Sensitive Detection of Intracellular Sumoylation via SNAP Tag

Jan 13, 2012 - method for intracellular sumoylation assay based on SNAP tag (a ... covalent conjugation of SNAP tag with its substrate benzyl guanidin...
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Sensitive Detection of Intracellular Sumoylation via SNAP TagMediated Translation and RNA Polymerase-Based Amplification Yong Yang and Chun-yang Zhang* Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China ABSTRACT: The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, defined as sumoylation, is an important post-translational modification that regulates diverse cellular processes and many human diseases. However, functional analysis of sumo modification is usually hampered by the lack of sensitive methods for measuring extremely low abundance of specific sumoylated target in the cells. Here, we develop an ultrasensitive method for intracellular sumoylation assay based on SNAP tag (a mutant of O6-alkylguanine-DNA alkyltransferase)-mediated translation and RNA polymerase-based amplification. Intracellular sumo modification is first converted to the double-stranded DNA (dsDNA) containing the specific T7 promoter sequence via the covalent conjugation of SNAP tag with its substrate benzyl guanidine derivate; then, the dsDNA is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the RNA intercalating dye RiboGreen and a standard fluorometer. This method exhibits excellent specificity and high sensitivity and can detect as little as 5 pg of sumoylated p53 proteins, which has improved by as much as 1000-fold than that in the conventional Western blotting assay. Moreover, this method can measure intracellular sumoylation under different physiological conditions. Due to the common translation and amplification module, this method can be further extended to detect a variety of sumoylated proteins and other ubiquitin-like modifications in the cells and might provide a powerful tool for comprehensive analysis of the functions of sumoylation and other ubiquitin-like modifications in the fundamental biological processes and many human diseases.

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small fraction of specific target is allowed to be sumoylated at a given time,19,21 making the functional analysis of sumoylation quite challenging. So far, several analytical approaches have been developed for sumoylation assay, including mass spectrometry-based proteomic method,23,24 in vitro sumoylation reaction,25,26 and computation-based sumoylation predication.27 However, a mass spectrometry-based proteomic method usually requires an expensive instrument and sophisticated operation to obtain the accurate results. The in vitro reconstitution reaction relies on the preparation of active substrate proteins and cannot measure sumo modification in the natural surroundings of living cells.28 The outcomes of computation-based sumoylation predication are theoretical outputs which need additional experimental validation.27 Recently, Jakobs et al. have fabricated a UBC9-fusion directed sumoylation (UFDS) system to analyze the protein sumoylation.21 The UFDS system can increase the amount of one specific sumoylated protein in the cells; however, the UFDS system bypasses the requirements of sumo ligases completely. In addition, artificial fusion of UBC9 with the substrate protein might lead to false results.28 Therefore, an ultrasensitive and

umoylation, the conjugation of the small ubiquitin-like modifier (SUMO) to specific lysine residues in target proteins, is involved in the regulation of a wide variety of cellular processes, including DNA-damage response,1 gene transcription,2,3 subcellular localization,4,5 cell-cycle regulation,6 protein−protein interaction,7 genome integrity,8,9 signal transduction,10 and telomere maintenance.11,12 Sumoylation is also implicated in the pathogenesis of neurodegenerative disorders,13,14 type 1 diabetes,15 liver disease,16 cancer development,17 and heart diseases.18 Although diverse cellular and pathologic processes are affected by this modification, the sumo system utilizes common enzymatic cascade known as E1activating enzyme, E2-conjugating enzyme, and/or E3 ligase to form an isopeptide bond between the sumo entity and the target proteins.10 The attachment of sumo with the substrate proteins either alters the substrate surface or results in the conformational change of substrates19 and, consequently, influences the substrate protein’s intracellular localization,4,5 interactions,19 stability, and activity.3 Sumo modification itself is also regulated by the sumo-specific proteases,10,20 and sumo entity can be efficiently removed from the target proteins by the isopeptidase activity of sumo proteases, thereby providing an important regulatory mechanism to control the amount of modified substrates and the availability of free sumo proteins.8 Due to the existence of both sumoylation−desumoylation equilibrium21 and the abundant substrates in the cells,22 only a © 2012 American Chemical Society

