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Visualizing and Quantifying Protein PolySUMOylation at the SingleMolecule Level Yong Yang and Chun-yang Zhang* Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China S Supporting Information *
ABSTRACT: Protein polySUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is associated with a variety of physiological processes. However, the analysis of protein polySUMOylation is often complicated by the heterogeneity of SUMO−target conjugates. Here, we develop a new strategy to visualize and quantify polySUMOylation at the single-molecule level by integrating the tetracysteine (TC) tag labeling technology and total internal reflection fluorescence (TIRF)-based singlemolecule imaging. As a proof-of-concept, we employ the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can specifically translate the SUMO-mediated modification into visible fluorescence signal without disturbing the function of SUMO-2. The SUMO monomers display homogeneous fluorescence spots at the single-molecule level, whereas the mixed SUMO chains exhibit nonuniform fluorescence spots with a wide range of intensities. Analysis of the number and the brightness of fluorescence spots enable quantitative measurement of the polySUMOylation degree inside the cells under different physiological conditions. Due to the frequent occurrence of posttranslational modification by polymeric chains in cells, this single-molecule strategy has the potential to be broadly applied for studying protein posttranslational modification in normal cellular physiology and disease etiology.
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heterogeneous populations of SUMO−target conjugates with variable stoichiometries, each of which has its own molecular weight.10,16 Several methods, such as Western blotting and in vitro SUMOylation, have been developed for the polySUMOylation assay.10,17 Although these methods can measure protein polySUMOylation in bulk, they are usually time-consuming and often inconclusive.18 Recently, Matic et al. developed a mass spectrometry-based strategy to detect SUMO chains by linearizing the branched peptide which consisted of the C terminus of modified branch and the N terminus of substrate peptide,19 wherein the SUMO chains can be analyzed by a standard database search engine without either special software20 or mutation of SUMO.21,22 However, this method requires artificial construction and incorporation of virtual peptides to the database.18 Due to the low-abundance of SUMOylated target23,24 and the heterogeneity of SUMO chains in the cells, a sensitive and direct method capable of delineating the full view of self-modification events is highly desired. Recently, we demonstrated a SNAP/CLIP-tag-based strategy to measure multiple SUMOylations at the single-molecule level.25 The integration of SNAP/CLIP-tag-mediated translation with single-molecule detection has the capability of
rotein SUMOylation is a kind of post-translational modification that closely involves numerous physiological and pathological processes,1 e.g., gene transcription,2 DNAdamage response,3 neurodegenerative disorders,4,5 and heart diseases.6 The human genome encodes at least three small ubiquitin-like modifier (SUMO) paralogues, namely, SUMO-1, SUMO-2, and SUMO-3.7,8 Although these SUMO isoforms utilize the same enzyme cascades to form an isopeptide bond with the target proteins, each isoform can act in its own manner. The vast majority of SUMO-1 in cells is found in conjugates (SUMO-1 is able to assemble a polymeric chain in vitro as well9), whereas there is a large pool of free cellular SUMO-2 and SUMO-3.7 More interestingly, SUMO-2 and SUMO-3, which are nearly identical with an internal consensus site for SUMOylation, can form chains on substrates both in vivo and in vitro.10 As a special form of SUMO-mediated modification, polySUMOylation plays essential roles in promyelocytic leukemia protein (PML) localization,11 chromosome segregation, DNA-damage response, and meiosis.12,13 Especially, SUMO chains can function as a platform to recruit E3 ubiquitin ligase, enabling the degradation of SUMOmodified target.14,15 Meanwhile, the molecular mechanism underlying polySUMOylation occurrence is being revealed.16 Knipscheer et al. demonstrated that the noncovalent interaction between Ubc9 and SUMO promoted the formation of SUMO chains. The synergy of Ubc9 and the residue lysine 11, which is required for SUMO modification, results in the formation of © 2014 American Chemical Society
