Cell extracts and Western blotting analysis
For preparation of total protein lysate, cells were harvested by trypsinization and centrifuged at 250 × g for 5 min at 4°C. Supernatant was removed, and the pellet was washed once with ice-cold PBS and then centrifuged again as described. The cell pellet was resuspended in Lysis buffer solution (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) at a density of 107 cells/mL and incubated for 30 min at 4°C. After centrifugation at 12,000 × g for 30 min at 4°C, the supernatant was collected as total cell lysate. Protein concentration was determined using Bio-Rad protein assay reagent (Bio-Rad, Milan, Italy). For preparation of nuclear enriched fractions, cells were harvested as described, and resuspended in T1 Solution (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA pH 8.0) at a cell density of 2 × 107 cells/mL. Nuclei were isolated by centrifugation at 800 × g for 10 min at 4°C, while the supernatant was collected as cytoplasmic cell lysate. Nuclei were then washed with 500 mL of T1 solution and collected by centrifugation as described. Nuclei were subsequently lysed with T2 Solution (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA pH 8.0, 5% glycerol), incubated for 30 min at 4°C, and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was collected as nuclear cell lysate and protein concentration was determined using Bio-Rad protein assay reagent (Bio-Rad). All cell lysis solutions were supplemented with Protease inhibitor cocktail (Sigma Aldrich), 0.5 mM PMSF, and DTT 1 mM. The reported amount of total or nuclear protein extracts was separated onto a 12% SDS-PAGE followed by Western blotting. The presence of APE1 and APE1-Dendra2 fusion protein was detected using an anti-APE1 monoclonal antibody (1:2000) (Novus, Littleton, CO), and anti-Actin rabbit polyclonal antibody (1:2000) (Sigma Aldrich) was used as a loading control. Endonuclease assay
A 28-mer oligonucleotide containing a tetrahydrofuran (THF) residue mimicking an abasic site was labeled in vitro with 32P and annealed with its complementary sequence (25). APE1 recognizes the THF abasic site and cleaves the phosphodiester backbone immediately 5′ to the AP site creating a nick. Reactions were separated in a urea 6 M denaturing 20% polyacrylamide gel, which allowed discrimination of unprocessed substrate (28-mer) and cleaved product (14-mer) by exposing the gel to autoradiography film (Sigma Aldrich). Results and Discussion Dendra2 does not alter APE1 subcellular localization
HeLa cells were used as a cellular model to generate an inducible reconstituted knock-in (KI) clone expressing an ectopic recombinant form of APE1 protein in fusion with Dendra2 at the C terminus. Endogenous expression of APE1 was knocked-down by siRNA technology in a conditional manner through a doxycycline-responsive promoter (19). Ectopic expression was achieved by stably cloning an siRNA-resistant cDNA APE1 in fusion with Dendra2 sequence (APE1-Dendra2). As a control, we used the empty vector expressing only Dendra2 (Dendra2). Inducible expression of specific APE1 siRNA sequences by doxycycline treatment efficiently promoted endogenous APE1 down-regulation in both control and KI clones. After 10 days of treatment, endogenous APE1 expression was almost undetectable (less than 10%) as compared with untreated cells, while ectopic APE1-Dendra2 fusion protein levels were slightly affected (Figure 1A). This time point was therefore chosen for further experiments. Comparison of APE1 protein amounts between the endogenous form in the Dendra clone at point zero and the ectopic fusion protein in the APE1-Dendra clone after 10 days of doxycycline treatment showed similar expression levels (1.8 ± 0.6 -fold).
