punctuated equ...

  16. The phosphatase activity of the isolated H4-H5 loop of Na(+)/K(+) ATPase resides outside its ATP binding site

    Czech Academy of Sciences Publication Activity Database

    Krumscheid, R.; Ettrich, Rüdiger; Sovová, Žofie; Sušánková, Klára; Lánský, Zdeněk; Hofbauerová, Kateřina; Linnertz, H.; Teisinger, Jan; Amler, Evžen; Schoner, W.

    2004-01-01

    Roč. 271, č. 19 (2004), s. 3923-3936. ISSN 0014-2956 R&D Projects: GA MŠk(CZ) LN00A141; GA ČR(CZ) GA204/01/0254; GA ČR(CZ) GA204/01/1001; GA ČR(CZ) GP206/03/D082; GA ČR(CZ) GA309/02/1479 Grant ostatní: Deutsche Forschungsgemeinschaft(DE) Bonn Scho 139/21-2+3; CZ-DE(CZ) TSR-088-97; CZ-DE(CZ) CZE 00/33 Institutional research plan: CEZ:AV0Z5011922 Keywords : Na(+)/K(+) ATPase * p-nitrophenylphosphate * H4-H5 loop Subject RIV: CE - Biochemistry Impact factor: 3.260, year: 2004

  17. Modulation and elimination of yeast prions by protein chaperones and co-chaperones

    OpenAIRE

    Reidy, Michael; Masison, Daniel C.

    2011-01-01

    The yeast system has provided considerable insight into the biology of amyloid and prions. Here we focus on how alterations in abundance or function of protein chaperones and co-chaperones affect propagation of yeast prions. In spite of a considerable amount of information, a clear understanding of the molecular mechanisms underlying these effects remains wanting.

  18. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

    of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able......  The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  19. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  20. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Science.gov (United States)

    Chintagari, Narendranath Reddy; Mishra, Amarjit; Su, Lijing; Wang, Yang; Ayalew, Sahlu; Hartson, Steven D; Liu, Lin

    2010-01-01

    Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H(+) into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+) chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca(2+) release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+) pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+) mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion. PMID:20169059

  1. Heat shock protein 90: the cancer chaperone

    Indian Academy of Sciences (India)

    Len Neckers

    2007-04-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.

  2. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe;

    2011-01-01

    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...... transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na...

  3. The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.

    Directory of Open Access Journals (Sweden)

    Richie Khanna

    Full Text Available Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER-retained P545L mutant form of human GAA (P545L GAA. AT2220 increased the specific activity of P545L GAA toward both natural (glycogen and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As

  4. A deleterious mutation in DNAJC6 encoding the neuronal-specific clathrin-uncoating co-chaperone auxilin, is associated with juvenile parkinsonism.

    Directory of Open Access Journals (Sweden)

    Simon Edvardson

    Full Text Available Parkinson disease is caused by neuronal loss in the substantia nigra which manifests by abnormality of movement, muscle tone, and postural stability. Several genes have been implicated in the pathogenesis of Parkinson disease, but the underlying molecular basis is still unknown for ∼70% of the patients. Using homozygosity mapping and whole exome sequencing we identified a deleterious mutation in DNAJC6 in two patients with juvenile parkinsonism. The mutation was associated with abnormal transcripts and marked reduced DNAJC6 mRNA level. DNAJC6 encodes the HSP40 Auxilin, a protein which is selectively expressed in neurons and confers specificity to the ATPase activity of its partner Hcs70 in clathrin uncoating. In Auxilin null mice it was previously shown that the abnormally increased retention of assembled clathrin on vesicles and in empty cages leads to impaired synaptic vesicle recycling and perturbed clathrin mediated endocytosis. Endocytosis function, studied by transferring uptake, was normal in fibroblasts from our patients, likely because of the presence of another J-domain containing partner which co-chaperones Hsc70-mediated uncoating activity in non-neuronal cells. The present report underscores the importance of the endocytic/lysosomal pathway in the pathogenesis of Parkinson disease and other forms of parkinsonism.

  5. Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

    Directory of Open Access Journals (Sweden)

    Colin A Cooper

    2010-02-01

    Full Text Available Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2 is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2 and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

  6. Regulation of conformation and activity of nuclear NF-kappaBeta p65 by phosphorylation, chaperones and p65 DNA-binding

    OpenAIRE

    Milanovic, Maja

    2014-01-01

    The TF NF-kB is an important regulator of immunity, stress responses as well as apoptosis, cell cycle progression and oncogenesis. NF-kB is activated by various stimuli and regulates expression many different target genes. The first part of this work shows that stimulation of cells with the cytokines TNF or IL-1 results in a profound conformational switch of the NF-kB subunit p65, as revealed by limited proteolysis assays. The cytokine-triggered reconfiguration of p65 mainly occurs for p65 co...

  7. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

    OpenAIRE

    Drakou, Christina E.; Malekkou, Anna; Hayes, Joseph M.; Carsten W Lederer; Leonidas, Demetres D.; Oikonomakos, Nikos G.; Lamond, Angus I.; Santama, Niovi; Zographos, Spyros E.

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. D...

  8. Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function

    Institute of Scientific and Technical Information of China (English)

    Ho Byoung Chae; Jeong Chan Moon; Mi Rim Shin; Yong Hun Chi; Young Jun Jung; Sun Yong Lee; Ganesh M.Nawkar

    2013-01-01

    Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase,type C (NTRC) in the light.Here we show overexpression of NTRC in Arabidopsis (NTRCoE) resulting in enhanced tolerance to heat shock,whereas NTRC knockout mutant plants (ntrcl) exhibit a temperature sensitive phenotype.To investigate the underlying mechanism of this phenotype,we analyzed the protein's biochemical properties and protein structure.NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase,a foldase chaperone,and as a holdase chaperone.The multiple functions of NTRC are closely correlated with protein structure.Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone,while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities.Heat shock converted LMW proteins into HMW complexes.Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions,but conferred only a slight decrease in its holdase chaperone function.The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRCoE plants.However,after prolonged incubation under heat shock,NTRCoE plants tolerated the stress to a higher degree than C217/454S-NTRCoE plants.The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.

  9. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

  10. The effect of inhibitors of plasma membrane H+ - ATPase and oxidoreductases on NH4+ uptake by Pisum arvense roots

    Directory of Open Access Journals (Sweden)

    Genowefa Kubik-Dobosz

    2014-02-01

    Full Text Available The effect of inhibitors of plasma membrane oxidoreductases (quinacrine and dicumarol and H+-ATPase (dicyclohexylcarbodiimide and orthovanadate on ammonium uptake by Pisum arvense seedlings and the activities of H+-ATPase and NADH-ferricyanide oxidoreductase was investigated. The uptake solution contained 50 µM NH4+. In I h experiments, quinacrine and dicumarol depressed strongly and irreversibly the rate of NH4+ uptake and markedly inhibited the activity of NADH-ferri-cyanide oxidoreductase in the plasma membrane vesicles prepared from root cells. Simultaneously, sodium orthovanadate inhibited the activity of plasma membrane H+-ATPase increased the rate of NH4+ uptake. Dicyclohexylcarbodiimide inhibited H+-ATPase activity and increased efflux of NH4+ from roots to ambient solution. The results indicate on the lack of direct connection between uptake rate of 50 µM NH4+ and H+-ATPase activity, and suggest that membrane redox systems play a predominant role in this process.

  11. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura;

    2011-01-01

    The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma...... membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  12. Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity

    Institute of Scientific and Technical Information of China (English)

    Bhagavathy S; Sumathi P

    2012-01-01

    Objective: To identify the alteration of the membrane potential and the effect of carotenoid extracts from Chlorococcum humicola (C. humicola) on membrane bound ATPases and lipid peroxidation. Methods: The total carotenoids were extracted from C. humicola. Four groups of Swiss albino mice were treated as control, Benzo(a)pyrene [B(a)P], total carotenoids, B(a)P +total carotenoids respectively for a period of 60 days. Membrane lipid peroxidation and ATPases (Total ATPases, Ca2+- ATPases, Mg2+ - ATPases, Na+K+ - ATPase) were determined in lung, liver and erythrocyte samples. Results: The activity of total ATPase was found to be significantly increased in the B(a)P treated liver and lung tissue. Erythrocyte membrane also showed higher ATPase activity which was significantly reverted on total carotenoid treatment. Conclusions:It can be concluded that the changes in membrane potential favour the functional deterioration of physiological system. The overall findings demonstrates that the animals post treated with carotenoid extract from C. humicola may maintains the alterations in membrane bound ATPase and lipid peroxidation in tissues against the carcinogenic chemical and hence aid in establishing the membrane potential action. Therefore C. humicola can be further extended to exploits its possible application for various health benefits as neutraceuticals and food additives.

  13. Chaperone binding at the ribosomal exit tunnel

    DEFF Research Database (Denmark)

    Kristensen, Ole; Gajhede, Michael

    2003-01-01

    The exit tunnel region of the ribosome is well established as a focal point for interaction between the components that guide the fate of nascent polypeptides. One of these, the chaperone trigger factor (TF), associates with the 50S ribosomal subunit through its N-terminal domain. Targeting of TF...

  14. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  15. Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Mervi Sepp

    Full Text Available The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA and plasmalemma Na+/K+-ATPase (NKA. While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK, ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

  16. Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells.

    Science.gov (United States)

    Dayan, J; Deguingand, S; Truzman, C

    1985-07-01

    Several pharmaceutical drugs show strong hepatotoxicity during therapeutic use. We have studied 6 of them: aminophenazone, clofibrate, nifuroxazide, oxamniquine, perhexiline maleate, tienilic acid. Their mutagenicity was assessed in the Ames test on 6 strains of Salmonella typhimurium, and in V79 Chinese hamster lung cells using a rat-hepatocyte-mediated metabolic activation system and the HGPRT and Na+/K+ ATPase assay. Nifuroxazide was positive in the Ames test in two Salmonella strains (TA100, and TA100 Fr1). In the hepatocyte-mediated mammalian V79 cell system, nifuroxazide, clofibrate and aminophenazone were negative; oxamniquine and tienilic acid were positive with and without metabolic activation in tests looking for ouabain and 6-thioguanine resistance. Perhexiline maleate was negative for the direct induction of 6-thioguanine resistance without metabolic activation, and positive after metabolisation mediated by primary rat's hepatocytes. These results suggest the need for some caution in the use of some pharmaceutical drugs because of hepatotoxicity and because 3 out of 6 drugs were shown to be slightly mutagenic in mammalian cells. PMID:2989681

  17. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus Emanuel; Liu, Xiang-Yu; Skjørringe, Tina; Morth, J Preben; Møller, Lisbeth Birk; Pedersen, Bjørn Panyella; Nissen, Poul

    2011-01-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in...... a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a...

  18. The role of individual domains and the significance of shedding of ATP6AP2/(prorenin receptor in vacuolar H(+-ATPase biogenesis.

    Directory of Open Access Journals (Sweden)

    Kenichiro Kinouchi

    Full Text Available The ATPase 6 accessory protein 2 (ATP6AP2/(prorenin receptor (PRR is essential for the biogenesis of active vacuolar H(+-ATPase (V-ATPase. Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD and transmembrane domain (TM of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M and causes X-linked mental retardation Hedera type (MRXSH and X-linked parkinsonism with spasticity (XPDS in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  19. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P; Cramb, Gordon

    2001-01-01

    seawater challenge test (35 ppt). Gill Na+,K+-ATPase alpha (1) and beta (1) subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased...

