Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and

Articles pubs.acs.org/acschemicalbiology

Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs Stephan M. Hacker, Annette Buntz, Andreas Zumbusch,* and Andreas Marx* Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany S Supporting Information *

ABSTRACT: Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

A

based on the complex formation of phosphate with molybdate have been developed.9 The resulting anion is detected either by reduction to molybdenum blue (PMo12O407−)10 or by complexation with organic dyes like malachite green, which results in a change of the absorption characteristics of the dye.11 These methods require additional reagents or separation steps that prohibit continuous monitoring of ATP turnover. A possible alternative is the detection of phosphate released in ATP turnover with an engineered fluorogenic phosphate sensor based on a phosphate-binding protein of E. coli.12 Obviously, however, this method requires strictly phosphate-free conditions, which limits its applicability. A second approach for the detection of ATP turnover in vitro relies on processing of primary reaction products of ATP by subsequent enzymatic reactions.13,14 One example is the reconversion of adenosine diphosphate (ADP) into ATP by, for example, pyruvate kinase, which is accompanied by the formation of pyruvate from phosphoenolpyruvate.13 Pyruvate is next reduced to lactate with the help of lactate dehydrogenase and NADH yielding NAD+. The depletion of NADH can be monitored spectroscopically by the loss of its absorption at 340

denosine-containing nucleotides are of pivotal importance in a number of basic biological processes. It is well-known that adenosine triphosphate (ATP) is the universal energy currency of every living organism where it couples endergonic and exergonic processes.1 ATP is also a key player in posttranslational protein modification.2−4 In this context, it acts, for example, as cofactor for the phosphate transfer by protein kinases2 and for the transfer of an adenosine monophosphate moiety to target proteins by adenylylating enzymes.3,4 Furthermore, ATP is the starting material for the formation of other important adenosine-based nucleotides like the second messengers cyclic adenosine monophosphate (cAMP),5 cyclic di-AMP (c-diAMP),6 diadenosine triphosphate (Ap3A),7 and diadenosine tetraphosphate (Ap4A).7 Due to the significance of all of these adenosine nucleotides, it would be very desirable to have methods at hand that allow studying their processing in complex environments like cell extracts or intact human cells. Consequently, numerous research efforts have lately been dedicated to the development of such methods, especially for ATP. One approach is based on the direct detection of the reaction products of ATP processing. This can be performed, for example, using radioactively labeled ATP.8 In this case, the products of ATP cleavage are separated by chromatographic means and detected by autoradiography. In order to circumvent the hazards of radioactive compounds, alternative methods © XXXX American Chemical Society

Received: June 15, 2015 Accepted: August 14, 2015

A

DOI: 10.1021/acschembio.5b00459 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology nm. In a similar manner, different enzyme-based reactions that convert phosphate generated in ATP turnover are employed.14 Thus, purine-nucleoside phosphorylase transforms the synthetic substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) to 2-amino-6-mercapto-7-methylpurine and ribose-1-phosphate in a phosphate-dependent fashion.14 Here, the change of absorption at 360 nm that is characteristic for 2amino-6-mercapto-7-methylpurine is used for the detection of ATP cleavage. However, since phosphate or ADP are intrinsically formed by endogenous sources and therefore are present in cells or cell extracts, applications of these approaches are limited. Methods based on similar detection systems have also been developed for other adenosine-based nucleotides like cAMP,15 Ap3A,16 and Ap4A.17 Studying the turnover of diadenosine polyphosphates is especially interesting because these signaling molecules are involved in cellular stress response. A better understanding of the human Ap3A hydrolase Fhit18 and the human Ap4A hydrolase NudT219 might thus help to gain novel insights into this cellular protection mechanism. Additionally to the described approaches, fluorogenic methods were applied to these enzymes20−23 and showed their potential especially in the case of Fhit, where they were used to detect the activity of endogenous Fhit in cell lysates.20 In addition to the known fluorogenic dinucleoside polyphosphate analogs, fluorogenic nucleotide analogs based on ATP would be highly desirable. Compounds of this type would allow the direct detection of their cleavage without the use of external enzymes or reagents and might act as substrates for various enzymes that cleave adenosine nucleotides. We have recently reported the synthesis of novel fluorogenic ATP analogs (Scheme 1a), which have demonstrated their usefulness in several applications, for example, employing recombinant enzymes or bacterial cell lysates.24−26 In this concept, ATP is equipped with two fluorescent dyes, a donor fluorophore (D) and an acceptor fluorophore (A). One of these dyes is attached to the adenosine moiety of ATP and the other to the phosphate chain. In this way, excitation of the donor dye leads to Förster resonance energy transfer (FRET) to the acceptor dye. Therefore, the fluorescence of the donor is reduced, and fluorescence of the acceptor is observed. After cleavage of the ATP analog, the two fluorophores are separated, FRET is impeded, and the direct emission of the donor is restored. So far, this concept has been used to detect the activity of the human ubiquitin-activating enzyme UBA124 and the phosphodiesterase I of Crotalus adamanteus (SVPD),25 as well as to shed light on a new ATP-dependent metabolic pathway in extracts of Desulfococcus biacutus.26 Nevertheless, in contrast to the specialized Ap3A probe, up to now the potential of these ATP analogs for studying processes in lysates of human cells has not been explored. Furthermore, it has not been examined whether this concept can be extended to studies in intact human cells. Here we report on the observation of turnover of doubly dye-modified ATP analogs in human cell lysates as well as in human cells. For these investigations, we chose to employ an O2′-modified ATP analog 1 and an O2′-modified adenosine tetraphosphate (Ap4) analog 2 since it was demonstrated that several ATP-cleaving enzymes tolerate modifications at these sites.24,27 Furthermore, the attachment of the modifications at the phosphate chain via ester bonds is advantageous due to the stability over a wide range of conditions.28 For comparison, a control (3) that does not contain a nucleotide-based cleavage