Next, we evaluated if the presence of Dendra2 at the C terminus of APE1 affected the localization of the ectopic protein within the different subcellular compartments. Live confocal microscopy analysis of the Dendra2 clone (Figure 1B) showed green fluorescence corresponding to Dendra2 before photoconversion in both the cytoplasmic and nuclear compartments, while nucleoli were completely excluded (red arrows). In contrast, expression of APE1 fused to Dendra2 showed green fluorescence in nuclei and the accumulation of the ectopic fusion protein within nucleolar structures (red arrows: Figure 1B), results consistent with our previous work (19, 26). Similar results were also obtained when transiently transfecting pDendra2-N and pDendra2-N-APE1 vectors into the human neuroblastoma cell line SF767 (Supplementary Figure S1). Taken together, these data lead to the conclusion that the presence of Dendra2 does not alter the subcellular localization of APE1. Next, we evaluated if a fixation protocol commonly used for immunofluorescence analysis would alter Dendra2 subcellular localization. Dendra2 displays uniform cytoplasmic/nuclear staining and does not localize to nucleoli (Figure 1B), but cell fixation with PFA 4% for 20 min before confocal analysis caused Dendra2 to accumulate in the nucleus and nucleolar compartment (Figure 1C). With the APE1-Dendra2 clone, we observed a more robust signal corresponding to nucleoli following fixation. These data confirm that mild fixation can alter protein localization (Figure 1C), while strong fixation ensures protein immobilization and ultrastructure preservation but may interfere with epitope recognition and penetration of antibodies (1). Therefore, we conclude that studies of APE1 subcellular trafficking could be performed more reliably by adopting a live microscopy analysis rather than an immunofluorescence-based approach. APE1-Dendra2 re-expression in APE1 knocked down cell clone rescues the loss of AP-endonuclease activity
A standard assay to measure APE1 endonuclease activity uses a dsDNA oligonucleotide containing one 32P radio labeled 5′ strand with a central tetrahydrofuran (THF) residue that mimics an AP site. APE1 is able to recognize the AP site and cleave the phosphodiester bond to generate a shorter oligonucleotide (25). We quantified APE1 endonuclease activity by incubating a cell extract with such a dsDNA probe, separating the reaction mixture in a denaturing polyacrylamide gel, and exposing the gel to a radiographic film (Figure 2A).
In order to evaluate if the presence of the fusion fluorescence protein altered APE1 endonuclease activity, Dendra2 and APE1-Dendra2 KI clones were treated (+) or not (-) with doxycycline for 10 days, and nuclear protein extracts were isolated under native conditions. Western blotting analysis confirmed the absence of endogenous APE1 in both clones after doxycycline treatment and the presence of the ectopic recombinant Dendra2-APE1 fusion protein in the KI clone. Densitometric analysis highlighted a 2.4-fold higher amount of APE1-Dendra2 protein in the KI clone with respect to the amount of endogenous APE1 protein in the Dendra2 untreated clone (Figure 2B). Therefore, to compare the endonuclease activity between endogenous and ectopic recombinant APE1 100 ng nuclear extracts of Dendra2 and 42 ng of APE1-Dendra2 containing equal amounts of APE1 protein were used in the assay. The graph in Figure 2C shows the percentage of product formation at different time points: loss of APE1 expression led to a significant reduction (∼2.7-fold after 10 min) of AP endonuclease activity (Dendra2 +). Notably, re-expression of APE1-Dendra2 ectopic protein completely rescued APE1 enzymatic activity (Figure 2C). Therefore we conclude that the presence of Dendra2 in fusion at the C terminus of APE1 does not alter the endonuclease activity of the protein, thus confirming the reliability of the cellular model generated. Dendra2 allows the calculation of APE1 protein half-life
The conventional method for calculating the half-life of a protein of interest is to inhibit protein biosynthesis by treating cells with CHX (9) and then measure the reduction in expression levels of the protein of interest by Western blotting. For this purpose, we treated HeLa cells with CHX or DMSO as control at the final concentration of 100 µg/mL for up to 10 h. After the reported times, cells were harvested and 15 µg of total protein extracts were separated onto a 12% SDS-PAGE gel and analyzed by Western blotting for the presence of APE1 (Figure 3A). Densitometric analysis as shown in the graph represents APE1 relative levels during CHX treatment normalized to untreated cells. After 9 h, the total protein amount of APE1 decreased by 50%, but we did not observe any significant reduction of APE1 during the first 2 h of treatment. This phenomenon could be ascribed to the time required for CHX