  20. Quantitative Proteomics Reveals That the Inhibition of Na(+)/K(+)-ATPase Activity Affects S-Phase Progression Leading to a Chromosome Segregation Disorder by Attenuating the Aurora A Function in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Xu, Zhongwei; Wang, Fengmei; Fan, Fengxu; Gu, Yanjun; Shan, Nana; Meng, Xiangyan; Cheng, Shixiang; Liu, Yingfu; Wang, Chengyan; Song, Yueying; Xu, Ruicheng

    2015-11-01

    Many studies have shown the Na(+)/K(+)-ATPase (NKA) might be a potential target for anticancer therapy. Cardiac glycosides (CGs), as a family of naturally compounds, inhibited the NKA activity. The present study investigates the antitumor effect of ouabain and elucidates the pharmacological mechanisms of CG activity in liver cancer HepG2 cell using SILAC coupled to LC-MS/MS method. Bioinformatics analysis of 330 proteins that were changed in cells under treatment with 0.5 μmol/L ouabain showed that the biological processes are associated with an acute inflammatory response, cell cycle, oxidation reduction, chromosome segregation, and DNA metabolism. We confirmed that ouabain induced chromosome segregation disorder and S-cell cycle block by decreasing the expression of AURKA, SMC2, Cyclin D, and p-CDK1 as well as increasing the expression of p53. We found that the overexpression or inhibition of AURKA significantly reduced or enhanced the ouabain-mediated the anticancer effects. Our findings suggest that AURKA is involved in the anticancer mechanisms of ouabain in HepG2 cells. PMID:26491887

  1. Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin

    Directory of Open Access Journals (Sweden)

    Younes-Ibrahim M.

    1997-01-01

    Full Text Available On the basis of our report that a glycolipoprotein fraction (GLP extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1 GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2 Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3 GLP inhibits Na,K-ATPase from intact cells, and 4 GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 µg protein GLP/ml all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 µg protein GLP/ml for rat and rabbit, respectively, indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ± SEM; control: 23.8 ± 1.8; GLP, 88 µg protein/ml: 8.2 ± 0.9, demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis

  2. Osmotic Stress and Viscous Retardation of the Na,K-ATPase Ion Pump

    Science.gov (United States)

    Esmann, Mikael; Fedosova, Natalya U.; Marsh, Derek

    2008-01-01

    The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K+-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers' theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl+ (a congener for K+), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl+. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5–6 nm3 (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl+ is in the region of 9 nm3. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22 nm3 between the E1-nucleotide bound forms and the E2-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E1 and E2 conformations of the Ca-ATPase. PMID:18055532

  3. "Oxygen Sensing" by Na,K-ATPase: These Miraculous Thiols.

    Science.gov (United States)

    Bogdanova, Anna; Petrushanko, Irina Y; Hernansanz-Agustín, Pablo; Martínez-Ruiz, Antonio

    2016-01-01

    Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its "oxygen-sensitivity" is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation, and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidized glutathione are the signaling messengers that make the Na,K-ATPase "oxygen-sensitive." This very ancient signaling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the "optimal" level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterize the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summary on (i) the sources of free radical production in hypoxic cells, (ii) localization of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzyme to a variety of stimuli (hypoxia, receptors' activation) (iii) redox-sensitive regulatory phosphorylation, and (iv) the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate. Better understanding of the processes

  4. Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

    International Nuclear Information System (INIS)

    Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes

  5. Water and molecular chaperones act as weak links of protein folding networks: energy landscape and punctuated equilibrium changes point towards a game theory of proteins.

    Science.gov (United States)

    Kovács, István A; Szalay, Máté S; Csermely, Peter

    2005-04-25

    Water molecules and molecular chaperones efficiently help the protein folding process. Here we describe their action in the context of the energy and topological networks of proteins. In energy terms water and chaperones were suggested to decrease the activation energy between various local energy minima smoothing the energy landscape, rescuing misfolded proteins from conformational traps and stabilizing their native structure. In kinetic terms water and chaperones may make the punctuated equilibrium of conformational changes less punctuated and help protein relaxation. Finally, water and chaperones may help the convergence of multiple energy landscapes during protein-macromolecule interactions. We also discuss the possibility of the introduction of protein games to narrow the multitude of the energy landscapes when a protein binds to another macromolecule. Both water and chaperones provide a diffuse set of rapidly fluctuating weak links (low affinity and low probability interactions), which allow the generalization of all these statements to a multitude of networks. PMID:15848154

  6. The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

    DEFF Research Database (Denmark)

    Mollerup, Jens; Berchtold, Martin Werner

    2005-01-01

    The heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFa-receptor activation as well as staurosporine...

  7. Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

    Directory of Open Access Journals (Sweden)

    Lisa Cadavez

    Full Text Available In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP. The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR, perturbing endoplasmic reticulum (ER homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP or protein disulfite isomerase (PDI, and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA or 4-phenylbutyrate (PBA, alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

  8. V-ATPase regulates communication between microvascular endothelial cells and metastatic cells.

    Science.gov (United States)

    Sennoune, S R; Arutunyan, A; del Rosario, C; Castro-Marin, R; Hussain, F; Martinez-Zaguilan, R

    2014-01-01

    To metastasize distant organs, tumor cells and endothelial cells lining the blood vessels must crosstalk. The nature of this communication that allows metastatic cells to intravasate and travel through the circulation and to extravasate to colonize different organs is poorly understood. In this study, we evaluated one of the first steps in this process—the proximity and physical interaction of endothelial and metastatic cells. To do this, we developed a cell separator chamber that allows endothelial and metastatic cells to grow side by side. We have shown in our previous studies that V-ATPases at the cell surface (pmV-ATPase) are involved in angiogenesis and metastasis. Therefore, we hypothesized that the physical proximity/interaction between endothelial and metastatic cells expressing pmV-ATPase will increase its activity in both cell types, and such activity in turn will increase pmV-ATPase expression on the membranes of both cell types. To determine pmV-ATPase activity we measured the proton fluxes (JH+) across the cell membrane. Our data indicated that interaction between endothelial and metastatic cells elicited a significant increase of JH+ via pmV-ATPase in both cell types. Bafilomycin, a V-ATPase inhibitor, significantly decrease JH+. In contrast, JH+ of the non-metastatic cells were not affected by the endothelial cells and vice-versa. Altogether, our data reveal that one of the early consequences of endothelial and metastatic cell interaction is an increase in pmV-ATPase that helps to acidify the extracellular medium and favors protease activity. These data emphasize the significance of the acidic tumor microenvironment enhancing a metastatic and invasive phenotype. PMID:24606724

  9. The role of Na(+), K(+)-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery.

    Science.gov (United States)

    Shen, Haitao; Liang, Peng; Qiu, Suhua; Zhang, Bo; Wang, Yongli; Lv, Ping

    2016-06-01

    Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na(+), K(+)-ATPase affects the regulation of intracellular Ca(2+) concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na(+), K(+)-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na(+), K(+)-ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na(+)-Ca(2+) exchanger with Ni(2+) (10(-3)M) or the L-type Ca(2+) channel with nimodipine (10(-8)M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na(+), K(+)-ATPase activity and increased [Ca(2+)]i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α2 isoform of Na(+), K(+)-ATPase in SMCs in vitro. We used a low concentration of the Na(+), K(+)-ATPase inhibitor ouabain, which possesses a high affinity for the α2 isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na(+), K(+)-ATPase activity in isolated basilar artery and the increased [Ca(2+)]i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na(+), K(+)-ATPase activity in SMCs through the α2 isoform contributing to vasoconstriction in the rat basilar artery. PMID:26924456

  10. Mitochondrial ATPase: a target for paracetamol-induced hepatotoxicity.

    Science.gov (United States)

    Parmar, D V; Ahmed, G; Khandkar, M A; Katyare, S S

    1995-10-01

    We examined the effect of paracetamol treatment (650 mg/kg) on the function of ATPase from rat hepatic mitochondria. The drug treatment caused an overall 35% decrease in ATPase activity, with a complete loss of the high affinity component as determined by substrate kinetic studies. The Km for the intermediate and low affinity components decreased by about 30% without change in Vmax, which may represent a compensatory mechanism. The drug treatment also resulted in a dramatic decrease in the phase transition temperature by about 19 degrees C without affecting the energies of activation of the enzyme. Mitochondrial total phospholipid content increased significantly with a reciprocal decrease in the cholesterol content. The total phospholipid/cholesterol molar ration increased by 50% after paracetamol treatment. However, phospholipid composition (as % of total) of the mitochondria was unaltered. PMID:8666039

  11. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  12. Small intestinal mucosa expression of putative chaperone fls485

    Directory of Open Access Journals (Sweden)

    Raupach Kerstin

    2010-03-01

    Full Text Available Abstract Background Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. Methods fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. Results fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. Conclusions Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

  13. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  14. The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function.

    Science.gov (United States)

    Raimunda, Daniel; González-Guerrero, Manuel; Leeber, Blaise W; Argüello, José M

    2011-06-01

    Cu(+)-ATPases play a key role in bacterial Cu(+) homeostasis by participating in Cu(+) detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P(1B-1) type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu(+) with high affinity in a trigonal planar geometry. The cytoplasmic Cu(+) chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu(+) is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu(+)-ATPases drive cytoplasmic Cu(+) efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu(+)-efflux pumps responsible for Cu(+) tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments. PMID:21210186

  15. Oryza sativa H+-ATPase (OSA) is Involved in the Regulation of Dumbbell-Shaped Guard Cells of Rice.

    Science.gov (United States)

    Toda, Yosuke; Wang, Yin; Takahashi, Akira; Kawai, Yuya; Tada, Yasuomi; Yamaji, Naoki; Feng Ma, Jian; Ashikari, Motoyuki; Kinoshita, Toshinori

    2016-06-01

    The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species. PMID:27048369

  16. The emerging structure of vacuolar ATPases.

    Science.gov (United States)

    Drory, Omri; Nelson, Nathan

    2006-10-01

    Bioenergetics and physiology of primary pumps have been revitalized by new insights into the mechanism of energizing biomembranes. Structural information is becoming available, and the three-dimensional structure of F-ATPase is being resolved. The growing understanding of the fundamental mechanism of energy coupling may revolutionize our view of biological processes. The F- and V-ATPases (vacuolar-type ATPase) exhibit a common mechanical design in which nucleotide-binding on the catalytic sector, through a cycle of conformation changes, drives the transmembrane passage of protons by turning a membrane-embedded rotor. This motor can run in forward or reverse directions, hydrolyzing ATP as it pumps protons uphill or creating ATP as protons flow downhill. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as an ATP-dependent proton pump. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. V- and F-ATPases have similar structure and mechanism of action, and several of their subunits evolved from common ancestors. Electron microscopy studies of V-ATPase revealed its general structure at low resolution. Recently, several structures of V-ATPase subunits, solved by X-ray crystallography with atomic resolution, were published. This, together with electron microscopy low-resolution maps of the whole complex, and biochemistry cross-linking experiments, allows construction of a structural model for a part of the complex that may be used as a working hypothesis for future research. PMID:16990452

  17. Copper-transporting P-type ATPases use a unique ion-release pathway

    DEFF Research Database (Denmark)

    Andersson, Magnus; Mattle, Daniel; Sitsel, Oleg; Klymchuk, Tetyana; Nielsen, Anna Marie; Møller, Lisbeth Birk; White, Stephen H; Nissen, Poul; Gourdon, Pontus

    2014-01-01

    Heavy metals in cells are typically regulated by PIB-type ATPases. The first structure of the class, a Cu(+)-ATPase from Legionella pneumophila (LpCopA), outlined a copper transport pathway across the membrane, which was inferred to be occluded. Here we show by molecular dynamics simulations that...... extracellular water solvated the transmembrane (TM) domain, results indicative of a Cu(+)-release pathway. Furthermore, a new LpCopA crystal structure determined at 2.8-Å resolution, trapped in the preceding E2P state, delineated the same passage, and site-directed-mutagenesis activity assays support a...... functional role for the conduit. The structural similarities between the TM domains of the two conformations suggest that Cu(+)-ATPases couple dephosphorylation and ion extrusion differently than do the well-characterized PII-type ATPases. The ion pathway explains why certain Menkes' and Wilson's disease...

  18. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

    Science.gov (United States)

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  19. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    Science.gov (United States)

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  20. CrATP as a new inhibitor of ecto-ATPases of trypanosomatids.