Received: December 3, 2011 Accepted: January 13, 2012 Published: January 13, 2012 1229

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facile method capable of measuring intracellular sumo modification is highly required. Herein, we develop an ultrasensitive method for sumoylation assay based on SNAP tag (a mutant of O6-alkylguanine-DNA alkyltransferase)-mediated translation and RNA polymerasebased amplification. The SNAP tag is a mutant of DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylguanine (BG) derivatives.29 As shown in Scheme 1, genetic fusion of SNAP Scheme 1. Scheme for Sumoylation Assay Based on SNAP Tag-Mediated Translation and RNA Polymerase-Based Amplificationa

Figure 1. Characterization of BG-dsDNA. (A) Schematic structure of BG-dsDNA. The dsDNA contains both the T7 promoter sequence and the T7 terminator sequence to facilitate the synthesis of RNA. (B) Sequences of the primers used for the preparation of BG-dsDNA. The specific T7 promoter sequence is underlined. (C) Gel electrophoresis of purified BG-dsDNA. Lane 1: DNA marker; Lane 2: purified BGdsDNA.

BG-dsDNAs were prepared using the BG-coupled 5′ primer and a 3′ primer that reversely complemented with the T7 terminator (Figure 1B) in a standard polymerase chain reaction with pET28a vector as the template. The PCR product was then isolated by gel electrophoresis to remove the unbound 5′ primers. The purified BG-dsDNA was verified by gel electrophoresis (Figure 1C). To assess whether this method could amplify intracellular sumoylation specifically, we chose tumor suppressor p53 proteins as the model target.21 Wild-type p53 expression plasmid was transfected into HEK293T cells either alone or together with SNAP-tagged sumo-1 (an isoform of sumo family). The Western blotting assay was performed in parallel to validate the expression and modification of p53 proteins in the cells. As shown in Figure 2A, expression of p53 alone produced negligible fluorescence signal, suggesting no conversion of sumoylation into the RNA. Coexpression of p53 with SNAP-tagged sumo-1 resulted in a remarkable fluorescence signal, which had increased by as much as 32-fold in comparison with p53 alone (Figure 2B). Further experiments demonstrated that the obtained fluorescence signal (Figure 2A) was proportional to the sumo modification in the cells measured by the Western blotting assay (Figure 2C, upper panel). To further confirm that the change of fluorescence was attributed to the sumo-mediated modification of p53, we mutated residue lysine 386 which was the main sumo acceptor site to arginine.21 As shown in Figure 2B, coexpression of p53K386R mutant with SNAP-tagged sumo-1 yielded relatively weak fluorescence signal as compared to the coexpression of SNAP-tagged sumo-1 with wild-type p53. The reduction of fluorescence intensity was in agreement with the decrease of sumo-1 modification in vivo (Figure 2C, upper panel). The above results clearly indicated that the developed method could amplify intracellular sumoylation efficiently and specifically. It had been reported that less than 1% of specific substrate was sumoylated at any given time,10 and such low abundance of modification restricted the analysis of sumoylation in vivo;21 thus, the sensitivity of developed method was essential for intracellular sumoylation assay. To this end, 2 μg of p53 proteins containing the SNAP-sumo-1 conjugated p53 were diluted at various ratios to determine the sensitivity of the developed sumoylation assay. As shown in Figure 3A, the fluorescence intensity declined with the dilution of p53 proteins as a result of the decrease of sumoylated p53 proteins (Figure

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After sequential reaction of E1, E2, and/or E3, SNAP-sumo fusion protein forms an isopeptide bond with the target protein in the cell. The sumo moiety acts as a bridge to link the SNAP tag and the substrate protein, and the SNAP moiety acts as an adapter to convert the sumo modification to dsDNA through the reaction with its substrate benzylguanine-coupled dsDNA (BG-dsDNA). The dsDNA with essential elements for transcription is then transcribed by T7 RNA polymerase to yield large amounts of RNAs, leading to significant amplification of sumoylation. The RNAs are assessed by RiboGreen dye with a standard fluorometer, and a remarkable fluorescence signal is observed in the presence of sumoylation.