to reach an intracellular concentration sufficient to block protein neo-synthesis. In parallel, we tested the cellular model to calculate APE1 half-life in vivo by following the reduction of Dendra2 fluorescence after photoconvertion from green to red in response to violet light irradiation (12). Using a 405 nm diode laser at 100% of power for a single scan at 1.27 µs pixel dwell time, it was possible to photoconvert almost all (more than 80 ± 7%) Dendra2 from green to red in a region of interest overlapping an entire cell within the field (Supplementary Figure S2). To reduce the damaging effect of irradiation, we reduced the laser power to 30% and obtained a partial photoconversion (24 ± 6%) of APE1-Dendra2 protein in the cell. In each experiment, some cells presented within the field were photoconverted while others were not irradiated and served as control. We did not observe any different behavior between these two populations in terms of cell morphology and motility, suggesting that the process of photoconversion was not harmful to the cell (Supplementary Movie S1). The absence of photobleaching, crosstalk between fluorophores, and stability of Dendra 2 have been also evaluated (data not shown). To calculate APE1 half-life after photoconversion, cells were grown in a confocal microscopy life station and imaged each hour (Figure 3B). Red and green fluorescence signals were quantified before and after photoconversion and then each hour for 14 consecutive hours. The graph in Figure 3C represents the mean fluorescence intensity of fifteen cells measured each hour in three independent experiments. The fluorescence half-life of Dendra2 after photoconversion was estimated at 7 h, about 2 h shorter than estimates obtained using CHX treatment. Similarly, APE1 half-life was estimated at 7 h in SF767 cells transiently expressing pDendra2-N-APE1 (Supplementary Figure S3). In contrast to the CHX treatment, we observed a gradual reduction of the red signal starting in the first hour of the in vivo analysis. Remarkably, this 2 h gap between the half-lives measured with the different methods fits with the delay in protein degradation during CHX treatment (Figure 3A). In addition, it has to be mentioned that CHX is a cytotoxic and pro-apoptotic agent that may also cause the onset of an adaptive mechanism involving the protein of interest, and therefore we cannot exclude that it could alter the half-life of APE1, a protein involved in cellular response to DNA damage and oxidative stress. In the Figure 3C, it is also possible to observe APE1-Dendra2 neo-synthesis. This supports our conclusion that our experimental procedures are not harmful to the cells, and therefore the half-life is accurate and not affected by experimental bias. Based on our data, we can conclude that the in vivo confocal microscopy approach is more reliable than using CHX to calculate protein life-time.
In conclusion, we generated and characterized a cellular model where endogenous APE1 was replaced by an ectopic recombinant form of the protein expressed in fusion with Dendra2. Moreover, we demonstrated that the presence of the fluorescent protein altered neither APE1 localization nor its fundamental DNA repair activity on abasic DNA. Finally, we calculated APE1 half-life using both the APE1-Dendra2 cellular model and the classical approach using the protein synthesis inhibitor CHX. This comparison provided us with a proof-of-concept that using PCFP in fusion with a DNA repair protein such as APE1 is a reliable approach for evaluating in vivo protein dynamics. It will be interesting to evaluate the practical application of this method in cancer research, where the rational combination of DNA damaging agents with DNA repair protein inhibitors offers the most promising perspective for clinical utility (27), and where methodological tools are required to characterize the effects of inhibitors on protein stability, localization, and enzymatic activity. Author Contributions
G.T. and C.V. defined the research theme, designed methods and carried out the laboratory experiments, analyzed the data, interpreted the results and wrote the paper. M.D.P. and M.M.K. contributed to the microscopy analysis, data interpretation and results discussion.
The authors would like to thank José Artacho and Freddy Radtke for providing technical and logistical support at the Ecole Polytechnique Fédérale de Lausanne. This work was supported by a grant from AIRC (IG10269) to G.T. and also by the Regione Friuli Venezia Giulia for the Project ‘MINA’ under the program entitled: “Programma per la Cooperazione Transfrontaliera Italia-Slovenia 2007-2013.”
The authors declare no competing interests.
Address correspondence to Carlo Vascotto, Department of Medical and Biological Sciences, University of Udine, Udine, Italy. E-mail: [email protected]
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