Scheme 1. Concept of the Fluorogenic Nucleotide Analogs Used in This Studya

a

(a) In the noncleaved state of the molecules, FRET occurs between the donor fluorophore (D) sulfo-Cy3 and the acceptor fluorophore (A) sulfo-Cy5. After enzymatic turnover, FRET is impeded. The decrease of FRET efficiency is a measure for enzymatic activity. (b) Compounds used in this study.

motif (Scheme 1b) was included in this study. As FRET pair, we chose the well-established cyanine dyes sulfo-Cy3 and sulfoCy5.25 We find that a single enzyme, namely, NudT2, is responsible for processing of 2 in the investigated human cell lysates. Additionally, we report on the successful internalization of the ATP analogs into HeLa cells by electroporation and their turnover in cells. This study underlines the potential of the depicted ATP analogs as powerful tools for exploring nucleotide turnover in the context of complex systems like cell lysates and whole cells.

■

RESULTS AND DISCUSSION Synthesis of ATP Analogs. Whereas ATP analog 1 was synthesized before,25 the syntheses for Ap4 analog 2 and the control 3 were established (Scheme 2). For the synthesis of 2, we started with O2′-(6-trifluoroacetamido)-adenosine triphosphate (4)27 and reacted it with 6-azidohexyl phosphate28 in order to obtain the doubly modified Ap4 analog 5 in 34% yield. The trifluoroacetamide of this compound was cleaved with sodium hydroxide, and the resulting amine was reacted in situ with the N-hydroxysuccinimide (NHS) ester of sulfo-Cy5 to give 6 in a yield of 53%. The azide of 6 was reduced using the Staudinger reaction29,30 resulting in a yield of 76% of 7. Finally, 7 was reacted with the sulfo-Cy3 NHS ester to give the final compound 2 in a yield of 39%. For the synthesis of the control compound 3, sulfo-Cy5 NHS ester was reacted with ethylene diamine to yield compound 8 in 57%. This molecule was B


Articles

ACS Chemical Biology Scheme 2. Synthesis of Molecules 2 and 3a

Conditions: (a) (i) EDC-HCl, DMF, RT, 2.5 h, (ii) methanol, RT, 3 h, (iii) 6-azido-hexyl phosphate, triethylamine, DMF, 40 °C, 12 h, 34%; (b) (i) 0.1 M aqueous NaOH, RT, 4 h, (ii) sulfo-Cy5-NHS ester, 0.1 M aqueous NaHCO3 (pH 8.7), DMF, RT, 12 h, 53%; (c) TCEP-HCl, water, methanol, triethylamine, RT, 12 h, 76%; (d) sulfo-Cy3-NHS ester, 0.1 M aqueous NaHCO3 (pH 8.7), DMF, RT, 12 h, 39%; (e) ethylene diamine, 0.1 M NaHCO3 (pH 8.7), DMF, RT, 12 h, 57%; (f) sulfo-Cy3-NHS ester, 0.1 M NaHCO3 (pH 8.7), DMF, RT, 12 h, 83%. a

To better understand the turnover of Ap4 analog 2, we next investigated the effect of 14 molecules known to inhibit nucleotide-dependent processes24,31−43 in HEK 293T and HeLa lysates (Figure 1c and Figure S9). We observed that the turnover of 2 is completely inhibited by POM 1 (sodium polyoxotungstate, 3Na2WO4·9WO3·H2O) and that protoporphyrin IX (PPIX) and suramin show a partial inhibition at 100 μM. All other compounds exhibit no inhibition at this concentration. Furthermore, the inhibitor profile was almost identical in both cell lines. The complete inhibition by POM 1 shows that the turnover of 2 is indeed an enzymatic activity and does not result from intrinsic chemical instability of compound 2. Furthermore, it suggests that the turnover of 2 is caused by a limited number of similar enzymes or even a single enzyme. The inhibition of the turnover of analog 2 by the same inhibitors in both cell lines additionally indicates that the same enzymatic activity is observed in both cell lines. Therefore, we next set out to identify the enzyme(s) responsible for the turnover of compound 2 in lysates of human cell lines. The structural motif of a δ-modified Ap4, as present in compound 2, is rarely present in natural products. To our knowledge, it is solely present in the second messenger Ap4A7 and related dinucleoside tetraphosphates. We therefore reasoned that the key enzyme that is responsible for Ap4Acleavage in human cells, namely, the human Ap4A hydrolase NudT2,19 might be responsible for the cleavage of compound 2 in cell lysates. To investigate whether the observed hydrolysis can be attributed to this enzyme, we next checked for its expression in the used cell lines (Figure 2a). Indeed, all studied cell lines contain detectable levels of the NudT2 protein. Furthermore, whereas all other cell lines show comparable expression of NudT2 (Figure 2a) and comparable cleavage rates of compound 2 (Figure 1b), the U2OS cell line shows reduced NudT2 expression and reduced cleavage activity. The expression levels of NudT2 therefore corroborate the hypothesis that NudT2 is involved in the turnover of 2 in lysates of human cell lines.

