Fluorescent ATP Analogue Research Articles

Cy3-ATP labeling of unfixed, permeabilized mouse hair cells

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

ABSTRACTThe bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

The role of the super-relaxed state of myosin in human metabolism

BackgroundThe super-relaxed state of myosin (SRX) plays a fundamental role in maintaining the low resting metabolic rate of skeletal muscle. Our previous work on this state has been in animal models. Piperine is a small molecule that has been shown to destabilize the SRX in rabbit fast twitch fibers. MethodsHere we extend this work to human muscle obtained from biopsies of the vastus lateralis of both lean and obese subjects. The slow release of nucleotides by myosin in the SRX was measured by incubating permeable fibers in a fluorescent analog of ATP and chasing with ATP. ResultsThe fraction of myosin heads in the SRX was 0.48 ± 0.04 with a lifetime of 148 ± 5 s in lean subjects and a fraction of 0.41 ± 0.05 and a lifetime of 176 ± 7 s in obese subjects. Addition of 100 μM piperine decreased the SRX population by 25 ± 4% in lean subjects and 21 ± 4% in obese subjects, with little change in lifetimes. Addition of piperine to human cardiac cells had no effect on the SRX, a requirement for a drug to treat metabolic diseases. ConclusionsIn human muscle the SRX and its responses to piperine are similar to those seen previously, with no significant differences between muscles from lean and obese subjects. Thus analogs of piperine that have greater specificity could provide effective treatment for metabolic diseases. The SRX provides a potential mechanism contributing to the large dynamic range of metabolic rate.

ATP-binding affinity of the ε subunit of thermophilic F1-ATPase under label-free conditions

The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem.282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2′(3′)-O-N′-methylaniloyl-aminoadenosine-5′-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.

In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD.

One of the major histopathological hallmarks of Parkinson's disease are Lewy bodies (LBs) -cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction in α-syn fibrillation in vitro upon posttranslational modification (PTM) by the Fic (Filamentation induced by cAMP) family adenylyltransferase HYPE/FICD (Huntingtin yeast-interacting protein E/FICD). Specifically, HYPE utilizes ATP to covalently decorate key threonine residues in α-syn's N-terminal and NAC (non-amyloid-β component) regions with AMP (adenosine monophosphate), in a PTM termed AMPylation or adenylylation. Status quo in vitro AMPylation reactions of HYPE substrates, such as α-syn, use a variety of ATP analogs, including radiolabeled α-32P-ATP or α-33P-ATP, fluorescent ATP analogs, biotinylated-ATP analogs (N6-[6-hexamethyl]-ATP-Biotin), as well as click-chemistry-based alkyl-ATP methods for gel-based detection of AMPylation. Current literature describing a step-by-step protocol of HYPE-mediated AMPylation relies on an α-33P-ATP nucleotide instead of the more commonly available α-32P-ATP. Though effective, this former procedure requires a lengthy and hazardous DMSO-PPO (dimethyl sulfoxide-polyphenyloxazole) precipitation. Thus, we provide a streamlined alternative to the α-33P-ATP-based method, which obviates the DMSO-PPO precipitation step. Described here is a detailed procedure for HYPE mediated AMPylation of α-syn using α-32P-ATP as a nucleotide source. Moreover, our use of a reusable Phosphor screen for AMPylation detection, in lieu of the standard, single-use autoradiography film, provides a faster, more sensitive and cost-effective alternative.

Biochemical characterization of the mouse ABCF3 protein, a partner of the flavivirus-resistance protein OAS1B

Mammalian ATP-binding cassette (ABC) subfamily F member 3 (ABCF3) is a class 2 ABC protein that has previously been identified as a partner of the mouse flavivirus resistance protein 2',5'-oligoadenylate synthetase 1B (OAS1B). The functions and natural substrates of ABCF3 are not known. In this study, analysis of purified ABCF3 showed that it is an active ATPase, and binding analyses with a fluorescent ATP analog suggested unequal contributions by the two nucleotide-binding domains. We further showed that ABCF3 activity is increased by lipids, including sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine. However, cholesterol inhibited ABCF3 activity, whereas alkyl ether lipids either inhibited or resulted in a biphasic response, suggesting small changes in lipid structure differentially affect ABCF3 activity. Point mutations in the two nucleotide-binding domains of ABCF3 affected sphingosine-stimulated ATPase activity differently, further supporting different roles for the two catalytic pockets. We propose a model in which pocket 1 is the site of basal catalysis, whereas pocket 2 engages in ligand-stimulated ATP hydrolysis. Co-localization of the ABCF3-OAS1B complex to the virus-remodeled endoplasmic reticulum membrane has been shown before. We also noted that co-expression of ABCF3 and OAS1B in bacteria alleviated growth inhibition caused by expression of OAS1B alone, and ABCF3 significantly enhanced OAS1B levels, indirectly showing interaction between these two proteins in bacterial cells. As viral RNA synthesis requires large amounts of ATP, we conclude that lipid-stimulated ATP hydrolysis may contribute to the reduction in viral RNA production characteristic of the flavivirus resistance phenotype.