tag with the sumo entity affords sumo modifier additional alkyltransferase activity in vivo. Once the sumo entity forms a covalent conjugation with its substrate, the SNAP tag acts as an adapter to convert the modification signal to nucleic acids through the reaction with benzylguanine derivative which carries a double-stranded DNA (BG-dsDNA). As the dsDNA contains both the T7 promoter sequence and the T7 terminator sequence for effective transcription, large amounts of RNAs will be produced by T7 RNA polymerase,30 resulting in significant amplification of limited sumo modification. The synthesized RNAs can be easily monitored using the RNA intercalating dye RiboGreen and a standard fluorometer.30 This method exhibits excellent specificity and high sensitivity and can detect as little as 5 pg of sumoylated p53 proteins, which has improved by as much as 1000-fold than that in the conventional Western blotting assay. Moreover, this method can measure intracellular sumoylation under different physiological conditions. In order to achieve the amplification of sumo modification, benzylguanine-coupled dsDNA (BG-dsDNA) must be a bifunctional molecule that acts as both a substrate of SNAP tag and a template for T7 RNA polymerase (Figure 1A). To this end, BG-GLA-NHS was first coupled to the 5′ end of a 33bp oligonucleotide which contains the specific T7 promoter sequence (Figure 1B) to prepare BG-coupled 5′ primer. The 1230

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Figure 2. Specificity of sumoylation assay based on SNAP tag-mediated translation and RNA polymerase-based amplification. (A) Fluorescence spectra of nonsumoylated (blue), adequately sumoylated (red), and poorly sumoylated (green) p53 proteins. (B) Quantification of fluorescence intensity in (A). Error bars show the standard deviation of three experiments. (C) Validation of the fluorescence measurement in (A) by the Western blotting assay. The upper panel probed with anti-SNAP antibody shows the amount of sumoylated p53 measured; the lower panel probed with antiFLAG antibody shows the same amount of total p53 proteins loaded.

cells, did not produce noticeable change in both the fluorescence assay (Figure 4A) and the Western blotting assay (Figure 4B), indicating that oxidative stress did not alter the sumoylation level of p53 proteins under this condition.37 The addition of Calyculin A, a potent inhibitor of serine/ threonine phosphatases 1 and 2A, abrogated the sumo-1 modification of p53 proteins in the Western blotting assay (Figure 4B, upper panel).38 Surprisingly, weak fluorescence signal was still observed in the sumoylation assay based on SNAP tag-mediated translation and RNA polymerase-based amplification (Figure 4A). This discrepancy might be attributed to the high sensitivity of this sumoylation assay which could sense extremely low abundance of sumoylated p53 proteins that was under the detection limit of the Western blotting assay (Figure 3). More importantly, the consistency of the fluorescence intensity (Figure 4A) with the amount of sumoylated p53 proteins (Figure 4B) indicated that this sumoylation assay could detect sumoylation at various physiological conditions with high accuracy. In conclusion, we have developed for the first time an ultrasensitive method for intracellular sumoylation assay based on SNAP tag-mediated translation and RNA polymerase-based amplification. The specific conjugation of SNAP tag with its substrate as well as the specific recognition of promoter sequence by T7 RNA polymerase ensure the modification to be amplified with high fidelity. Moreover, the synergy of SNAP tag and T7 RNA polymerase can magnify the modification effectively. The high sensitivity of this sumoylation assay can