reacted with sulfo-Cy3 NHS ester to yield 83% of 3. Measurements of the fluorescence characteristics of the analogs (Figures S1−S3) before and after treatment with phosphodiesterase I from C. adamanteus (snake venom phosphodiesterase, SVPD)25 reveal that both nucleotide analogs (1 and 2) undergo significant changes in their fluorescence characteristics upon cleavage of the phosphate chain and are therefore useful tools to study enzymatic nucleotide hydrolysis. Investigations of Nucleotide Analogs in Cell Lysates. Having compounds 1−3 in hand, we set out to explore their action in lysates of human cell lines. We decided to investigate the human cell lines HEK 293T, HeLa, HaCaT, H1299, and U2OS to gain insight into the generality of the findings. All cell lines were grown to confluence, harvested, and lysed using sonication. In order to monitor the turnover of the analogs, we incubated them with the cell lysates and continuously measured the fluorescence intensity of the fluorescence donor sulfo-Cy3, which rises upon cleavage. All data points were used to calculate turnover of the compounds in comparison to a completely intact and completely cleaved control (Figure 1a and Figure S4 − S8). Turnover rates were calculated by linear fitting of the amount of turnover plotted against the time (Figure 1b). Compound 3, serving as a control that does not contain a nucleotide-based cleavage motif, is stable under the applied conditions. This shows that the amide bonds used for the attachment of the dyes and the dyes themselves are stable. Therefore, any turnover observed with the other analogs is attributed to cleavage of the nucleotide motif. Interestingly, compound 1 and compound 2 showed markedly different behavior in these experiments. Whereas ATP analog 1 is stable under the conditions applied, Ap4 analog 2 is rapidly turned over. This finding was observed in all cell lines studied and indicates that this difference in stability is of general nature. Furthermore, this result shows that the ATP analog 1 is not processed to a significant degree by any enzyme present in the lysate. C


Articles

ACS Chemical Biology

Figure 2. Investigation of the role of the enzyme NudT2 in the turnover of 2 in lysates: (a) Western blot of the lysates of different cell lines stained either by Coomassie or by immunoblotting against NudT2 (α-NudT2). Numbers underneath the respective panels depict the relative intensity in each lane. (b) Turnover of ATP analog 1, Ap4 analog 2, and the control 3 by recombinant and purified NudT2. (c) Inhibition of the turnover of Ap4 analog 2 by purified recombinant NudT2 by a selection of compounds known to inhibit nucleotidedependent processes.24,31−43 Indicated compounds at 10 μM concentration were used in the experiments. All data represent mean ± standard error of triplicates.

Figure 1. Turnover of ATP analog 1, Ap4 analog 2, and the control 3 in lysates of human cell lines: (a) representative time courses for compounds 1−3 (10 μM) in the lysate of HEK293T cells (0.25 mg mL−1 protein concentration); (b) specific activity (SA) of the turnover of the molecules in lysates of five different cell lines; (c) inhibition of the turnover of Ap4 analog 2 in lysates of the cell lines HEK293T and HeLa by a selection of compounds known to inhibit nucleotidedependent processes.24,31−43 Indicated compounds at 100 μM concentration were used in the experiments. All data represent mean ± standard error of triplicates.

(Figure 2c and Figure S11) resembles the inhibition profile obtained from cell lysates. This indicates that NudT2 is the main enzyme responsible for turnover of 2 in lysates. In addition to the inhibitors identified before, β-lapachone was identified to inhibit recombinant NudT2-promoted cleavage of 2, but not the cleavage of 2 in cell lysates. The free concentration of β-lapachone in cell lysates might be reduced by other processes, explaining why the inhibition of NudT2 is not observed in cell lysates. Earlier studies demonstrated that also the inhibition of UBA1 by β-lapachone in cell lysates

In order to obtain further insight concerning whether compound 2 is a substrate of NudT2, we next investigated the activity of recombinant NudT2 (Figure 2b and Figure S10). Whereas ATP-analog 1 and the control 3 withstand NudT2 treatment, the Ap4 analog 2 is rapidly turned over by NudT2. The inability of NudT2 to cleave the ATP analog 1 is in line with earlier findings reporting that NudT2 is unable to hydrolyze Ap3A. It cleaves nucleotides between the γ- and δphosphate,44 which is not present in Ap3A and in analog 1. The inhibitor profile for the turnover of 2 by recombinant NudT2 D