Received: November 14, 2013 Accepted: January 2, 2014 Published: January 2, 2014 967
dx.doi.org/10.1021/ac403753r | Anal. Chem. 2014, 86, 967−972
Analytical Chemistry
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Recombinant Protein Production. Purification of recombinant SUMO-2 and TC-SUMO2 was carried out as described previously.36 Briefly, 400 mL of LB media was inoculated with bacteria BL21 (DE3) transformed with either pET-28a-SUMO2 or pET-28a-TC-SUMO2. The protein expression was initiated by the addition of 1 mM isopropyl 1-thio-β-d-galactopyranoside and incubation at 37 °C for 4 h. Bacteria were then collected by centrifugation and resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM TCEP) containing the protease inhibitor cocktail, followed by sonication and clarification. The 6× histidine fusion protein in the soluble fraction was purified by nickel affinity chromatography. After sequential washing with the washing buffer I (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM TCEP) and washing buffer II (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 40 mM imidazole, 1 mM TCEP), the purified proteins were eluted with elution buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 200 mM imidazole, 1 mM TCEP). The concentration of eluted proteins was determined by the BCA protein assay kit according to the manufacturer’s protocol. In Vitro SUMOylation. In vitro SUMOylation reaction was carried out following the manufacturer’s instruction. Briefly, 50 ng of SUMO-2 or TC-SUMO2 was incubated in 10 μL of reaction solution containing SUMO activating enzyme, SUMO conjugating enzyme, Mg-ATP solution, and specific SUMOylation buffer at 37 °C for 2 h. The reaction products were either used for Western blotting assay with SUMO2-specific antibody or denatured immediately with 8 M urea followed by 2-fold dilution with phosphate buffered saline for single-molecule detection. Cell Culture, Transfection, and Nickel Affinity Chromatography to Purify Sp100-SUMO2 Conjugates. HEK293T cells obtained from American Type Culture Collection (Manassas, VA) were maintained as subconfluent monolayers in DMEM (Invitrogen) with 10% fetal bovine serum (Invitrogen) containing 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen) at 37 °C with 5% CO2. Cells growing on 60 mm Petri dishes were transfected with 2.5 μg of pcDNA3.1-TC-SUMO2 construct along with 2.5 μg of pcDNA3.1-His-Sp100 expression construct using the standard calcium phosphate precipitation method. After transfection for 36 h, the cells were collected for either Western blotting assay or immunoprecipitation experiments. For the drug treatment experiments in Figure 3, the cells were either treated with 400 ng/mL doxorubicin for 16 h37 or treated with 100 mM H2O2 for 20 min7 before lysis. To prevent the deSUMOylation of Sp100 by SUMO-specific protease, the cells were lysed under denaturing conditions (100 mM NaH2PO4, 10 mM Tris·HCl, 8 M urea, 1 mM TCEP, pH = 8.0) followed by nickel affinity chromatography.38 After extensive washing with washing buffer (100 mM NaH2PO4, 10 mM Tris·HCl, 4 M urea, 1 mM TCEP, pH = 6.3), the Sp100-SUMO conjugates were finally eluted with elution buffer (100 mM NaH2PO4, 10 mM Tris·HCl, 2 M urea, 1 mM TCEP, pH = 4.5).The elutes were either directly used for Western blotting assay or diluted 10-fold with phosphate buffered saline for single-molecule detection. Total Internal Reflection Fluorescence Imaging. An inverted Olympus IX71 microscope (Olympus, Japan) was equipped with an electric XY-piezo-Z stage (Applied Scientific Instrumentation, Eugene, OR) and a UAPON100XOTIRF objective (1.49 NA, Olympus). A sapphire 488-nm laser (50 mW, Coherent, USA) was used for biarsenical dye excitation.
simultaneously detecting protein SUMOylations with high sensitivity.25 For protein polySUMOylation, the targets are modified by polymeric chains among which the SUMO entities are attached in a compact manner.13,16 Thus, the fusion of SNAP/CLIP tag (size of ∼20 kDa)26 with the SUMO (size of ∼11 kDa)23 might sterically hinder the SUMO chain formation. To overcome the potential steric hindrance, we employed a small tetracysteine tag (