The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.

Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter

Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a Kd of 4.8 μM. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH.

The archaellum, the rotating motility structure of archaea, is best studied in the crenarchaeon Sulfolobus acidocaldarius. To better understand how assembly and rotation of this structure is driven, two ATP-binding proteins, FlaI and FlaH of the motor complex of the archaellum of the euryarchaeon Pyrococcus furiosus, were overexpressed, purified and studied. Contrary to the FlaI ATPase of S. acidocaldarius, which only forms a hexamer after binding of nucleotides, FlaI of P. furiosus formed a hexamer in a nucleotide independent manner. In this hexamer only 2 of the ATP binding sites were available for binding of the fluorescent ATP-analog MANT-ATP, suggesting a twofold symmetry in the hexamer. P. furiosus FlaI showed a 250-fold higher ATPase activity than S. acidocaldarius FlaI. Interaction studies between the isolated N- and C-terminal domains of FlaI showed interactions between the N- and C-terminal domains and strong interactions between the N-terminal domains not previously observed for ATPases involved in archaellum assembly. These interactions played a role in oligomerization and activity, suggesting a conformational state of the hexamer not observed before. Further interaction studies show that the C-terminal domain of PfFlaI interacts with the nucleotide binding protein FlaH. This interaction stimulates the ATPase activity of FlaI optimally at a 1:1 stoichiometry, suggesting that hexameric PfFlaI interacts with hexameric PfFlaH. These data help to further understand the complex interactions that are required to energize the archaellar motor.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires’ Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the ‘open’ conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

Probing Protein Kinase-ATP Interactions Using a Fluorescent ATP Analog.

Eukaryotic protein kinases are an intensely investigated class of enzymes which have garnered attention due to their usefulness as drug targets. Determining the regulation of ATP binding to a protein kinase is not only critical for understanding function in a cellular context but also for designing kinase-specific molecular inhibitors. Here, we provide a general procedure for characterizing ATP binding to eukaryotic protein kinases. The protocol can be adapted to identify the conditions under which a particular kinase is activated. The approach is simple, requiring only a fluorescent ATP analog such as TNP-ATP or MANT-ATP and an instrument to monitor changes in fluorescence. Although the interaction kinetics between a kinase and a given ATP analog may differ from that of native ATP, this disadvantage is offset by the ease of performing and interpreting this assay. Importantly, it can be optimized to probe a large variety of conditions under which the kinase-nucleotide binding might be affected.

Kinetic Characterization of Novel Rice Plant Kinesin E11

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures. In this study, we characterized biochemical and kinetic properties of another rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E.coli expression system and purified with Co-chelate column. The fluorescent ATP analogue, Mant-ATP was employed for the kinetic study. We observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters were analyzed by monitoring the FRET using stopped flow apparatus. The binding rate and dissociation rate were measured, and compared with those of other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are faster than those of other rice plant specific kinesin. We also examined the motility activity of E11 with in vitro microtubule gliding assay. The motility assay revealed that E11 has relatively higher motor activity than other rice kinesins.

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase

Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.

Abstract 1173: Extracellular ATP is internalized through not only macropinocytosis but also other endocytic process and promotes growth of human lung cancer cells with Ras mutations