Articles


found in certain vesicles. Electroporation is therefore a suitable method for delivery of the nucleotide analogs into cells and opens way to study their behavior in a cellular context. Next, we investigated whether FRET within 1−3 can be observed intracellularly. For this purpose, we performed an acceptor photobleaching experiment.48 In this experiment, the FRET efficiency is determined by the increase of donor fluorescence after acceptor photobleaching. For all analogs, high FRET efficiencies of more than 60% were observed in cells (Figure 4a and Figure 5). This demonstrates that monitoring of FRET is well suited for studying the behavior of the analogs in cellular environments and that after the recovery time of the electroporation a large fraction of the analogs is still not cleaved. In order to further investigate the fate of 1−3 in cells after internalization, we subsequently performed a time-dependent experiment. For this purpose, the cells were kept in an incubator for indicated times after the recovery period of the electroporation before fixation (Figures 4b and 5). For the control 3, the FRET efficiency is unaltered over time. This demonstrates that the dyes and the amide bonds used for their connection are also stable in the intracellular environment. Therefore, a decrease in FRET efficiency can be attributed to cleavage of the nucleotide motif also under these conditions. For the ATP analog 1 and the Ap4 analog 2, a decrease of the FRET efficiency is observed over time. Cleavage of the nucleotide motif is therefore also detectable in cells. Also in intact cells, turnover of 2 is the fastest. However, 1 is also cleaved to a significant extent, as opposed to the experiments in cell lysates. Thus, a decrease in FRET efficiency can be detected for both nucleotide-based analogs but not for the control 3. This finding demonstrates that the analogs are indeed excellent tools for studying nucleotide hydrolysis in cells. The fact that cleavage of the Ap4 analog 2 is the fastest process in cells indicates that NudT2 also contributes here to the turnover of 2 as it does in lysates. Nevertheless, the hydrolysis of 1 in this context demonstrates that the cell lysate studies do not reveal all activities that contribute to turnover of the nucleotide analogs in an intact cell. This might on the one hand be due to loss of some enzymes or some activities during the preparation of the cell lysates. On the other hand, it may also be a consequence of the different microenvironments in the cell and the strongly varying conditions found in different cellular compartments. In line with the hypothesis that the difference in behavior in lysates and in cells is at least partly caused by compartmentalization, we observed that with increasing incubation time the analogs accumulate in vesicular structures (Figure 4b). Earlier studies have shown that certain cells can store ATP into acidic vesicles after uptake.49 To investigate whether the analogs used here resemble the behavior of ATP in this instance, we performed a colocalization experiment (Figure 6) with two dyes, namely, quinacrine dihydrochloride50 and Lysotracker green,51 which show fluorescence in acidic compartments. Indeed, colocalization of the acceptor fluorescence of the analog 2 with both of these dyes is observed. The used analogs therefore localize from the cytosol to acidic vesicles over time after electroporation. These findings also underline the importance of designing the analogs in a way that they are stable to nonenzymatic cleavage over a wide range of pH conditions.28 To date, we cannot distinguish whether intact analog 2 is transported into these vesicles and cleaved there or

required significantly higher concentrations than inhibition of the isolated enzyme.24 To further corroborate the responsibility of NudT2 for the cleavage of Ap4 analog 2 in cell lysates, we finally performed siRNA experiments to knock down NudT2 levels in HeLa and HEK 293T cells (Figure 3 and Figure S12). Indeed, in both cell

Figure 3. siRNA experiment for NudT2. (a) Western blot of lysates of HeLa cells treated with either siRNA directed against the mRNA of NudT2 or control siRNA. Blots were stained by immunoblotting against tubulin (α-tubulin) and NudT2 (α-NudT2). Numbers underneath the respective panels depict the relative intensity in each lane. (b) Specific activity of the turnover of Ap4 analog 2 in the same lysates as used in part a. All data represent mean ± standard error of triplicates.

lines a reduced level of NudT2 expression was observed after treatment with the specific siRNA directed against the mRNA for NudT2 (Figure 3a and Figure S12a). The degree of the reduction of NudT2 protein levels correlates with the degree of reduction in the cleavage rate of 2 (Figure 3b and Figure S12b). Taken together, the acceptance of 2 as substrate of recombinant NudT2 together with the similar inhibitor profiles of its turnover by recombinant NudT2 and in cell lysates as well as siRNA experiments strongly suggest that NudT2 is responsible for the cleavage of compound 2 in lysates of human cell lines. These findings demonstrate that compound 2 is a very valuable tool to understand the activity of endogenous NudT2 in complex systems like cell lysates of human cell lines. This tool will contribute to understanding Ap4A-metabolism in humans and in this way to obtain insights into the functions of this important second messenger. Furthermore, NudT2 has been shown to be a prognostic factor for human breast cancer.45 Thus, analog 2 will therefore be suitable to identify inhibitors of this enzyme that might have a potential in cancer therapy. Investigations of Nucleotide Analogs in Cells. Having clarified the behavior of the analogs 1−3 in lysates of human cell lines, we explored whether these analogs can be internalized into human cells. In order to deliver the large and hydrophilic molecules 1−3 into the cytoplasm, we performed electroporation of HeLa cells.46,47 After the electroporation and the subsequent recovery time, the cells were directly fixed with formaldehyde and studied using fluorescence microscopy (Figure 4a). Indeed, monitoring the fluorescence intensity of the fluorescence acceptor sulfo-Cy5 reveals that 1−3 are internalized into these cells after electroporation. Fluorescence intensity of sulfo-Cy5 was used to monitor cellular uptake, because it is almost independent of the cleavage status of the compounds. We furthermore detected that after electroporation the analogs are distributed throughout the whole cell and that a higher concentration of these compounds can be E