3B, upper panel). Notably, distinct fluorescence signal was observed even in 1:5000 dilutions (Figure 3A). In contrast, 5fold dilution led to nearly zero signal in the Western blotting assay (Figure 3B, upper panel). These results indicated that the sensitivity of this sumoylation assay had improved by as much as 1000 times in comparison with the Western blotting assay. As compared to the detection limit of 5 ng per load in the Western blotting assay,31 this method can detect as little as 5 pg of sumoylated protein experimentally. The improved sensitivity of this sumoylation assay might be attributed to two factors: (1) The covalent binding of SNAP tag with the BG-dsDNA29 can translate the modification into nucleic acids which are easily amplified by the polymerase; (2) The transcription property of T7 RNA polymerase, which is characterized by high rate of RNA chain elongation (∼200 nucleotides per second)32 and extensive iteration of transcription cycle,33 can produce RNAs with high efficiency. Sumo conjugation was a reversible and dynamic process that can be dramatically affected by the desumoylation enzymes19 and various cellular stress.34,35 To demonstrate the wide applicability of the developed strategy, we further employed this method to measure p53 sumoylation under various physiological conditions. As shown in Figure 4A, the treatment of cells with DNA-damaging agent doxorubicin increased the fluorescence intensity as compared with the control, suggesting that DNA damage caused an increase in the sumo-1 conjugated form of p53 proteins in the cells.36 Unlike DNA damage stress, H2O2 treatment, which could produce oxidative stress in the 1231

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break up the bottleneck of measuring low abundance of sumoylated proteins in the cells, thereby facilitating subsequent functional studies. More importantly, the common translation and amplification module allow this sumoylation assay to be widely applicable, and all intracellular sumo modification can be amplified in the same manner through the coexpression of SNAP-SUMO and different substrates within the cells. In addition, this strategy might be further extended to other ubiquitin-like modifications by just genetic fusion of the SNAP tag with the ubiquitin-like modifier.39,40 Therefore, this method might provide a valuable tool to reveal the functions of sumoylation and other ubiquitin-like modifications in the fundamental biological processes and many human diseases.

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EXPERIMENTAL SECTION Materials. All the chemical reagents unless otherwise stated were purchased from Sigma-Aldrich commercial suppliers (USA) and used without purification. The anti-FLAG M2 affinity resin and anti-FLAG antibody were obtained from Sigma co, Ltd. (USA). The RNA interacting dye RiboGreen and all reagents used for cell culture were purchased from Invitrogen (USA) and were used according to the instructions. Full length cDNA of human p53 and sumo-1 were obtained from Proteintech Group Inc. (Wuhan, China branch). The pSNAP-tag (m) vector, anti-SNAP antibody, and BG-GLANHS were purchased from New England Biolabs, Inc. (USA). All the primers used for cloning were synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The BG-coupled 5′ primer was synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China). Cloning and Mutagenesis. Full-length human p53 cDNA was amplified by PCR and subsequently inserted into p3XFLAG-myc-CMV-24 vector (a kind gift from Dr. Guanghui Wang at University of Science and Technology of China) with EcoR I and SalI double digestion. The p53 mutant p53K386R was generated by site-directed mutagenesis using PCR-strategy. To facilitate the sumoylation of target protein in vivo, active version of SUMO-1 cDNA, whose C terminal diglycine motif was exposed,41 was amplified and inserted into pSNAP-tag (m) vector (New England Biolabs, Inc., USA) at the BamH I and Xho I sites. All the clones were verified by DNA sequencing. Cell Culture, Transfection, and Treatments. HEK293T cells from American Type Culture Collection (Manassas, VA) were maintained as subconfluent monolayers in Dulbecco's modified eagle medium (DMEM; Invitrogen) with 10% fetal bovine serum (Invitrogen, USA) and 100 units/mL penicillin plus 100 μg/mL streptomycin (Invitrogen, USA) at 37 °C with 5% CO2. Cells growing on 60-mm Petri dishes were either transfected with 2.5 μg of FLAG-p53 construct or the mutant FLAG-p53K386R alone or along with 2.5 μg of SNAP-SUMO-1 expression construct using the standard calcium phosphate precipitation method. After 36 h of transfection, the cells were collected for the Western blotting assay or an immunoprecipitation experiment. For the drug treatment experiments in Figure 4, the cells cotransfected with FLAG-p53 and SNAPSUMO-1 were either treated with 400 ng/mL doxorubicin for 16 h,36 treated with 5 μM H2O2 for 20 min,37 or treated with 0.1 μM calyculin A for 45 min before lysis.38 Immuoprecipitation and BG-dsDNA Coupling. Transfected and treated cells were sonicated in the lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween-20, 1 mM DTT, 20 mM N-ethylmaleimide, and protease inhibitors), and the cell debris was removed by centrifugation. After centrifugation, the