Abstract Within a tumor, extracellular ATP (exATP) concentrations are in the range of several hundred micromolars or higher, 103-104 times higher than those in normal tissues1. We recently showed that extracellular ATP, within the intratumoral concentrations, was internalized by Ras-mutated (Rasmut) A549 human lung cancer cells through macropinocytosis, elevated intracellular ATP levels and rescued cancer cells under various stresses2. However, macropinocytosis3 alone could not account for all the elevated intracellular ATP increase. Here, we show that other endocytic processes, in addition to macropinocytosis, also contributed to the internalization of exATP in A549 and H1299 human lung cancer cells. Cell viability assays, intracellular ATP luminescence measurement assays, fluorescent microscopy were used in this study. First, high-molecular-weight (HMW) dextran uptake assay indicated that A549 and H1299, both Rasmut cancer cells, exhibited a phenotype of macropinocytosis. The colocalization of the red fluorescent HMW dextran and green nonhydrolyzable ATP analog inside the cells indicated that extracellular ATP was internalized by macropinocytosis. In contrast, significantly fewer macropinosomes were found in NL-20 noncancerous lung cells. In addition, the colocalization of low-molecular-weight (LMW) dextran, an endocytic tracer, and ATP analog indicated the internalization of exATP through non-macropinocytic-endocytosis in both lung cancer cell lines. Interestingly, when both red HMW dextran and green LMW dextran were applied to cancer cells, not only colocalization, but also some green-only endosomes were observed, strongly suggesting that both endocytic processes were responsible for exATP internalization. Collectively, these data indicated for the first time that other endocytic processes in addition to macropinocytosis contributed to the internalization of exATP in human lung cancer cells with Rasmut. This finding may significantly enhance our understanding of energy hemostasis within cancer cells and the Warburg effect, which in turn facilitates the development of anticancer therapeutics targeting the endocytic processes and Ras mutations as well as novel anticancer strategies. 1. Pellegatti, et al. (2008). PLoS ONE 3, e2599. 2. Qian, et al. (2014). Cancer Letters 351, 242-251. 3. Commisso, et al. (2013). Nature 497, 633-637. Citation Format: Yanrong Qian, Xuan Wang, Yunsheng Li, Yanyang Cao, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP is internalized through not only macropinocytosis but also other endocytic process and promotes growth of human lung cancer cells with Ras mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2015-1173

Kinetic Characterization of Rice Plant Specific Kinesin E11 using Fluorescent ATP Analogue

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures.In this study, we characterized rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E. coli expression system and purified with Co-chelate column in order to characterize biochemical and ATPase kinetic properties. The fluorescent ATP analogues, Mant-ATP was employed for the kinetic characterization. We have successfully observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters of initial binding of Mant-ATP to E11 and release of Mant-ADP from E11 were analyzed by monitoring the FRET using stopped flow apparatus and compared with other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are slower than those of other rice plant specific kinesin.

Abstract 5257: Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis

Abstract Cancer cells are known to exhibit reprogrammed metabolism and prefer to use glycolygsis for their ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. However, cancer cells are also known for more rapid growth and proliferation, therefore requiring more ATP than normal cells of the same tissues. How cancer cells manage to use low efficiency glycolysis but still meet their higher ATP demands is enigmatic. In our previous study, we found that extracellular ATP induced large intracellular ATP increase and drug resistance to tyrosine kinase inhibitors (TKIs) that function as ATP analogs / competitors in cancer cells. The induced ATP increase was from neither OXPHOS nor glycolysis, and was not dependent on AMPK, the master ATP regulator. Furthermore, the increase did not involve new gene expression or new protein synthesis. Recently, it was reported that cells transformed with an oncogenic form of KRas gene (KRas+) exhibit a phenotype of macropinocytosis, a type of endocytosis. KRas-induced macropinocytosis nonspecifically takes up extracellular proteins. All these led us to hypothesize that the increased intracellular ATP induced by extracellular ATP is, at least in part, not produced intracellularly but is transported into cancer cells. To test this hypothesis, high molecular weight fluorescent dextran was use to determine if KRas+ human non-small cell lung cancer A549 cells exhibit macropinocytosis. Nonhydrolyzable fluorescent ATP analog was used to visualize if the ATP analog can be transported into A549 cells along with dextran. Fluorescence microscopy studies revealed that A549 cells exhibit the phenotype of macropinocytosis, which can be reduced by a macropinocytosis inhibitor EIPA. HMW dextran and the nonhydrolysable fluorescent ATP analog were found co-localized inside A549 cells, demonstrating that they were transported into A549 cells via macropinosomes. It was also found that the ATP increase cannot be explained by purinergic receptor signaling alone. In comparison, KRas- human breast cancer MCF7 cells do not take up HMW dextran, indicating that they do not exhibit macropinocytosis. All these provide strong and visual evidence that ATP can be transported into KRas+ A549 cancer cells by macropinocytosis. Other experiments using radioactive ATP or purinergic receptor inhibitors also strongly support this conclusion. This is the first time that ATP (ATP analog) is shown to be taken into cancer cells. This finding will have major impact on how we view cancer cells in terms of their energy exchange with other cancer or noncancer cells in the tumor and on the Warburg effect. It will also have profound implications on how to more effectively inhibiting KRas+ or KRas- cancer cells by targeting ATP transport and ATP metabolism. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Xiaozhuo Chen. Cancer cells have not only sweet tooth but also large mouths: Evidence for uptake of ATP into KRas oncogene-expressing cancer cells by macropinocytosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5257. doi:10.1158/1538-7445.AM2014-5257

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.