Articles


Figure 4. Behavior of ATP analog 1, Ap4 analog 2, and control 3 in HeLa cells. Cells were electroporated in the presence of the indicated molecule, fixed using para-formaldehyde after the given time, and imaged using confocal fluorescence microscopy. After an initial measurement of the fluorescence intensity of the donor (Donor pre) and acceptor (Acceptor pre), the acceptor was bleached in the indicated area at high laser intensity, and both fluorescence intensities were measured again (Donor post, Acceptor post). The FRET efficiency was calculated from the increase of donor fluorescence using the formula given in the Methods section. A transmission image of all cells was additionally recorded. (a) Images of cells fixed directly after the recovery period of the electroporation process. (b) Images of fixed cells that were incubated for 60 min at 37 °C after the recovery period. Scale bar is 20 μm.

and in human cells. Studies of these analogs in cell lysates exhibit that the two analogs show very different behavior despite the fact that they differ only by one phosphate group. Whereas ATP analog 1 is stable, Ap4 analog 2 is processed rapidly in all investigated cell lysates. To understand the turnover of compound 2 in lysates, we performed in depth analysis of its cleavage in this environment. We found that the observed activity can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Compound 2 is therefore an interesting tool to study the activity of endogenous NudT2 even in complex environments like cell extracts. Because NudT2 has been shown to be a prognostic factor for breast cancer,45 this tool might contribute to a better understanding of its involvement in cancerogenesis and furthermore allow the straightforward screening for inhibitors, which may be valuable leads for anticancer drugs. In the second part of this work, we studied the properties of the nuceleotide analogs in the intracellular context. We found that electroporation is a suitable method to deliver 1−3 into human epithelial cells. Using this setup, we show that high FRET efficiencies can be detected for all analogs using

Figure 5. Quantification of the FRET efficiencies for ATP analog 1, Ap4 analog 2, and control 3 after different times of incubation. Data were obtained from 15 cells per condition from three independent experiments. Bars represent the median. Statistical significance is given for the indicated pair of analogs at the time of the respective column.

whether the cleavage occurs in the cytosol and the cleavage products are then stored in these vesicles. Summary. In conclusion, we present investigations of two different fluorogenic nucleotide analogs in human cell lysates F


Articles


10 μM of the respective analog, and the fluorescence of the fluorescence donor was monitored using an infinite f500 plate reader (TECAN) with excitation at 535 nm and emission at 595 nm. The turnover of compound was calculated relative to a noncleaved control (10 μM analog in hypotonic buffer) and a cleaved control (10 μM analog in hypotonic buffer pretreated with 0.01 units μL−1 SVPD (Worthington) for 10 min). Turnover rates were calculated by fitting the linear portion of the time traces. For inhibitor experiments, the same setup was used, except that prior to addition of the analog, indicated amounts of the respective inhibitor were added and incubated at RT for 1 h. Inhibition efficiency was calculated relative to a positive control (DMSO or water instead of the compound). Electroporation. HeLa cells were electroporated using the ElectroCell S20 electropulsator (βtech, Toulouse). Ten square-wave electric pulses of 100 μs length and electric field strength of 0.4 kV cm−1 were delivered at a 1 Hz frequency. Stainless-steel electrodes (megaStil, Ljubljana) were used in order to avoid electrochemical reactions of the metal. The interelectrode distance was 4 mm such that the electrodes could fit into the microscopy chamber. During electroporation, the cells were maintained in 100 μL of a low conductivity buffer (10 mM K2HPO4/KH2PO4, pH 7.4, 1 mM MgCl2, 250 mM sucrose) to minimize temperature increase. After the application of the electric field, the cells were maintained in the pulsation buffer for 5 min at RT to allow for membrane resealing. Afterward the cells were washed 3−5 times with 100 μL of PBS. Thereafter, the cells were either directly fixed with 4% PFA (w/v) for 20 min in the dark or incubated at 37 °C with 5% CO2 (v/v) for indicated times and then fixed. After fixation, the cells were washed once with PBS and kept in PBS for subsequent microscopy experiments. Colocalization. For colocalization experiments, cells were incubated with 2 μM quinacrine dihydrochloride 30 min before electroporation and 30 min after electroporation. For colocalization with LysoTracker, cells were incubated for 5 min with 50 nM Lysotracker Green DND-26 (Life Technologies) 25 min after electroporation. Thirty minutes after electroporation, the cells were fixed with 4% PFA (w/v) as described above. Confocal Microscopy and FRET Analysis. Fluorescence microscopy was performed on a TCS SP5 confocal laser scanning microscope (Leica, Wetzlar) using a 63×, 1.4 NA PLAPO oil immersion objective lens. Fluorescence images of the donor sulfo-Cy3 and the acceptor sulfo-Cy5 were acquired with excitation at 561 and 633 nm, respectively. Quinacrine dihydrochloride and Lysotracker Green were excited at 488 nm. For acceptor photobleaching experiments, the implemented FRET acceptor bleaching wizard of the Leica TCS SP5 was used. Bleaching of the acceptor sulfo-Cy5 was performed with high laser intensity at 633 nm. Prebleach and postbleach images of the donor sulfo-Cy3 and the acceptor sulfo-Cy5 were acquired using low laser intensities to avoid acquisition bleaching. The total fluorescence intensity within the bleached area was measured in the donor channel before and after photobleaching the acceptor, and the apparent FRET efficiency was calculated as