    Science.gov (United States)

    Moreira, O C; Rios, P F; Esteves, F F; Meyer-Fernandes, J R; Barrabin, H

    2009-01-01

    Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites. PMID:19126268

  1. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

    Directory of Open Access Journals (Sweden)

    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  2. Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

    Science.gov (United States)

    Reis, M; Farage, M; de Souza, A C; de Meis, L

    2001-11-16

    The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production. PMID:11544263

  3. Effects of M-cresol on Activity of Total ATPase During the Early Development of Cyprinus carpio var.color%间甲酚对瓯江彩鲤早期发育总 ATP 酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    朱俊华; 梁琍; 姚俊杰; 冯亚楠

    2014-01-01

    Activity of total adenosine triphosphatase ( ATPase) was determined during the early development of Cyp-rinus carpio var.color exposed to 7 mg/L, 13 mg/L and 19 mg/L m-cresol at the temperature of (20 ±2)℃.The influence of m-cresol on the total ATPase was analyzed through biochemical and static toxicological methods .The results showed that the activity of total ATPase had no significant difference in the fertilized eggs and the mature eggs of Cyprinus carpio var.color( P>0.05) .However, the activities of total ATPase in all treatment groups dem-onstrated obvious dose-effect relationship and stage-effect relationship in response to m-cresol during the early de-velopment of Cyprinus carpio var.color.In general, the activity of total ATPase decreased with the extension of de-velopmental time and increasing of m-cresol concentration from gastrula stage of the early development of Cyprinus carpio var.color.%在水温(20±2)℃条件下,设置7 mg/L、13 mg/L、19 mg/L的间甲酚(m-cresol)浓度梯度,研究了瓯江彩鲤(Cyprinus carpio var.color)早期发育过程中总ATP酶活性变化及间甲酚对总ATP酶影响。结果表明,瓯江彩鲤总ATP酶活性在成熟卵和受精卵中无显著差异(P>0.05);各浓度组总ATP酶活性表现为良好的剂量-效应和发育时期-效应关系;总体上看,瓯江彩鲤早期发育从原肠胚期开始,总ATP酶活性随着发育时间的延长和暴露浓度的增加而降低。

  4. Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

    DEFF Research Database (Denmark)

    Haagensen, L.; Jensen, D.H.; Gesser, Hans

    2008-01-01

    The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the...... pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

  5. Na+/K+-ATPase α1 mRNA expression in the gill and rectal gland of the Atlantic stingray, Dasyatis sabina, following acclimation to increased salinity

    OpenAIRE

    Evans, Andrew N.; Lambert, Faith N

    2015-01-01

    Background The salt-secreting rectal gland plays a major role in elasmobranch osmoregulation, facilitating ion balance in hyperosmotic environments in a manner analogous to the teleost gill. Several studies have examined the central role of the sodium pump Na+/K+-ATPase in osmoregulatory tissues of euryhaline elasmobranch species, including regulation of Na+/K+-ATPase activity and abundance in response to salinity acclimation. However, while the transcriptional regulation of Na+/K+-ATPase in ...

  6. Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide.

    Science.gov (United States)

    Macri, Christophe; Wang, Fengjuan; Tasset, Inmaculada; Schall, Nicolas; Page, Nicolas; Briand, Jean-Paul; Cuervo, Ana Maria; Muller, Sylviane

    2015-01-01

    The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired. PMID:25719862

  7. Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans

    Science.gov (United States)

    Morales-Rios, Edgar; Watt, Ian N.; Zhang, Qifeng; Ding, Shujing; Fearnley, Ian M.; Montgomery, Martin G.; Wakelam, Michael J. O.; Walker, John E.

    2015-01-01

    The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F1-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex. PMID:26423580

  8. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

    Directory of Open Access Journals (Sweden)

    Hongjie Xia

    2015-07-01

    Full Text Available RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71, which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16, another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings

  9. Multiple functions of the histone chaperone Jun dimerization protein 2.

    Science.gov (United States)

    Tsai, Ming-Ho; Wuputra, Kenly; Lin, Yin-Chu; Lin, Chang-Shen; Yokoyama, Kazunari K

    2016-09-30

    The Jun dimerization protein 2 (JDP2) is part of the family of stress-responsible transcription factors such as the activation protein-1, and binds the 12-O-tetradecanoylphorbol-13-acetateresponse element and the cAMP response element. It also plays a role as a histone chaperone and participates in diverse processes, such as cell-cycle arrest, cell differentiation, apoptosis, senescence, and metastatic spread, and functions as an oncogene and anti-oncogene, and as a cellular reprogramming factor. However, the molecular mechanisms underlying these multiple functions of JDP2 have not been clarified. This review summarizes the structure and function of JDP2, highlighting the specific role of JDP2 in cellular-stress regulation and prevention. PMID:27041241

  10. Characterization of Branchial Na,K-ATPase from Three Freshwater Fish Species (Oreochromis niloticus, Cyprinus carpio, and Oncorhynchus mykiss)

    OpenAIRE

    Atli, Gülüzar; CANLI, Mustafa

    2008-01-01

    Branchial Na,K-ATPase activity was characterized in 3 freshwater fish species (Oncorhynchus mykiss, Oreochromis niloticus, and Cyprinus carpio) with different ecological needs. Na+, K+, and Cl- concentrations in the gills were also measured. The maximal Na,K-ATPase activity was observed at 100 mM Na+, 20-40 mM K+, 3-4 mM Mg2+, and 1 mM ouabain. The maximal velocity (Vmax) of Na,K-ATPase isolated from O. mykiss (1.07 µmol Pi/mg prot/h) was lower than that isolated from O. niloticus (7.25 µmol ...

  11. V-ATPase as an effective therapeutic target for sarcomas

    International Nuclear Information System (INIS)

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity

  12. V-ATPase as an effective therapeutic target for sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Perut, Francesca, E-mail: francesca.perut@ior.it [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)

    2014-01-01

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

  13. Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design.

    Science.gov (United States)

    AhYoung, Andrew P; Koehl, Antoine; Cascio, Duilio; Egea, Pascal F

    2015-09-01

    Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. PMID:26130467

  14. Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type

    OpenAIRE

    Jorge-Finnigan, Ana; Brasil, Sandra; Underhaug, Jarl; Ruíz-Sala, Pedro; Merinero, Begoña; Banerjee, Ruma; Desviat, Lourdes R; Ugarte, Magdalena; Martinez, Aurora; Pérez, Belén

    2013-01-01

    Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of o...

  15. Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models.

    Science.gov (United States)

    Sanchez-Martinez, Alvaro; Beavan, Michelle; Gegg, Matthew E; Chau, Kai-Yin; Whitworth, Alexander J; Schapira, Anthony H V

    2016-01-01

    GBA gene mutations are the greatest cause of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase) but the mechanisms by which loss of GCase contributes to PD remain unclear. Inhibition of autophagy and the generation of endoplasmic reticulum (ER) stress are both implicated. Mutant GCase can unfold in the ER and be degraded via the unfolded protein response, activating ER stress and reducing lysosomal GCase. Small molecule chaperones that cross the blood brain barrier help mutant GCase refold and traffic correctly to lysosomes are putative treatments for PD. We treated fibroblast cells from PD patients with heterozygous GBA mutations and Drosophila expressing human wild-type, N370S and L444P GBA with the molecular chaperones ambroxol and isofagomine. Both chaperones increased GCase levels and activity, but also GBA mRNA, in control and mutant GBA fibroblasts. Expression of mutated GBA in Drosophila resulted in dopaminergic neuronal loss, a progressive locomotor defect, abnormal aggregates in the ER and increased levels of the ER stress reporter Xbp1-EGFP. Treatment with both chaperones lowered ER stress and prevented the loss of motor function, providing proof of principle that small molecule chaperones can reverse mutant GBA-mediated ER stress in vivo and might prove effective for treating PD. PMID:27539639

  16. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may

  17. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    Directory of Open Access Journals (Sweden)

    Picking Wendy L

    2010-07-01

    Full Text Available Abstract Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155 of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151. Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II

  18. Molecular chaperones: The modular evolution of cellular networks

    Indian Academy of Sciences (India)

    Tamás Korcsmáros; István A Kovács; Máté S Szalay; Péter Csermely

    2007-04-01

    Molecular chaperones play a prominent role in signaling and transcriptional regulatory networks of the cell. Recent advances uncovered that chaperones act as genetic buffers stabilizing the phenotype of various cells and organisms and may serve as potential regulators of evolvability. Chaperones have weak links, connect hubs, are in the overlaps of network modules and may uncouple these modules during stress, which gives an additional protection for the cell at the network-level. Moreover, after stress chaperones are essential to re-build inter-modular contacts by their low affinity sampling of the potential interaction partners in different modules. This opens the way to the chaperone-regulated modular evolution of cellular networks, and helps us to design novel therapeutic and anti-aging strategies.

  19. Enabling the (3 + 2) cycloaddition reaction in assembling newer anti-tubercular lead acting through the inhibition of the gyrase ATPase domain: lead optimization and structure activity profiling.

    Science.gov (United States)

    Jeankumar, Variam Ullas; Reshma, Rudraraju Srilakshmi; Janupally, Renuka; Saxena, Shalini; Sridevi, Jonnalagadda Padma; Medapi, Brahmam; Kulkarni, Pushkar; Yogeeswari, Perumal; Sriram, Dharmarajan

    2015-02-28

    DNA gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis, is absent in humans and is a well validated target for anti-tubercular drug discovery. In this study, a moderately active inhibitor of Mycobacterium tuberculosis GyrB, the pharmaceutically unexploited domain of DNA gyrase, was reengineered using a combination of molecular docking and medicinal chemistry strategies to obtain a lead series displaying considerable in vitro enzyme efficacy and bacterial kill against the Mycobacterium tuberculosis H37Rv strain. Biophysical investigations using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain. Furthermore, the molecules were completely devoid of hERG toxicity up to 30 μM, as evaluated in a zebra fish model with a good selectivity index, and from a pharmaceutical point of view, turned out as potential candidates against TB. PMID:25569565

  20. Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

    Science.gov (United States)

    Hossain, Mohammad Arif; Higaki, Katsumi; Saito, Seiji; Ohno, Kazuki; Sakuraba, Hitoshi; Nanba, Eiji; Suzuki, Yoshiyuki; Ozono, Keiichi; Sakai, Norio

    2015-09-01

    Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant GM1 β-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations. PMID:26108143

  1. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation in the...... affordable alternative using a microscope-based cytometer. This system can simultaneously provide information on flippase activity and expression levels. Taken together, the findings described in this thesis provide new tools for P4-ATPase characterization and valuable insights into the regulation and the...

  2. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    Science.gov (United States)

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  3. Pantothenate is required in Neurospora crassa for assembly of subunit peptides of cytochrome c oxidase and ATPase/ATP synthase.

    OpenAIRE

    Brambl, R; Plesofsky-Vig, N

    1986-01-01

    One polypeptide subunit of cytochrome c oxidase (EC 1.9.3.1) and two subunits of the ATPase/ATP synthase (EC 3.6.1.34) in mitochondria of Neurospora crassa are covalently modified with a derivative of pantothenic acid. In asexual spores of a pantothenate auxotroph of Neurospora, deprivation of pantothenic acid blocked the increase of the specific activities of cytochrome c oxidase and the ATPase above the basal activities in the dormant spores. Under cellular panthothenate deprivation, all th...

  4. The effect of inhibitors of plasma membrane H+ - ATPase and oxidoreductases on NH4+ uptake by Pisum arvense roots

    OpenAIRE

    Genowefa Kubik-Dobosz; Aleksandra Turska; Halina Lekacz; Waldemar Karcz; Józef Buczek

    2014-01-01

    The effect of inhibitors of plasma membrane oxidoreductases (quinacrine and dicumarol) and H+-ATPase (dicyclohexylcarbodiimide and orthovanadate) on ammonium uptake by Pisum arvense seedlings and the activities of H+-ATPase and NADH-ferricyanide oxidoreductase was investigated. The uptake solution contained 50 µM NH4+. In I h experiments, quinacrine and dicumarol depressed strongly and irreversibly the rate of NH4+ uptake and markedly inhibited the activity of NADH-ferri-cyanide oxidoreductas...