Figure 3. Sensitivity of sumoylation assay based on SNAP tagmediated translation and RNA polymerase-based amplification. (A) Variance of the fluorescence intensity with the dilution of sumoylated p53 proteins. Error bars show the standard deviation of three experiments. (B) The Western blotting assay of SNAP-sumo-1 conjugated p53 proteins in (A). The Western blotting assay was performed in parallel with the fluorescence assay. The upper panel probed with anti-SNAP antibody shows the amount of sumoylated p53 measured; the lower panel probed with anti-FLAG antibody shows the amount of total p53 proteins loaded.

Figure 4. Detection of p53 sumoylation under different physiological conditions. (A) Fluorescence measurement of p53 sumoylation under DNA damage (doxorubicin), oxidative stress (H2O2), and phosphatase inhibition (Calyculin A) conditions. Cells without any treatment were used as the control. Error bars show the standard deviation of three experiments. (B) Detection of sumoylated p53 proteins in (A) by the Western blotting assay. 1232

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supernatants were incubated with anti-FLAG M2 affinity resin (Sigma, USA) at 4 °C for 1 h. After washing 3 times with cold lysis buffer, the beads were incubated with BG-dsDNA in SNAP-tag reaction buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20, 1 mM DTT) at 25 °C for 1 h. After extensive washing, the p53-SUMO-1-SNAP-dsDNA conjugates were eluted from the beads with 0.1 M glycine (pH 2.6) for two times; the elution was neutralized immediately with the neutralization buffer (0.5 M Tris, 1 M NaCl) to prevent the denaturation of the conjugates. Western Blotting Assay. Two aliquots of neutralized elutates (2 μL per spot) were dipped onto a nitrocellulose membrane: one was used for the detection of total protein, and the other was for the sumoylation assay. The membrane was then blocked with nonfat powdered milk at room temperature for 1 h. After washing, the blot was incubated with anti-SNAP antibody (New England Biolabs, Inc., USA) and anti-FLAG antibody (Sigma, USA) for 60 min, respectively. Horseradish peroxidase-conjugated antimouse and antirabbit IgGs (Jackson ImmunoResearch, USA) were used as the secondary antibodies. Immunoreactive spots were visualized by enhanced chemiluminescence with ECL detection kit (Amersham Biosciences, USA) according to the instruction of the manufacturer. The images were acquired by Kodak 4000 MM (Kodak, Japan). RNA Amplification and Measurement. In addition to the Western blotting assay, the same amount of neutralized elutates was used as the template to transcribe RNA in vitro. Briefly, 2 μL of elutate was added into 18 μL of reaction mixture which contained 10 units of T7 RNA Polymerase, 20 units of RNase inhibitor, 5 mM DTT, 2 mM NTP mixture, and 1× T7 RNA Polymerase buffer (TaKaRa Biotechnology Co., Ltd., China). RNA amplification was performed at 37 °C for 1 h, followed by the addition of RNase-free TE to reach 50 μL. The RNA intercalating dye RiboGreen (Invitrogen, USA) was then added to the reaction mixture (50 μL, 1:200 diluted in RNase-free TE buffer) and incubated for 5−10 min in the dark. The photoluminescence spectra were obtained by a photoluminescence spectrometer (FSP920, Edinburgh Instruments Ltd., UK) with an excitation wavelength of 492 nm. The fluorescence intensity at emission wavelength of 525 nm was used for quantitative measurements.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected].

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ACKNOWLEDGMENTS

The authors are grateful for the gift of p3XFLAG-myc-CMV-24 vector from Dr. Guanghui Wang at University of Science and Technology of China. This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the Award for the Hundred Talent Program of the Chinese Academy of Science, the Chinese Natural Science Foundation (Grant Nos. 21075129 and 31000599), the Award for the Innovation Team Project of Guangdong Province, the Natural Science Foundation of Guangdong Province (Grant No. 10478922035-X004914), and the Natural Science Foundation of Shenzhen City (Grant Nos. JC201005270327A and JC201005270355A). 1233

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