Figure 6. Colocalization of Ap4 analog 2 with markers for acidic vesicles. Thirty minutes after internalization of analog 2 via electroporation, cells were fixed and monitored by fluorescence microscopy. Acidic vesicles were stained with either Quinacrine or Lysotracker. Analog 2 was monitored using the acceptor fluorescence. Scale bar is 20 μm.

acceptor-photobleaching in fixed cells. In time-dependent experiments, we detect that both compounds 1 and 2 are processed intracellularly. This demonstrates that the analogs are valuable tools for studying nucleotide turnover in cells. Furthermore, the control compound 3, which does not bear a nucleotide-specific cleavage motif, is completely stable in this context over the investigated time. This indicates that the analogs are stable to nonenzymatic cleavage also inside cells and that any turnover of the FRET-based nucleotide analogs can be attributed to specific nucleotide cleavage. In this way, these analogs for the first time enable the detection of enzymatic nucleotide turnover in complex cellular environments by fluorescence microscopy. We show that after prolonged incubation the analogs are stored in acidic vesicles as also reported for ATP in several examples.49 Although further research will be needed to fully understand the behavior of these analogs in complex systems, this study demonstrates that the depicted analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

■

Eapp = (I(Donor‐post) − I(Donor‐pre))/I(Donor‐post) Donor and acceptor pre- and postbleach images were processed with ImageJ (http://rsb.info.nih.gov/ij/, U.S. National Institutes of Health). The images were background corrected and smoothed by applying a filter that replaces each pixel with the average of its 3 × 3 neighborhood. The FRET images were calculated using the FRETcalc ImageJ plugin (http://rsb.info.nih.gov/ij/plugins/fret/fret-calc. html).52 An intensity threshold was applied to circumvent erroneous FRET efficiency calculations for pixels with very low intensity. The “siemens” look-up-table was used for false-color representation of the FRET efficiencies. Statistical Analysis. Statistical analysis was performed using Prism GraphPad Software, La Jolla, CA, USA. In total, 15 single cells were imaged per time point and ATP analog in three independent experiments. Bars represent the median. The statistical significance of differences between FRET efficiencies of different ATP analogs at

METHODS

Monitoring Turnover of the Nucleotide Analogs in Vitro. For monitoring of the enzymatic turnover either the prepared lysates (0.25 mg mL−1 protein concentration) or recombinant NudT2 (2 nM) diluted with hypotonic buffer (50 mM HEPES (pH 6.8), 10% glycerol (v/v), 5 mM MgCl2) were used. Reactions were started by addition of G


Articles


(13) Wo, M. A. (1999) in Methods ih Molecular Biology (Osheroff, N., and Bjornsti, M. A., Eds.), Vol. 95, pp 311−325, Humana Press Inc., New York. (14) Webb, M. R. (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl. Acad. Sci. U. S. A. 89, 4884−4887. (15) Zhu, S., Gan, Z., Li, Z., Liu, Y., Yang, X., Deng, P., Xie, Y., Yu, M., Liao, H., Zhao, Y., Zhao, l., and Liao, F. (2009) The measurement of cyclic nucleotide phosphodiesterase 4 activities via the quantification of inorganic phosphate with malachite green. Anal. Chim. Acta 636, 105−110. (16) Pace, H. C., Garrison, P. N., Robinson, A. K., Barnes, L. D., Draganescu, A., Roesler, A., Blackburn, G. M., Siprashvili, Z., Croce, C. M., Huebner, K., and Brenner, C. (1998) Genetic, biochemical, and crystallographic characterization of Fhit−substrate complexes as the active signaling form of Fhit. Proc. Natl. Acad. Sci. U. S. A. 95, 5484− 5489. (17) Prescott, M., Milne, A. D., and McLennan, A. G. (1989) Characterization of the bis(5′-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia. Biochem. J. 259, 831−838. (18) Siprashvili, Z., Sozzi, G., Barnes, L. D., McCue, P., Robinson, A. K., Eryomin, V., Sard, L., Tagliabue, E., Greco, A., Fusetti, L., Schwartz, G., Pierotti, M. A., Croce, C. M., and Huebner, K. (1997) Replacement of Fhit in cancer cells suppresses tumorigenicity. Proc. Natl. Acad. Sci. U. S. A. 94, 13771−13776. (19) Thorne, N. M., Hankin, S., Wilkinson, M. C., Nuñez, C., Barraclough, R., and McLennan, A. G. (1995) Human diadenosine 5′,5‴-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases. Biochem. J. 311, 717− 721. (20) Hacker, S. M., Mortensen, F., Scheffner, M., and Marx, A. (2014) Selective monitoring of the enzymatic activity of the tumor suppressor Fhit. Angew. Chem., Int. Ed. 53, 10247−10250. (21) Mateo, J., Rotllan, P., and Miras-Portugal, M. T. (1996) Suramin - a powerful inhibitor of neural ecto-diadenosine polyphosphate hydrolase. Br. J. Pharmacol. 119, 1−2. (22) Rotilan, P., Ramos, A., Pintor, J., Torres, M., and Miras-Portugal, M. T. (1991) Di(1,N6 -ethenoadenosine)5′,5‴-P1,P4-tetraphosphate, a fluorescent enzymatically active derivative of Ap4A. FEBS Lett. 280, 371−374. (23) Draganescu, A., Hodawadekar, S. C., Gee, K. R., and Brenner, C. (2000) Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates. J. Biol. Chem. 275, 4555−4560. (24) Hacker, S. M., Pagliarini, D., Tischer, T., Hardt, N., Schneider, D., Mex, M., Mayer, T. U., Scheffner, M., and Marx, A. (2013) Novel fluorogenic ATP analogues for online monitoring of ATP consumption: observing ubiquitin activation in real-time. Angew. Chem., Int. Ed. 52, 11916−11919. (25) Hardt, N., Hacker, S. M., and Marx, A. (2013) Synthesis and fluorescence characteristics of ATP-based FRET probes. Org. Biomol. Chem. 11, 8298−8305. (26) Gutiérrez Acosta, O. B., Hardt, N., Hacker, S. M., Strittmatter, T., Schink, B., and Marx, A. (2014) Thiamine pyrophosphate stimulates acetone activation by desulfococcus biacutus as monitored by a fluorogenic ATP analogue. ACS Chem. Biol. 9, 1263−1266. (27) Hacker, S. M., Hardt, N., Buntru, A., Pagliarini, D., Möckel, M., Mayer, T. U., Scheffner, M., Hauck, C. R., and Marx, A. (2013) Fingerprinting differential active site constraints of ATPases. Chem. Sci. 4, 1588−1596. (28) Hacker, S. M., Mex, M., and Marx, A. (2012) Synthesis and stability of phosphate modified ATP analogues. J. Org. Chem. 77, 10450−10454. (29) Staudinger, H., and Meyer, J. (1919) Ü ber neue organische Phosphorverbindungen III. Phosphinmethylenderivate und Phosphinimine. Helv. Chim. Acta 2, 635−646. (30) Ranjitkar, P., Perera, B. G. K., Swaney, D., Hari, S. B., Larson, E. T., Krishnamurty, R., Merritt, E. A., Villén, J., and Maly, D. J. (2012)