  5. Effects of Na/K-ATPase and its ligands on bone marrow stromal cell differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Sayed

    2014-07-01

    Full Text Available Endogenous ligands of Na/K-ATPase have been demonstrated to increase in kidney dysfunction and heart failure. It is also reported that Na/K-ATPase signaling function effects stem cell differentiation. This study evaluated whether Na/K-ATPase activation through its ligands and associated signaling functions affect bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells differentiation capacity. BMSCs were isolated from male Sprague–Dawley rats and cultured in minimal essential medium alpha (MEM-α supplemented with 15% Fetal Bovine serum (FBS. The results showed that marinobufagenin (MBG, a specific Na/K-ATPase ligand, potentiated rosiglitazone-induced adipogenesis in these BMSCs. Meanwhile, it attenuated BMSC osteogenesis. Mechanistically, MBG increased CCAAT/enhancer binding protein alpha (C/EBPα protein expression through activation of an extracellular regulated kinase (ERK signaling pathway, which leads to enhanced rosiglitazone-induced adipogenesis. Inhibition of ERK activation by U0126 blocks the effect of MBG on C/EBPα expression and on rosiglitazone-induced adipogenesis. Reciprocally, MBG reduced runt-related transcription factor 2 (RunX2 expression, which resulted in the inhibition of osteogenesis induced by β-glycerophosphate/ascorbic acid. MBG also potentiated rosiglitazone-induced adipogenesis in 3T3-L1 cells and in mouse BMSCs. These results suggest that Na/K-ATPase and its signaling functions are involved in the regulation of BMSCs differentiation.

  6. RIN4 functions with plasma membrane H+-ATPases to regulate stomatal apertures during pathogen attack

    DEFF Research Database (Denmark)

    Liu, Jun; Elmore, James M.; Fuglsang, Anja Thoe;

    2009-01-01

    purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H+-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines......Abstract Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4- mediated immune signal transduction, we...... exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its...

  7. Absence of influence of strong permanent magnetic field on isolated membrane preparations of Na, K-dependent ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Savich, M.L.; Nazarova, N.M.; Raykhman, L.M.; Kuznetsov, A.N.

    A study is made of the effect of a permanent magnetic field with an induction of 10 T on isolated membrane preparations of Na, K-dependent ox brain ATPase. The 10 T field was not found to have any influence on the Na, K-ATPase activity under any of the conditions tested. The insensitivity of isolated Na, K-ATPase preparations to permanent magnetic field even at great field strength may result from insufficient size of cooperative areas of membrane lipids in small lipoprotein vesicles. The data obtained can therefore only be extended with caution to larger membrane formations functioning in vivo. 5 references, 1 figure.

  8. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  9. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ. PMID:27112153

  10. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-04-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  11. Expression and variability of molecular chaperones in the sugarcane expressome.

    Science.gov (United States)

    Borges, Júlio C; Cagliari, Thiago C; Ramos, Carlos H I

    2007-04-01

    Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study. PMID:16687190

  12. 黄芪有效部位对糖尿病大鼠Na+-K+-ATP酶活性及AMPK蛋白表达的影响%Effects of astragalus active ingredients on Na+-K+-ATPase and AMP-activated protein kinase protein expression in diabetic rats

    Institute of Scientific and Technical Information of China (English)

    范颖; 李楠; 孙云峰; 马哲; 林庶茹

    2012-01-01

    目的:通过研究糖尿病模型大鼠胰腺组织钠-钾-ATP酶(Na+-K+-ATP)活性、腺苷酸激活蛋白激酶(AMPK)活性及肝、骨骼肌组织AMPK蛋白表达的变化,探讨黄芪有效部位对糖尿病大鼠能量代谢的影响.方法:SD大鼠随机分为正常组、模型组、中药对照组、黄芪组、黄酮组、多糖组、皂苷组、酮糖组、酮苷组、糖苷组、酮糖苷组,共11组,每组14只.由链脲佐菌素( 52mg/kg)诱导糖尿病大鼠模型,造模同日给予黄芪及其有效部位进行干预.观测30日,检测血糖,生化法分析胰腺组织Na+-K+-ATP酶的活性,ELISA法分析胰腺AMPK活性,Western Blot法分析肝、骨骼肌组织AMPK蛋白表达.结果:糖尿病模型大鼠血糖显著升高(P<0.01),胰腺Na+-K+-ATP酶活性、AMPK水平以及肝、骨骼肌AMPK蛋白表达均显著降低(P<0.01);与模型组比较,黄芪组、黄酮组、酮糖组、酮糖苷组血糖降低( P<0.05,P<0.01),胰腺Na+-K+-ATP酶活性、AMPK水平显著升高(P<0.01),酮苷组血糖下降、胰腺Na+-K+-ATP酶活性升高(P<0.01),皂苷组、糖苷组胰腺Na+-K+-ATP酶活性升高(P<0.01).结论:黄芪、黄芪黄酮及含黄芪黄酮的有效部位能够降低糖尿病大鼠的血糖,其机制可能与改善Na+-K+-ATP酶活性、上调AMPK水平及蛋白表达有关.%Objective: This study is designed to evaluate the effect of astragalus active ingredients on energy metabolism in diabetic rats by studying Na+-K+-ATPase and AMPK in pancreas and AMPK protein expression in liver and skeletal muscle. Methods: Diabetes rats were induced by STZ (52mg/kg, peritioneal injection). Diabetic rats were administered by Astragalux radix and its active ingredients for 30 days from the day of STZ pj. Astragalus radix active ingredients were astragalux radix group (AS), astragalus flavonoids group (ASF), astragalus polysaccharides group (ASP), and astragalosides group (ASS), astragalus flavonolids and polysaccharides group (ASF

  13. 云芝多糖对运动训练大鼠脑组织抗氧化能力和ATPase活性的影响%Effect of Krestin Polysaccharide on Antioxidant Capability and ATPase Activity in Brain Tissues of Rats with High Intensity Exercise

    Institute of Scientific and Technical Information of China (English)

    习雪峰; 王单一; 熊正英; 张林

    2012-01-01

    目的:探讨云芝多糖对力竭运动大鼠脑组织的部分抗氧化酶活性和Na^2+,K^+-ATP酶(Na^+,K^+.ATPasc),Ca^2+,Mg^2+-ATP酶(Ca^2+,Mg^2+.ATPase)活性影响。方法:选取成年雄性SD大鼠24只。将大鼠随机分为3组:安静对照组8只,运动对照组8只,运动加药组8只,进行为期8周的大强度耐力跑台训练。测定大鼠脑组织部分抗氧化酶和Na^+,K^+-ATPase,Ca^2+,Mg^2+.ATPase活性的变化。结果:与安静对照组相比,运动对照组脑组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)、Na^+,K^+-ATPase、Can,Mg^2+-ATPase活性和血糖含量显著下降(P〈0.01或P〈0.05),MDA生成显著增多(P〈0.05);云芝多糖提高了力竭运动大鼠脑组织SOD、CAT、GSH.Px、T-AOC、Na^+,K^+-ATPase、Ca^2+,Mg^2+.ATPase活性和血糖含量(P〈0.01或P〈0.05),减少MDA生成(P〈0.05)。结论:云芝多糖可以提高力竭运动大鼠脑组织中抗氧化酶活性和Na^+,D.ATPase和Ca^2+,Mg^2+-ATPase的活性,这对于维持运动中能量的供应和抗氧化能力,从而提高大鼠的运动能力具有重要意义。%Objective: To explore the effect of Krestin polysaechatide (PSK) on the activities of antioxidant enzymes and Na^+- K^+-ATPase as well as Ca^2 +-Mg^2+-ATPase in brain tissues of rats after exhaustive exercise. Methods: 24 SD rats were divided into three groups including the control group without exercise, exercise group and exercise plus PSK group. Each group included 8 rats. The rats from the exercise and exercise plus PSK groups were subjected to high-intensity endurance running for 8 consecutive weeks. The changes in the activities of antioxidant enzymes, Na^+-K^+-ATPase and Ca^2+-Mg^2+-ATPase were measured. Results: The activities of SOD

  14. Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

    Science.gov (United States)

    Sichting, Martin; Mokranjac, Dejana; Azem, Abdussalam; Neupert, Walter; Hell, Kai

    2005-01-01

    Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones. PMID:15719019

  15. Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

    Directory of Open Access Journals (Sweden)

    H.L. Santos

    2002-03-01

    Full Text Available SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM and low (K0.5 = 1.4 mM affinity sites for ATP, with negative cooperativity. Ouabain (5 mM, oligomycin (1 µg/ml and sodium vanadate (3 µM inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß2 association.

  16. Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1

    OpenAIRE

    O'Sullivan, Roderick J; Arnoult, Nausica; Daniel H Lackner; Oganesian, Liana; Haggblom, Candy; Corpet, Armelle; Almouzni, Genevieve; Karlseder, Jan

    2014-01-01

    The mechanism of activation of the Alternative Lengthening of Telomeres (ALT) pathway of mammalian chromosome end maintenance has remained an unresolved issue. We have discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT associated PML bodies (APBs), extra-chromosomal telomeric DNA species an elevated frequency of telomeric sister chromatid exchanges (t-SCE) even...

  17. Lipase Maturation Factor 1: a lipase chaperone involved in lipid metabolism

    OpenAIRE

    Péterfy, Miklós

    2011-01-01

    Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomer...

  18. The influence of temperature on the distribution and intensity of the reaction product in rat muscle fibers obtained with the histochemical method for myosin ATPase

    DEFF Research Database (Denmark)

    Kirkeby, S; Tuxen, A

    1989-01-01

    was raised and in others was depressed by alteration of the incubation temperature. There was no obvious correlation between the temperature sensitivity of ATPase in the muscle fibers and their activity for succinic dehydrogenase. It is proposed that the histochemical method for myosin ATPase can be...

  19. Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?

    Directory of Open Access Journals (Sweden)

    Helena Öhrvik

    2015-07-01

    Full Text Available The human copper (Cu chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

  20. Stratified analysis of lectin-like chaperones in the folding disease-related metabolic syndrome rat model.

    Science.gov (United States)

    Hirano, Makoto; Imagawa, Ayami; Totani, Kiichiro

    2016-09-01

    The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels. PMID:27425249

  1. GRP75 upregulates clathrin-independent endocytosis through actin cytoskeleton reorganization mediated by the concurrent activation of Cdc42 and RhoA.

    Science.gov (United States)

    Chen, Hang; Gao, Zhihui; He, Changzheng; Xiang, Rong; van Kuppevelt, Toin H; Belting, Mattias; Zhang, Sihe

    2016-05-01

    Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptide-directed protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells. PMID:27090015

  2. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    Weihuan Cao; Mary Konsolaki

    2011-08-01

    The FK506-binding protein (FKBP) family of immunophilins consists of proteins with a variety of protein–protein interaction domains and versatile cellular functions. Analysis of the functions of immunophilins has been the focus of studies in recent years and has led to the identification of various molecular pathways in which FKBPs play an active role. All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of the immunosuppressant molecule FK506 to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of calcineurin. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration. This review will focus on recent studies involving FKBP immunophilins in Alzheimer’s-disease-related pathways.

  3. New ATPase regulators-p97 goes to the PUB

    DEFF Research Database (Denmark)

    Madsen, Louise; Seeger, Michael; Semple, Colin A; Hartmann-Petersen, Rasmus

    2009-01-01

    The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed to....... Recently, a small, conserved family of proteins, containing PUB domains, was found to function as p97 adaptors. Intriguingly, their association with p97 is regulated by tyrosine phosphorylation, suggesting that they act as a relay between signalling pathways and p97 functions. Here we give an overview of...

  4. Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts.

    Science.gov (United States)

    Cavieres, J D

    1984-04-11

    An average target size of 251 kDa has been obtained for the (Ca2+ + Mg2+)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer. PMID:6142728

  5. Structural features and interactions of substrates complexed with molecular chaperones

    OpenAIRE

    Ungelenk, Sophia Maria

    2015-01-01

    Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is consti...

  6. Effect of Turmeric, Turmerin and Curcumin on Ca2+, Na/K+ Atpases in Concanavalin A-Stimulated Human Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Suman K. Das

    2003-01-01

    Full Text Available Abstract: Ion transport enzymes may play an important role in T cell activation. This study investigates the role of turmeric and its individual components, turmerin-and curcumin-on Ca2+ and Na/K+ adenosine triphosphatases (ATPase in the course of T cell activation. Concanavalin A (Con A stimulated human blood mononuclear T cell proliferation paradigm was investigated for 3, 5 and 7 day periods with different concentrations of turmeric, curcumin and turmerin. Con A-stimulated cells treated with turmeric (250, 50, 5 μg/ml for 3 and 5 days inhibited ATPase levels when compared to base levels obtained by cells in media alone. At day 7, there was a 3-fold increase for Ca2+ATPase levels and a 2-fold increase for Na/K+ATPase. Curcumin (250, 50, 5 μg/ml showed the same pattern for ATPase activity as turmeric at 3 and 5 days with a 2-fold increase at day 7. Turmerin (2500, 1250, 250, 25 ng/ml for Na/K+ ATPase activity showed an increase at day 3, a decrease on day 5, and a 2-fold increase on day 7. Ca2+ ATPase activity in the presence of turmerin showed an increase in ATPase levels at day 3 (except at 2500ng/ml where it decreased and a decrease in day 5 (except at 25 ng/ml where it increased. Turmeric and curcumin generally inhibited Ca2+ATPase and Na/K+ATPases in early (day 3 and intermediate (day 5 stages of mitogen stimulation. However, the effect after 7 days incubation for turmeric, curcumin and turmerin showed a marked increase up to three fold.

  7. Oxidized LDL-bound CD36 recruits a Na+/K+-ATPase-Lyn complex in macrophages that promotes atherosclerosis

    Science.gov (United States)

    Chen, Yiliang; Kennedy, David J.; Ramakrishnan, Devi Prasadh; Yang, Moua; Huang, Wenxin; Li, Zhichuan; Xie, Zijian; Chadwick, Alexandra C.; Sahoo, Daisy; Silverstein, Roy L.

    2016-01-01

    One characteristic of atherosclerosis is the accumulation of lipid-laden macrophage foam cells in the arterial wall. We have previously shown that the binding of oxidized LDL (oxLDL) to the scavenger receptor CD36 activates the kinase Lyn, initiating a cascade that inhibits macrophage migration and is necessary for foam cell generation. Here, we identified the plasma membrane ion transporter Na/K-ATPase as a key component in the macrophage oxLDL-CD36 signaling axis. Using peritoneal macrophages isolated from Atp1a1 heterozygous or Cd36 null mice, we demonstrated that CD36 recruited a Na/K-ATPase-Lyn complex for Lyn activation in response to oxLDL. Macrophages deficient in the α1 Na/K-ATPase catalytic subunit did not respond to activation of CD36, showing attenuated oxLDL uptake and foam cell formation, and oxLDL failed to inhibit migration of these macrophages. Furthermore, Apoe-null mice, which are a model of atherosclerosis, were protected from diet-induced atherosclerosis by global deletion of a single allele encoding the α1 Na+/K+-ATPase subunit or reconstitution with macrophages that lacked an allele encoding the α1 Na+/K+-ATPase subunit.. These findings identify Na/K-ATPase as a potential target for preventing or treating anti-atherosclerotic therapy. PMID:26350901

  8. Analysis of the Inhibitory Effect of Gypenoside on Na+,K+-ATPase in Rats' Heart and Brain and Its Kinetics

    Institute of Scientific and Technical Information of China (English)

    HAN Xiao-yan; WEI Hong-bo; ZHANG Fu-cheng

    2007-01-01

    ObjectiYe: To study the effects of gypenoside (Gyp) on the activity of microsomal Na+,K+-ATPase in rat's heart and brain in vitro. Methods: The microsomal Na+, K+-ATPase was prepared from rat's heart and brain by differential centrifugation. The activity of microsomal Na+, K+-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na+, K+-ATPase of rats. Results: Gyp reversibly inhibited the brain and heart's microsomal Na+, K+-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07 ±6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na+, or K+ concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na+, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K+. Conclusion: Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain's microsomal Na+, K+-ATPase activities in rats.

  9. Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism.

    Science.gov (United States)

    Bremberger, C; Haschke, H P; Lüttge, U

    1988-10-01

    Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C3 and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C3 to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase. PMID:24221927

  10. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2010-05-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  11. The use of a chaperone in obstetrical and gynaecological practice.

    LENUS (Irish Health Repository)

    Afaneh, I

    2012-02-01

    The aim of this study was to assess the use of a chaperone in obstetrical and gynaecological practice in Ireland and to explore patients\\' opinions. Two questionnaires were designed; one for patients and the other one was sent to 145 gynaecologists in Ireland. One hundred and fifty two women took part in this survey of whom 74 were gynaecological and 78 were obstetric patients. Ninety five (65%) patients felt no need for a chaperone during a vaginal examination (VE) by a male doctor. On the other hand 34 (23%) participating women would request a chaperone if being examined by a female doctor. Among clinicians 116 (80%) responded by returning the questionnaire. Overall 60 (52%) always used a chaperone in public practice, in contrast to 24 (27%) in private practice. The study demonstrated that most patients do not wish to have a chaperone during a VE but a small proportion would still request one regardless of the examiner\\'s gender. Patients should be offered the choice of having a chaperone and their opinion should be respected and documented.

  12. Chaperoning Roles of Macromolecules Interacting with Proteins in Vivo

    Directory of Open Access Journals (Sweden)

    Baik L. Seong

    2011-03-01

    Full Text Available The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

  13. Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

    OpenAIRE

    Fendler, K; Grell, E; Haubs, M; Bamberg, E

    1985-01-01

    The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient ...

  14. Structure function relationship in P-type ATPases : a biophysical approach

    OpenAIRE

    Apell, Hans-Jürgen

    2003-01-01

    P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolut...

  15. Autoradiographic localization of Na+-K+-ATPase with 3H-ouabain

    International Nuclear Information System (INIS)

    Using 3H-ouabain as an inhibitor, the site of the Na+-K+-ATPase system in cells was determined autoradiographically. Experiments were performed woth guinea pig's kidney tissue. The application of light microscopical autoradiography to freeze-dried tissue showed that especially the distal tubule, and to a smaller extent the proximal tubule and the collecting tubule have Na+-K+-ATPase. Electron microscopical autoradiography showed that this activity is restricted to the baso-lateral plasmamembranes. The quantity of specific bound ouabain turns out to be correlated to the quantity of baso-lateral plasmamembrane's surface

  16. Alteration of alpha 1 Na+,K(+)-ATPase 86Rb+ influx by a single amino acid substitution

    International Nuclear Information System (INIS)

    The sodium- and potassium-dependent adenosine triphosphatase (Na+,K(+)-ATPase) maintains the transmembrane Na+ gradient to which is coupled all active cellular transport systems. The R and S alleles of the gene encoding the Na+,K(+)-ATPase alpha 1 subunit isoform were identified in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats, respectively. Characterization of the S allele-specific Na+,K(+)-ATPase alpha 1 complementary DNA identified a leucine substitution of glutamine at position 276. This mutation alters the hydropathy profile of a region in proximity to T3(Na), the trypsin-sensitive site that is only detected in the presence of Na+. This mutation causes a decrease in the rubidium-86 influx of S allele-specific sodium pumps, thus marking a domain in the Na+,K(+)-ATPase alpha subunit important for K+ transport, and supporting the hypothesis of a putative role of these pumps in hypertension

  17. Vacuolar-type H+-ATPase and Na+, K+-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

    DEFF Research Database (Denmark)

    Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo; Madsen, Steffen S

    2001-01-01

    Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days t...

  18. An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non–cell autonomous neurodegeneration

    Science.gov (United States)

    Gallardo, Gilbert; Barowski, Jessica; Ravits, John; Siddique, Teepu; Lingrel, Jerry B; Robertson, Janice; Steen, Hanno; Bonni, Azad

    2015-01-01

    Perturbations of astrocytes trigger neurodegeneration in several diseases, but the glial cell–intrinsic mechanisms that induce neurodegeneration remain poorly understood. We found that a protein complex of α2-Na/K ATPase and α-adducin was enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS). Knockdown of α2-Na/K ATPase or α-adducin in mutant SOD1 astrocytes protected motor neurons from degeneration, including in mutant SOD1 mice in vivo. Heterozygous disruption of the α2-Na/K ATPase gene suppressed degeneration in vivo and increased the lifespan of mutant SOD1 mice. The pharmacological agent digoxin, which inhibits Na/K ATPase activity, protected motor neurons from mutant SOD1 astrocyte–induced degeneration. Notably, α2-Na/K ATPase and α-adducin were upregulated in spinal cord of sporadic and familial ALS patients. Collectively, our findings define chronic activation of the α2-Na/K ATPase/α-adducin complex as a critical glial cell–intrinsic mechanism of non–cell autonomous neurodegeneration, with implications for potential therapies for neurodegenerative diseases. PMID:25344630

  19. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    Energy Technology Data Exchange (ETDEWEB)

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M [UMASS, MED; (UCB); (UW-MED)

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  20. The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding*

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J. H.; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C.

    2012-01-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  1. The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding.

    Science.gov (United States)

    Baaklini, Imad; Wong, Michael J H; Hantouche, Christine; Patel, Yogita; Shrier, Alvin; Young, Jason C

    2012-12-01

    DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70. PMID:23091061

  2. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  3. Effects of exogenous creatine phosphate on glutamic acid and Ca2+-ATPase activity in brain of mice after exhaustive exercise%外源性磷酸肌酸对游泳力竭小鼠大脑中谷氨酸和钙-ATP酶活力的影响

    Institute of Scientific and Technical Information of China (English)

    马集; 卢畅; 姜茜; 殷林波; 刘彦娜; 刘克敏

    2013-01-01

    Objective:To observe the effects of exogenous creatine phosphate on glutamic acid level and Ca2+-ATPase activity in brain of mice after exhaustive exercise and to further reveal the mechanism of exogenous creatine phosphate in allaying tiredness.Methods:All 36 mice,6-week-age,were divided into 4 groups:exhaustive swimming control group (group A); exhaustive swimming with medication group (group B); 8-min swimming control group (group C);and 8-min swimming with medication group (group D).The method of mice weight-loading swimming was used to sets up the model of exhaustive exercise,and each mouse loaded weight with 6% of the mass of itself.Thirty min before the experiment,mice in groups B and D were given the intraperitoneal injection with creatine phosphate sodium by the standard of 1000 mg/kg,and the mice in groups A and C were given the same proportionate normal saline as placebo.The exhaustive swimming time was recorded,and glutamic level and Ca2+-ATPase activity were measured by using biochemical kits.Results:After testing,the exhaustion time in group B was longer than that in group A (P<0.05).The Glu contents in groups B and D were significantly lower than in groups A and C (P<0.05).Ca2+-ATPase activity in groups B and group D was significantly higher than that in groups A and C (P<0.05).Conclusion:The mechanism of exogenous creatine phosphate in allaying tiredness may be closely related with increased Ca2+-ATPase activity and reduced glutamic level.%目的:研究外源性磷酸肌酸(PCr)对游泳力竭小鼠大脑中谷氨酸(Glu)和钙-ATP酶(Ca2+-ATPase)活力的影响,以进一步揭示PCr的抗疲劳机制.方法:将44只6周龄小鼠分为力竭对照组12只(A组)、力竭给药组12只(B组)、游泳8min对照组10只(C组)、游泳8min给药组10只(D组),采取小鼠负重游泳的力竭运动模型,每只小鼠负重量为自身体质量的6%.于游泳前30min,B、D组小鼠经腹腔注射磷酸肌酸钠溶液1000mg/kg;A、C组小鼠注

  4. Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons

    DEFF Research Database (Denmark)

    Tal, D M; Yanuck, M D; Van Hall, Gerrit;

    1989-01-01

    A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na...