different time points was evaluated using a two-way-ANOVA and Bonferroni post-tests. The degree of significance is given with the following labels: NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00459. Additional Figures S1−S12, synthetic details, general biochemical methods, and NMR spectra (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +49 7531 88 5140. Tel: +49 7531 88 5139. *E-mail: [email protected]. Fax: +49 7531 88 3139. Tel: +49 7531 88 2357. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support by the DFG (Grant SFB 969) and the Konstanz Research School Chemical Biology is gratefully acknowledged. S.M.H. also acknowledges the Studienstiftung des deutschen Volkes and the Zukunftskolleg of the University of Konstanz for stipends. A.B. also acknowledges the Telekom Stiftung for a stipend.

■

REFERENCES

(1) Lipmann, F. (1941) Metabolic generation and utilization of phosphate bond energy. Adv. Enzymol. 1, 99−162. (2) Manning, G., Whyte, D. B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) The protein kinase complement of the human genome. Science 298, 1912−1934. (3) Hedberg, C., and Itzen, A. (2015) Molecular perspectives on protein adenylylation. ACS Chem. Biol. 10, 12−21. (4) Roy, C. R., and Mukherjee, S. (2009) Bacterial FIC proteins: AMP up infection. Sci. Signaling 2, pe14. (5) Robinson, G. A., Butcher, R. W., and Sutherland, E. W. (1968) Cyclic AMP. Annu. Rev. Biochem. 37, 149−174. (6) Corrigan, R. M., and Gründling, A. (2013) Cyclic di-AMP: another second messenger enters the fray. Nat. Rev. Microbiol. 11, 513−524. (7) McLennan, A. G. (2000) Dinucleoside polyphosphates-friend or foe? Pharmacol. Ther. 87, 73−89. (8) Wegele, H., Muschler, P., Bunck, M., Reinstein, J., and Buchner, J. (2003) Dissection of the Contribution of Individual Domains to the ATPase Mechanism of Hsp90. J. Biol. Chem. 278, 39303−39310. (9) Bell, R. D., and Doisy, E. A. (1920) Rapid colorimetric methods for the determination of phosphorus in urine and blood. J. Biol. Chem. 44, 55−67. (10) Barrows, J. N., Jameson, G. B., and Pope, M. T. (1985) Structure of a heteropoly blue. The four-electron reduced β-12molybdophosphate anion. J. Am. Chem. Soc. 107, 1771. (11) Geladopoulos, T. P., Sotiroudis, T. G., and Evangelopoulos, A. E. (1991) A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192, 112−116. (12) Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994) Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33, 8262−8271. H