  5. NaCl effects on root plasma membrane ATPase of salt tolerant wheat

    NARCIS (Netherlands)

    Mansour, MMF; van Hasselt, PR; Kuiper, PJC

    2000-01-01

    Wheat seedlings of a salt tolerant cultivar were grown hydroponically in presence and absence of 100 mM NaCl. Roots were harvested, and the plasma membrane (PM) fraction was purified. PM ATPase required a divalent cations for activity (Mg > Mn > Ca > Co > Zn > Ni > Cu), and it was further stimulated

  6. Relations between erythrocyte Ca2+ , Mg2+ levels and cell membrane ATPase activities in patients with hypertension in obese children%肥胖儿童红细胞钙镁水平及ATP酶活性与高血压关系的探讨

    Institute of Scientific and Technical Information of China (English)

    朱树森; 符云峰; 卢振敏; 王素敏; 孙爱丽

    2001-01-01

    目的探讨细胞离子代谢紊乱在肥胖儿童高血压发病中的作用。方法测定125例(正常对照37例,正常血压肥胖34例,正常体重高血压21例,高血压肥胖33例)12~16岁中学生的红细胞膜ATP酶活性、血浆和红细胞胞浆Ca2+、Mg2+水平。结果正常血压肥胖组(ONT)儿童红细胞膜Na+-K+-ATP酶和Ca2+-ATP酶活性较正常对照组(NT)显著降低,正常体重高血压组(NOHT)和高血压肥胖组(OHT)两酶活性又显著低于ONT组。ONT组、NOHT组和OHT组红细胞胞浆Ca2+水平三组之间无显著差异,但均显著高于NT组。红细胞胞浆Mg2+,血浆Ca2+、Mg2+在NT组和ONT组之间无显著差异,在NOHT组和OHT组均显著降低。结论红细胞膜Na+-K+-ATP酶和Ca2+-ATP酶活性降低可能在肥胖儿童高血压的发病机制中有重要作用。%Objective To explore the roles of the metabolic disorders of cellular ions in pathogenesis of hypertension in obese children. Methods Erythrocyte membrane ATPase activities,plasma and cellular Ca2 + , Mg2 + levels were measured in 125(37 non-obese normotensives,34 obese normotensives,21 hypertensives and 33 obese hypertensives)middle school students aged 12~16 years. Results Erythrocyte membrane Na+ -K+-ATPase and Ca2+ -ATPase activities were significantly lower in obese children with normotension than those in non-obese normotensive children,and they were also significantly lower in two groups of hypertensive children than those in obese children with normotension. Erythrocyte Ca2+ in normotensive obese and two group of hypertensive children was significantly higher than that in non-obese normotensive children, but there were no significant differences between the three groups. There were no significant differences found in erythrocyte Mg2+ , plasma Ca2+ and Mg2+ between normotensive obese and normotensive non-obese children, but significant decreases were found in both groups of hypertensive children. Conclusion Decreased Na +-K+-ATPase

  7. Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Sarcoplasmic reticulum Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca2+-ATPase occurred within a few hours in the presence of ≤ 50 μM Ca2+. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high 45Ca2+ concentration (500 μM), monomeric Ca2+-ATPase was stable for several house. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca2+-ATPase was found to be 105-106 M-1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca2+-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca2+ and 48V vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. The results suggest that formation of Ca2+-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit

  8. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg

    2015-01-01

    the bacterial proteins LpCopA and SsZntA, which represent Cu+- and Zn2+-ATPases, respectively. The thesis first compares the recent pioneering P1B-ATPase structure of LpCopA to that of the well-described Ca2+-ATPase SERCA, showing how Cu+-ATPases have managed to adapt the general P-type ATPase...... Zn2+ homeostasis that belong to the superfamily of P-type ATPases, transmembrane proteins which are present in virtually all lifeforms, with functions ranging from membrane potential generation to muscle relaxation. The goal of this thesis is to improve our understanding of P1B-ATPases by focusing on...... crystal structure of LpCopA in a new conformational state is then presented and studied using a variety of methods, showing that Cu+-ATPases use an ion release pathway unique for the P-type ATPase superfamily. The next section introduces the two pioneering crystal structures of a Zn2+-ATPase, Ss...

  9. Histone Chaperone HIRA in Regulation of Transcription Factor RUNX1.

    Science.gov (United States)

    Majumder, Aditi; Syed, Khaja Mohieddin; Joseph, Sunu; Scambler, Peter J; Dutta, Debasree

    2015-05-22

    RUNX1 (Runt-related transcription factor 1) is indispensable for the generation of hemogenic endothelium. However, the regulation of RUNX1 during this developmental process is poorly understood. We investigated the role of the histone chaperone HIRA (histone cell cycle regulation-defective homolog A) from this perspective and report that HIRA significantly contributes toward the regulation of RUNX1 in the transition of differentiating mouse embryonic stem cells from hemogenic to hematopoietic stage. Direct interaction of HIRA and RUNX1 activates the downstream targets of RUNX1 implicated in generation of hematopoietic stem cells. At the molecular level, HIRA-mediated incorporation of histone H3.3 variant within the Runx1 +24 mouse conserved noncoding element is essential for the expression of Runx1 during endothelial to hematopoietic transition. An inactive chromatin at the intronic enhancer of Runx1 in absence of HIRA significantly repressed the transition of cells from hemogenic to hematopoietic fate. We expect that the HIRA-RUNX1 axis might open up a novel approach in understanding leukemogenesis in future. PMID:25847244

  10. The histone chaperone CAF-1 safeguards somatic cell identity.

    Science.gov (United States)

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Euong Ang, Cheen; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter J; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2015-12-10

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  11. Interaction of Spin-Labeled Inhibitors of the Vacuolar H+-ATPase with the Transmembrane Vo-Sector

    Science.gov (United States)

    Dixon, Neil; Páli, Tibor; Kee, Terence P.; Ball, Stephen; Harrison, Michael A.; Findlay, John B. C.; Nyman, Jonas; Väänänen, Kalervo; Finbow, Malcolm E.; Marsh, Derek

    2008-01-01

    The osteoclast variant of the vacuolar H+-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC50 in the 10-nanomolar range. PMID:17872954

  12. Diallyl tetrasulfide improves cadmium induced alterations of acetylcholinesterase, ATPases and oxidative stress in brain of rats

    International Nuclear Information System (INIS)

    Cadmium (Cd) is a neurotoxic metal, which induces oxidative stress and membrane disturbances in nerve system. The garlic compound diallyl tetrasulfide (DTS) has the cytoprotective and antioxidant activity against Cd induced toxicity. The present study was carried out to investigate the efficacy of DTS in protecting the Cd induced changes in the activity of acetylcholinesterase (AChE), membrane bound enzymes, lipid peroxidation (LPO) and antioxidant status in the brain of rats. In rats exposed to Cd (3 mg/kg/day subcutaneously) for 3 weeks, a significant (P +K+-ATPase, Mg2+-ATPase and Ca2+-ATPase) were observed in brain tissue. Oral administration of DTS (40 mg/kg/day) with Cd significantly (P < 0.05) diminished the levels of LPO and protein carbonyls and significantly (P < 0.05) increased the activities of ATPases, antioxidant enzymes, GSH and TSH in brain. These results indicate that DTS attenuate the LPO and alteration of antioxidant and membrane bound enzymes in Cd exposed rats, which suggest that DTS protects the brain function from toxic effects of Cd

  13. Evolution of Plant P-Type ATPases

    OpenAIRE

    Pedersen, Christian N. S.; Kristian B. Axelsen; Harper, Jeffrey F.; Palmgren, Michael G.

    2012-01-01

    Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae) were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauri and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a non-vascular moss), Selaginella moellendorffii (a primitive vascular plant), and Arabidopsis thaliana (a model flowering plant). Each organism contained sequences for all five...

  14. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

    Directory of Open Access Journals (Sweden)

    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24mg/kg (14 ppm rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS, heat shock factor 1 (HSF1, and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  15. Changes of mitochondrial structure, ATPase and Ca2+ concentration in spermatogenic cells of mouse testes induced by low dose radiation

    International Nuclear Information System (INIS)

    Objective: To observe the ultrastructure, ATPase activity and Ca2+ concentration ([Ca2+]i) of mitochondria in the sperematogenic cells of mouse testes 3-24 h after low dose radiation with 0.025-0.200 Gy X-rays, and illuminate the effects of mitochondrion structure and relative biological function on apoptosis. Methods: The ultrastructure changes of mitochondria in the spermatogenic cells were observed with transmission electron microscope; the ATPase activity was measured with protein enzymic method; [Ca2+]i was measured indirectly by flow cytometry with Fluo-3 probes. Results: The mitochondria swelled and vacuolizated, and their cristae were broken in the spermatogonia and spermatocytes 12 h after irradiation, and their nuclei were karyopyknosis, the acrosomal vesicle structure was ambiguity, the membrane structure was unclear, and the mitochondria in spermatids were vacuolization. The activities of Na+-K+-ATPase in mouse testis tissue 12 h after irradiated with 0.025-0.200 Gy decreased compared with those with 0 Gy, the Na+-K+-ATPase activities of the cells irradiated with 0.05-0.200 Gy decreased significantly compared with those with 0 Gy (P2+-ATPase of the cells irradiated with 0.025-0.200 Gy decreased significantly compared with those with 0 Gy (P2+]i in mouse testis spermatogenic cells had similar dose-response relationship, [Ca2+]i after irradiated with 0.075 Gy decreased compared with those with 0 Gy (P+-K+-ATPase in mouse testis tissues decreased obviously compared with those at 0 h (P2+-ATPase in mouse testis tissues increased slightly at 3 h, then decreased at 6-24 h compared with those at 0 h (P2+]i in mouse testis spermatogenic cells had similar time course-response relationship, [Ca2+]i at 12 h decreased significantly compared with at 0 h (P2+]i induced by low dose radiation. (authors)

  16. Contributions of the Na⁺/K⁺-ATPase, NKCC1, and Kir4.1 to hippocampal K⁺ clearance and volume responses

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Assentoft, Mette; Cotrina, Maria L; Hua, Susan Z; Nedergaard, Maiken; Kaila, Kai; Voipio, Juha; MacAulay, Nanna

    2014-01-01

    (+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2β2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during...... neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K...

  17. Hsp70-Hsp40 chaperone complex functions in controlling polarized growth by repressing Hsf1-driven heat stress-associated transcription.

    Directory of Open Access Journals (Sweden)

    Aleksandar Vjestica

    Full Text Available How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments.

  18. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    Science.gov (United States)

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. PMID:27208492

  19. Chaperone-assisted translocation of flexible polymers in three dimensions

    CERN Document Server

    Suhonen, P M

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain $\\beta \\approx 1.26$ for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be ...

  20. Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Brasil, Sandra; Underhaug, Jarl; Ruíz-Sala, Pedro; Merinero, Begoña; Banerjee, Ruma; Desviat, Lourdes R; Ugarte, Magdalena; Martinez, Aurora; Pérez, Belén

    2013-09-15

    Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations. PMID:23674520

  1. 逆灸关元、命门穴对极限耐力运动大鼠NOS、钠-钾-ATP酶活性的影响%Effect of Preventive Moxibustion at Guanyuan(CV-4) or Mingmen(DU-4) on NOS and Na-K-ATPase Activity of Extreme Endurance Exercise Rats

    Institute of Scientific and Technical Information of China (English)

    罗丽; 李晓泓; 孙志芳; 莫捷; 朱世鹏; 丁娜; 章庆庆; 王洋; 张露芬

    2014-01-01

    目的:观察逆灸关元、命门穴对大鼠极限运动时的运动耐力及心肌一氧化氮合酶(NOS)、骨骼肌及心肌钠-钾-ATP酶活性的影响。方法:将大鼠按游泳能力随机分为正常对照组、力竭对照组、逆灸关元组、逆灸命门组、逆灸关元力竭组、逆灸命门力竭组,分别干预20 d后进行力竭游泳,观察力竭时间并检测相应的酶活性。结果:与正常组比较,力竭组钠-钾-ATP酶活性明显降低,NOS活性明显升高(P0.05)。与力竭组比较,逆灸关元、命门均可使力竭大鼠游泳时间延长,钠-钾-ATP酶活性增高(P0.05). Compared with the exhaustion group,preventive moxibustion at Guanyuan(CV-4) or Mingmen(DU-4) significantly increased swimming time of exhausted rats and Na-K-ATPase activity also increased significantly (P<0.05,P<0.01),preventive moxibustion Mingmen (DU-4) was significantly decreased NOS activity and there were significant differences between preventive moxibustion at Guanyuan(CV-4)(P<0.05,P<0.01). Conclusion:Preventive moxibustion at both Mingmen(DU-4) and Guanyuan(CV-4) could improve the exer-cise tolerance of rats and prolong their swimming exhausted time. The mechanism may be adjusted NOS and excited Na-K-ATPase activity.

  2. Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts

    OpenAIRE

    1990-01-01

    Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)- transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclast...

  3. Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon.

    Science.gov (United States)

    Wakai, Satoshi; Ohmori, Asami; Kanao, Tadayoshi; Sugio, Tsuyoshi; Kamimura, Kazuo

    2005-10-01

    ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively. PMID:16244438

  4. A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-01-01

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1. PMID:27499168

  5. A pivotal role of vacuolar H+-ATPase in regulation of lipid production in Phaeodactylum tricornutum

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-01-01

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H+-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1. PMID:27499168

  6. Golgi-associated LC3 lipidation requires V-ATPase in noncanonical autophagy.

    Science.gov (United States)

    Gao, Ying; Liu, Yajun; Hong, Liang; Yang, Zuolong; Cai, Xinran; Chen, Xiaoyun; Fu, Yuanyuan; Lin, Yujie; Wen, Weijie; Li, Sitong; Liu, Xingguo; Huang, Heqing; Vogt, Andreas; Liu, Peiqing; Yin, Xiao-Ming; Li, Min

    2016-01-01

    Autophagy is an evolutionarily conserved catabolic process by which cells degrade intracellular proteins and organelles in the lysosomes. Canonical autophagy requires all autophagy proteins (ATGs), whereas noncanonical autophagy is activated by diverse agents in which some of the essential autophagy proteins are dispensable. How noncanonical autophagy is induced and/or inhibited is still largely unclear. In this study, we demonstrated that AMDE-1, a recently identified chemical that can induce canonical autophagy, was able to elicit noncanonical autophagy that is independent of the ULK1 (unc-51-like kinase 1) complex and the Beclin1 complex. AMDE-1-induced noncanonical autophagy could be specifically suppressed by various V-ATPase (vacuolar-type H(+)-ATPase) inhibitors, but not by disturbance of the lysosome function or the intracellular ion redistribution. Similar findings were applicable to a diverse group of stimuli that can induce noncanonical autophagy in a FIP200-independent manner. AMDE-1-induced LC3 lipidation was colocalized with the Golgi complex, and was inhibited by the disturbance of Golgi complex. The integrity of the Golgi complex was also required for multiple other agents to stimulate noncanonical LC3 lipidation. These results suggest that the Golgi complex may serve as a membrane platform for noncanonical autophagy where V-ATPase is a key player. V-ATPase inhibitors could be useful tools for studying noncanonical autophagy. PMID:27512951

  7. A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yanaun Chen; Shuo Lin; Xiaodong Shu; Duanqing Pei; Bin Wu; Liangliang Xu; Huapeng Li; Jianhong Xia; Wenguang Yin; Zhuo Li; Dawei Shi; Song Li

    2012-01-01

    Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro,but the in vivo functions of them remain largely unknown.We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells.We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells.In zebrafish,SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs.Mechanistically,SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated.Like SNX10,V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10.We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a,which is a critical regulator of ciliary membrane extension.These results identify an SNX10/V-ATPaseregulated vesicular trafficking pathway that is crucial for ciliogenesis,and reveal that SNX10/V-ATPase,through the regulation of cilia formation in various organs,play an essential role during early embryonic development.

  8. Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells

    OpenAIRE

    Wang, Wan; Sreekumar, Parameswaran G.; Valluripalli, Vinod; Shi, Pu; Wang, Jiawei; Lin, Yi-An; Cui, Honggang; Kannan, Ram; Hinton, David R.; MacKay, J. Andrew

    2014-01-01

    αB-crystallin is a protein chaperone with anti-apoptotic and anti-inflammatory activity that is apically secreted in exosomes by polarized human retinal pigment epithelium. A 20 amino acid mini-peptide derived from residues 73-92 of αB-crystallin protects human retinal pigment epithelial (RPE) cells from oxidative stress, a process involved in the progression of age related macular degeneration (AMD). Unfortunately, due to its small size, its development as a therapeutic requires a robust con...

  9. [The lack of the effect of a strong constant magnetic field on isolated membrane preparations of Na,K-dependent ATPase].

    Science.gov (United States)

    Savich, M L; Nazarova, N M; Raĭkhman, L M; Kuznetsov, A N

    1985-01-01

    Effect of constant magnetic field (CMF) with induction 10 T on membrane preparations of Na,K-dependent ATPase of bovine brain (lipoproteid vesicules with 300-500 A diameter) were studied. No CMF effect on the activity of Na,K-dependent ATPase was observed under different experimental conditions (three temperature points 15, 20 and 37 degrees C and great variation of Na+,K+ concentrations ratio). CMF also produced no effect on the preparations of Na,K-dependent ATPase immobilized by adsorption on millipore filters. PMID:2983778

  10. 女贞子提取物对大强度耐力训练大鼠不同组织Mg2+-ATPase活性的影响%Effect of Fructus Ligustri Lucidi Extracts on Mg2+-ATPase Activity in Different Tissues of Rats of High-Intensity Endurance Training and Exercise Capacity

    Institute of Scientific and Technical Information of China (English)

    马云慧; 熊正英

    2013-01-01

    To analyze the change of Mg2+-ATPase activity in different tissue of rats like heart,liver,brain,kidney and quadriceps after the rats were fed with Fructus Ligustri Lucidi extracts (FLLE) and trained with high-intensity endurance training.To study the effect of FLLE as the exercise nutrition tonifying formula on the exercise capacity in rats.SD rats were randomly divided Sedentary control group,exercise control group and exercise + FLLE group,n =8.Exercise control group received high-intensity treadmill training for 6 weeks,exercise+FLLE group was fed with 2mL 400 mg/kg FLLE extract besides high-intensity treadmill training daily.Sedentary control group and exercise control group was given with 2 mL 0.5 % Tween-80 solution.After 6 weeks,sedentary control group was in rest state,and after exercise control and exercise+FLLE group were given an exhaustive exercise,determination of the different tissue Mg2+-ATPase activity of each group.The results show the exercise control group and exercise+ FLLE group of the different tissue Mg2+-ATPase activity was significantly lower than the sedentary control group,exercise+FLLE group of the different tissue Mg2+-ATPase activity was higher than the exercise control group (P<0.01 or P<0.05); with the sedentary control group,exercise control rat heart,liver,brain,kidney and quadriceps Mg2+-ATPase activity decreased by 21.01%,10.06 %,11.15 %,19.89 % and 12.36 %; with the exercise control group,exercise+FLLE group rat heart,liver,brain,kidney and quadriceps Mg2+-ATPase activity were increased by 21.62%,9.21%,8.24%,21.94% and 7.05%; compared with that of rats in the control group,the time of exhaustive exercise of rats in the exercise+FLLE group was prolonged by 23.09 %.That indicates,complementing FLLE can increase the concentration of antioxidants in the body,preventing oxidative damage to cell membranes,maintaining the steady-state concentrations of intracellular ion,and ensuring the mitochondria

  11. Reaction sequence and molecular mass of a Cl(-)-translocating P-type ATPase.

    OpenAIRE

    Gerencser, G A; Zelezna, B

    1993-01-01

    The basolateral membranes of Aplysia californica foregut absorptive cells contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these orthovanadate-sensitive activities were reconstituted into proteoliposomes. The reaction sequence kinetics were determined by [gamma-32P]ATP-induced phosphorylation of the reconstituted Cl- pump. Rapid phosphorylation and dephosphorylation kinetics of acyl phosphate bonding were confirm...

  12. Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

    Directory of Open Access Journals (Sweden)

    Seguro L.F.B.C.

    2003-01-01

    Full Text Available Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

  13. Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

    Science.gov (United States)

    Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

    2006-03-01

    Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

  14. Chaperone-assisted translocation of flexible polymers in three dimensions

    Science.gov (United States)

    Suhonen, P. M.; Linna, R. P.

    2016-01-01

    Polymer translocation through a nanometer-scale pore assisted by chaperones binding to the polymer is a process encountered in vivo for proteins. Studying the relevant models by computer simulations is computationally demanding. Accordingly, previous studies are either for stiff polymers in three dimensions or flexible polymers in two dimensions. Here, we study chaperone-assisted translocation of flexible polymers in three dimensions using Langevin dynamics. We show that differences in binding mechanisms, more specifically, whether a chaperone can bind to a single site or multiple sites on the polymer, lead to substantial differences in translocation dynamics in three dimensions. We show that the single-binding mode leads to dynamics that is very much like that in the constant-force driven translocation and accordingly mainly determined by tension propagation on the cis side. We obtain β ≈1.26 for the exponent for the scaling of the translocation time with polymer length. This fairly low value can be explained by the additional friction due to binding particles. The multiple-site binding leads to translocation the dynamics of which is mainly determined by the trans side. For this process we obtain β ≈1.36 . This value can be explained by our derivation of β =4 /3 for constant-bias translocation, where translocated polymer segments form a globule on the trans side. Our results pave the way for understanding and utilizing chaperone-assisted translocation where variations in microscopic details lead to rich variations in the emerging dynamics.

  15. PpiD is a player in the network of periplasmic chaperones in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Behrens-Kneip Susanne

    2010-09-01

    Full Text Available Abstract Background The inner membrane-anchored periplasmic folding factor PpiD is described as a parvulin-like peptidyl prolyl isomerase (PPIase that assists in the maturation of the major beta-barrel outer membrane proteins (OMPs of Escherichia coli. More recent work however, calls these findings into question. Here, we re-examined the role of PpiD in the E. coli periplasm by analyzing its functional interplay with other folding factors that influence OMP maturation as well as general protein folding in the periplasmic compartment of the cell, such as SurA, Skp, and DegP. Results The analysis of the effects of both deletion and overexpression of ppiD on cell envelope phenotypes revealed that PpiD in contrast to prior observations plays only a minor role, if any, in the maturation of OMPs and cannot compensate for the lack of SurA in the periplasm. On the other hand, our results show that overproduction of PpiD rescues a surA skp double mutant from lethality. In the presence of increased PpiD levels surA skp cells show reduced activities of both the SigmaE-dependent and the Cpx envelope stress responses, and contain increased amounts of folded species of the major OMP OmpA. These effects require the anchoring of PpiD in the inner membrane but are independent of its parvulin-like PPIase domain. Moreover, a PpiD protein lacking the PPIase domain also complements the growth defects of an fkpA ppiD surA triple PPIase mutant and exhibits chaperone activity in vitro. In addition, PpiD appears to collaborate with DegP, as deletion of ppiD confers a temperature-dependent conditional synthetic phenotype in a degP mutant. Conclusions This study provides first direct evidence that PpiD functions as a chaperone and contributes to the network of periplasmic chaperone activities without being specifically involved in OMP maturation. Consistent with previous work, our data support a model in which the chaperone function of PpiD is used to aid in the early

  16. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16

  17. Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase

    DEFF Research Database (Denmark)

    Ebbesson, Lars O E; Tipsmark, Christian K; Holmqvist, Bo;

    2005-01-01

    cells. Double labeling for NOS immunoreactivity and NKA alpha-subunit mRNA revealed cellular colocalization of NKA alpha-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a...... via intracellular, possibly both auto/paracrine, inhibition of Na(+),K(+)-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na(+),K(+)-ATPase activity...

  18. Direct observation of proton pumping by a eukaryotic P-type ATPase.

    Science.gov (United States)

    Veshaguri, Salome; Christensen, Sune M; Kemmer, Gerdi C; Ghale, Garima; Møller, Mads P; Lohr, Christina; Christensen, Andreas L; Justesen, Bo H; Jørgensen, Ida L; Schiller, Jürgen; Hatzakis, Nikos S; Grabe, Michael; Pomorski, Thomas Günther; Stamou, Dimitrios

    2016-03-25

    In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters. PMID:27013734

  19. Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

    Science.gov (United States)

    Andrés, María T; Acosta-Zaldívar, Maikel; Fierro, José F

    2016-07-01

    Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events. PMID:27139463

  20. Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines.