Articles

ACS Chemical Biology Affinity-based probes based on type II kinase inhibitors. J. Am. Chem. Soc. 134, 19017−19025. (31) Rotllán, P., Rodríguez-Ferrer, C. R., Asensio, A. C., and Oaknin, S. (1998) Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin. FEBS Lett. 429, 143−146. (32) Müller, C. E., Iqbal, J., Baqi, Y., Zimmermann, H., Röllich, A., and Stephan, H. (2006) Polyoxometalates–a new class of potent ectonucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors. Bioorg. Med. Chem. Lett. 16, 5943−5947. (33) Cassé, C., Giannoni, F., Nguyen, V. T., Dubois, M. F., and Bensaude, O. (1999) The transcriptional inhibitors, actinomycin D and alpha-amanitin, activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain. J. Biol. Chem. 274, 16097−16106. (34) Andrews, W. J., Panova, T., Normand, C., Gadal, O., Tikhonova, I. G., and Panov, K. I. (2013) Old drug, new target. Ellipticines selectively inhibit RNA Polymerase I transcription. J. Biol. Chem. 288, 4567−4582. (35) Semmler, J., Gebert, U., Eisenhut, T., Moeller, J., Schönharting, M. M., Alléra, A., and Endres, S. (1993) Xanthine derivatives: comparison between suppression of tumour necrosis factor-alpha production and inhibition of cAMP phosphodiesterase activity. Immunology 78, 520−525. (36) Huyer, G., Liu, S., Kelly, J., Moffat, J., Payette, P., Kennedy, B., Tsaprailis, G., Gresser, M. J., and Ramachandran, C. (1997) Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J. Biol. Chem. 272, 843−851. (37) Crack, B. E., Pollard, C. E., Beukers, M. W., Roberts, S. M., Hunt, S. F., Ingall, A. H., McKechnie, K. C., IJzerman, A. P., and Leff, P. (1995) Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br. J. Pharmacol. 114, 475− 481. (38) Rüegg, U. T., and Gillian, B. (1989) Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol. Sci. 10, 218−220. (39) Freitag, A., Wessler, I., and Racké, K. (1998) Phosphodiesterase inhibitors suppress α2-adrenoceptor-mediated 5-hydroxytryptamine release from tracheae of newborn rabbits. Eur. J. Pharmacol. 354, 67− 71. (40) Gierse, J., Thorarensen, A., Beltey, K., Bradshaw-Pierce, E., Cortes-Burgos, L., Hall, T., Johnston, A., Murphy, M., Nemirovskiy, O., Ogawa, S., Pegg, L., Pelc, M., Prinsen, M., Schnute, M., Wendling, J., Wene, S., Weinberg, R., Wittwer, A., Zweifel, B., and Masferrer, J. (2010) A novel autotaxin inhibitor reduces lysophosphatidic acid levels in plasma and the site of inflammation. J. Pharmacol. Exp. Ther. 334, 310−317. (41) Hunt, N. H., and Evans, T. (1980) RMI 12330A, an inhibitor of cyclic nucleotide phosphodiesterases and adenylate cyclase in kidney preparations. Biochim. Biophys. Acta 613, 499−506. (42) Sun, N. J., Woo, S. H., Cassady, J. M., and Snapka, R. M. (1998) DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia. J. Nat. Prod. 61, 362−366. (43) Zawacka-Pankau, J., Kowalska, A., Issaeva, N., Burcza, A., Kwiek, P., Bednarz, N., Pramanik, A., Banecki, B., and Podhajska, A. J. (2007) Tumor suppressor Fhit protein interacts with protoporphyrin IX in vitro and enhances the response of HeLa cells to photodynamic therapy. J. Photochem. Photobiol., B 86, 35−42. (44) Ge, H., Chen, X., Yang, W., Niu, L., and Teng, M. (2013) Crystal structure of wild-type and mutant human Ap4A hydrolase. Biochem. Biophys. Res. Commun. 432, 16−21. (45) Oka, K., Suzuki, T., Onodera, Y., Miki, Y., Takagi, K., Nagasaki, S., Akahira, J.-I., Ishida, T., Watanabe, M., Hirakawa, H., Ohuchi, N., and Sasano, H. (2011) Nudix-type motif 2 in human breast carcinoma: a potent prognostic factor associated with cell proliferation. Int. J. Cancer 128, 1770−1782. (46) Rols, M.-P., and Teissie, J. (1998) Electropermeabilization of mammalian cells to macromolecules: Control by puls duration. Biophys. J. 75, 1415−1423.

(47) Golzio, M., Teissie, J., and Rols, M. P. (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc. Natl. Acad. Sci. U. S. A. 99, 1292−1297. (48) Kenworthy, A. K. (2001) Imaging protein protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289−296. (49) Sakaki, H., Tsukimoto, M., Harada, H., Moriyama, Y., and Kojima, S. (2013) Autocrine regulation of macrophage activation via exocytosis of ATP and activation of P2Y11 Receptor. PLoS One 8, e59778. (50) Inoue, K., Komatsu, R., Imura, Y., Fujishita, K., Shibata, K., Moriyama, Y., and Koizumi, S. (2014) Mechanism underlying ATP release in human epidermal keratinocytes. J. Invest. Dermatol. 134, 1465−1468. (51) Dou, Y., Wu, H. J., Li, H. Q., Qin, S., Wang, Y. E., Li, J., Lou, H. F., Chen, Z., Li, X. M., Luo, Q. M., and Duan, S. (2012) Microglial migration mediated by ATP-induced ATP release from lysosomes. Cell Res. 22, 1022−1033. (52) Stepensky, D. (2007) FRETcalc plugin for calculation of FRET in non-continuous intracellular compartments. Biochem. Biophys. Res. Commun. 359, 752−758.

I


Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and

Recommend Documents