    Directory of Open Access Journals (Sweden)

    Michael Reidy

    2014-10-01

    Full Text Available Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

  1. S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase

    DEFF Research Database (Denmark)

    Rizza, Salvatore; Montagna, Costanza; Cardaci, Simone;

    2016-01-01

    . We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its......-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 1-13. ©2016 AACR....

  2. The evolutionary history of sarco(endoplasmic calcium ATPase (SERCA.

    Directory of Open Access Journals (Sweden)

    Ianina Altshuler

    Full Text Available Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+/K(+ transporters, H(+/K(+ transporters, and plasma membrane Ca(2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endoplasmic reticulum calcium ATPase (SERCA, which maintains calcium homeostasis in the cell by actively pumping Ca(2+ into the sarco(endoplasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

  3. Blocking the chaperone kinome pathway: Mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

    International Nuclear Information System (INIS)

    Research highlights: → Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. → Binding of WA to Cdc37 cleft suppresses its kinase binding activity. → 17-DMAG binding to the association complex results in H-bonds with 60% clustering. → The ligands' bound complex was found structurally and thermodynamically stable. -- Abstract: The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 'client' proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactions between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands' bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.

  4. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  5. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2008-01-01

    . Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na+ cycle from an Na+/K+ cycle in the pump. The Na+ cycle possibly involves transport through the recently characterized Na+-specific site...... regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive 22Na+ influx into K+ loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump....... A shift to such uncoupled mode is believed to produce pumps mediating uncoupled Na+ efflux by modifying the transport stoichiometry of single pump units. Udgivelsesdato: February 5...

  6. The chaperone like function of the nonhistone protein HMGB1

    Energy Technology Data Exchange (ETDEWEB)

    Osmanov, Taner; Ugrinova, Iva [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria); Pasheva, Evdokia, E-mail: eva@bio21.bas.bg [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria)

    2013-03-08

    Highlights: ► The HMGB1 protein strongly enhanced the formation of nucleosome particles. ► The target of HMGB1 action as a chaperone is the DNA not the histone octamer. ► The acetylation of HMGB1 decreases the stimulating effect of the protein. -- Abstract: Almost all essential nuclear processes as replication, repair, transcription and recombination require the chromatin template to be correctly unwound and than repackaged. The major strategy that the cell uses to overcome the nucleosome barrier is the proper removal of the histone octamer and subsequent deposition onto DNA. Important factors in this multi step phenomenon are the histone chaperones that can assemble nucleosome arrays in vitro in the absence of ATP. The nonhistone protein HMGB1 is a good candidate for a chaperone as its molecule consists of two DNA binding motives, Box’s A and B, and a long nonstructured C tail highly negatively charged. HMGB1 protein is known as a nuclear “architectural” factor for its property to bind preferentially to distorted DNA structures and was reported to kink the double helix. Our experiments show that in the classical stepwise dialysis method for nucleosome assembly the addition of HMGB1 protein stimulates more than two times the formation of middle-positioned nucleosomes. The stimulation effect persists in dialysis free experiment when the reconstitution is possible only in the presence of a chaperone. The addition of HMGB1 protein strongly enhanced the formation of a nucleosome in a dose dependant manner. Our results show that the target of HMGB1 action as a chaperone is the DNA fragment not the histone octamer. One possible explanation for the stimulating effect of HMGB1 is the “architectural” property of the protein to associate with the middle of the DNA fragment and to kink it. The acquired V shaped DNA structure is probably conformationals more favorable to wrap around the prefolded histone octamer. We tested also the role of the post

  7. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    Energy Technology Data Exchange (ETDEWEB)

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.; (Harvard-Med); (Brandeis)

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  8. Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth

    Science.gov (United States)

    Lv, Lei; Li, Dong; Zhao, Di; Lin, Ruiting; Chu, Yajing; Zhang, Heng; Zha, Zhengyu; Liu, Ying; Li, Zi; Xu, Yanping; Wang, Gang; Huang, Yiran; Xiong, Yue; Guan, Kun-Liang; Lei, Qun-Ying

    2016-01-01

    SUMMARY Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA. PMID:21700219

  9. Interplay between Molecular Chaperones and the Ubiquitin-Proteasome System in Targeting of Misfolded Proteins for Degradation

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl

    have been used, both in the yeast model organism S. pombe and in mammalian tissue culture cells. The articles included in this thesis cover four studies. The first study was focused on finding the specific UPS components involved in degradation of ubiquitin-fusion proteins and misframed ubiquitin. This...... involved in the specific degradation pathway were identified. This work had its origin in epistasis mapping of two Hsp70 co-chaperone proteins, which also formed the basis for the fourth study. In this final study, the Hsp70 co-chaperones were overexpressed in S. pombe. This led to a cell growth defect...... which, by RNA sequencing, was shown to be caused by a broad cellular stress response. In addition to these studies, this thesis contains a review article, covering the protein quality control systems active in the nucleus of yeast model systems and higher eukaryotes. In conjunction with the first study...

  10. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    OpenAIRE

    Mahmmoud, Yasser A.

    2008-01-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K+. Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, ...

  11. A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae*

    OpenAIRE

    Adle, David J.; Sinani, Devis; Kim, Heejeong; Lee, Jaekwon

    2006-01-01

    Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpe...

  12. Mitochondrial ultrastructural and atpase changes during the life cycle of Ascaris Suum

    OpenAIRE

    Rodrick, G E; S. D. Long; W. A. Sodeman Junior; Smith, D. L.

    1982-01-01

    Ultrastructural morphology and ATPase specific activities of mitochondria isolated from 1-celled fertilized egg, 10-day embryo, 21-day infective larvae and adult body wall muscle of Ascaris suum and rat liver were determined and compared. Although cristae of both muscle and egg mitochondria contained numerous elementary particles with head pieces of conventional diameter (85 A), each muscle mitochondrion contained relatively few, short cristae with a diminished frequency of elementary particl...

  13. Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain sensitive Na/K-ATPase

    OpenAIRE

    Smolyaninova, Larisa V.; Dergalev, Alexander A.; Kulebyakin, Konstantin Y.; Carpenter, David O.; Boldyrev, Alexander A.

    2012-01-01

    It is known that ouabain, a selective inhibitor of Na/K-ATPase, can cause not only activation of signal cascades, which regulate the cell viability, but also can cause free radical accumulation, which can evoke the oxidative stress. We have shown that nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The incr...

  14. The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider; Lange, Marianne; Ahring, Birgitte Kiær

    1999-01-01

    response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a...

  15. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase.

    Science.gov (United States)

    Mahmmoud, Yasser A

    2008-02-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+). Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive (22)Na(+) influx into K(+)-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na(+) cycle from an Na(+)/K(+) cycle in the pump. The Na(+) cycle possibly involves transport through the recently characterized Na(+)-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na(+) efflux by modifying the transport stoichiometry of single pump units. PMID:18230728

  16. 不同冷敏感型甘蔗茎尖Ca2+和Ca2+-ATP酶活性对低温的响应%Response of Ca2+ and Ca2+-ATPase activity in the stem tip of sugarcane to low temperature stress

    Institute of Scientific and Technical Information of China (English)

    李素丽; 杨丽涛; 李志刚; 李杨瑞; 韩春旺; 梁兆宙

    2011-01-01

    为了探明不同冷敏感型甘蔗品种茎尖Ca2+和Ca2+-ATP酶活性对低温胁迫的响应机制,本研究利用植物组织化学技术结合电子显微镜观察了2个不同冷敏感型甘蔗品种茎尖在低温胁迫前后Ca2+和Ca2+-ATP酶的变化规律.供试品种为桂糖28号(抗冷型)和园林6号(冷敏感型),在0℃条件下,分别处理0、2、4和6 d后切取茎尖进行组织化学定位,获得如下结果:1)低温胁迫前后桂糖28号形态和细胞结构变化不明显,但园林6号发生质壁分离现象,线粒体空泡化,细胞崩溃,组织水溃状明显;2)低温胁迫开始,2个甘蔗品种细胞质和细胞核Ca2+沉淀颗粒均增多,但随着低温胁迫时间的延长桂糖28号细胞质和细胞核Ca2+沉淀颗粒减少,并维持在一个低稳态水平,而园林6号细胞质和细胞核一直维持在高Ca2+浓度水平;3)低温对桂糖28号Ca2+-ATP酶活性与分布影响不大,其活性一直维持在较高水平,而园林6号Ca2+-ATP酶活性随着低温胁迫时间的延长而变弱.结果说明,低温条件下细胞质和细胞核Ca2+浓度增高是导致甘蔗茎尖细胞受伤害的重要原因,维持较高Ca2+-ATP酶活性有助于避免Ca2+-中毒.%Low temperature in winter is an adverse abjotic stress to sugarcane industry,and caused significant lose. In order to investigate the response of Ca2+ and Ca2+-ATPase activity in the stem tip of different cold sensitivity sugarcane cultivars, Electron microscope (EMS) and phytohistochemistry technology were employed to detect the changes of Ca2+ level and Ca2+ -ATPase activity in the stem tips of two sugarcane varieties YL6 (cold sensitive) and GT28 (cold resistant) before and after low temperature treatment. The plants were treated at 0 ℃ in fridge and samples were taken after treatment at 0,2,4 and 6 d, respectively. The results showed that there was no significant morphological difference and cellular structures in the stem tip of GT28 before and after low

  17. Chaperone use during intimate examinations in primary care: postal survey of family physicians

    Directory of Open Access Journals (Sweden)

    Upshur Ross EG

    2005-12-01

    Full Text Available Abstract Background Physicians have long been advised to have a third party present during certain parts of a physical examination; however, little is known about the frequency of chaperone use for those specific intimate examinations regularly performed in primary care. We aimed to determine the frequency of chaperone use among family physicians across a variety of intimate physical examinations for both male and female patients, and also to identify the factors associated with chaperone use. Methods Questionnaires were mailed to a randomly selected sample of 500 Ontario members of the College of Family Physicians of Canada. Participants were asked about their use of chaperones when performing a variety of intimate examinations, namely female pelvic, breast, and rectal exams and male genital and rectal exams. Results 276 of 500 were returned (56%, of which 257 were useable. Chaperones were more commonly used with female patients than with males (t = 9.09 [df = 249], p Conclusion Clinical practice concerning the use of chaperones during intimate exams continues to be discordant with the recommendations of medical associations and medico-legal societies. Chaperones are used by only a minority of Ontario family physicians. Chaperone use is higher for examinations of female patients than of male patients and is highest for female pelvic exams. The availability of a nurse in the clinic to act as a chaperone is associated with more frequent use of chaperones.

  18. Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Tu, S.I.; Nagahashi, G.; Brouillette, J.N.

    1987-08-01

    A tonoplast-type vesicle preparation, substantially free from other subcellular membranes, was obtained from corn roots by equilibrium sucrose density gradient centrifugation. At pH 6.5 and in the presence of chloride ions, the tonoplast-type ATPase activity as measured by Pi release, was inhibited by nitrate ions. The ATPase activity was insensitive to molybdate and vanadate, indicating a minimum nonspecific phosphatase and plasma membrane contamination. The vesicles exhibited an ATP hydrolysis-supported proton uptake which was measured by the absorption change of acridine orange. The ATP hydrolysis supported uptake and the subsequent perturbant-induced release of protons (decay) was described by a kinetic model which was previously developed to evaluate the coupling between proton pumping and the primary energy yielding process for other biomembranes. The proton pumping activity was more sensitive to nitrate ions then was ATP hydrolysis. The differential effect and the kinetic analysis of nitrate inhibition led us to suggest that (i) the coupling between Pi release and proton pumping was indirect in nature and (ii) the primary inhibitory effect of nitrate ion was originated from an interaction with a protogenic protein domain which is functionally linked to the ATPase in the tonoplast-type membrane.

  19. Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor.

    Science.gov (United States)

    Goldgof, Gregory M; Durrant, Jacob D; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M; Manary, Micah J; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W; Slayman, Carolyn W; Amaro, Rommie E; Suzuki, Yo; Winzeler, Elizabeth A

    2016-01-01

    The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type