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A Fluorescence-Resonance-Energy-Transfer-Based Protease Activity Assay and Its Use to Monitor Paralog-Specific Small Ubiquitin-Like Modifier Processing

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro
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  See discussions, stats, and author profiles for this publication at: Fluorescence resonance energy transfer-basedmethod for detection of DNA binding activitiesof nuclear factor κ B  Article   in  BioTechniques · August 2007 DOI: 10.2144/000112475 · Source: PubMed CITATIONS 14 READS 57 7 authors , including:Hua-Jun HeNational Institute of Standards and Technolo… 42   PUBLICATIONS   462   CITATIONS   SEE PROFILE Anhong ZhouUtah State University 86   PUBLICATIONS   1,037   CITATIONS   SEE PROFILE Adolfas K GaigalasNational Institute of Standards and Technolo… 88   PUBLICATIONS   1,812   CITATIONS   SEE PROFILE Sige ZouNational Institutes of Health 76   PUBLICATIONS   2,262   CITATIONS   SEE PROFILE All content following this page was uploaded by Sige Zou on 22 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Vol. 43 ı No. 1 ı 2007 ı BioTechniques ı 93 Short Technical Reports INTRODUCTION Nuclear factor κ  B (NF- κ  B) plays an important role in the inducible transcriptional response to pathogenic signals, oxidative stresses, and pro-inflammatory cytokines (1). NF- κ  B binds directly to its target genes as a homo- or heterodimer formed with members of the Rel family proteins that include p50, p52, p65/Rel, Rel B, and cRel. In the resting cells, NF- κ  B is mostly localized at the cytosol in inactive forms bound by the inhibitory I κ  B proteins (2,3). After stimulation by cytokines, stressors, or pathogenic signals, signal transduction pathways are activated, which lead to the phosphorylation of I κ  B and result in ubiquitination and degradation of I κ  B by proteasomes (4). Consequently, NF- κ  B is released and then trans-located to the nucleus, followed by binding to the κ  B consensus sequence located in the promoter regions of a variety of genes to induce gene expres-sions (1,5,6). Hence, DNA binding activities of NF- κ  B reflect cellular responses to various signals. Simple and flexible methods for the detection of DNA binding activities of NF- κ  B are highly desirable.A number of techniques have been developed for studying protein-DNA interactions. A commonly used method, electrophoretic mobility shift assay (EMSA) (7,8), detects slower migration of protein-DNA complexes relative to free DNA molecules using a nondena-turing gel electrophoresis. A reporter assay has also been used to detect DNA binding activity (9). The luciferase or β -galactosidase gene is placed under the control of a promoter that contains the consensus sequence recognized by the DNA binding protein. In addition, a chromatin immunoprecipitation (CHIP) assay has been developed for studying protein-DNA interactions (10). However, these assays are laborious, time-consuming, and difficult to apply for high-throughput screening.Renard et al. (11) established a DNA binding assay on the basis of a modified enzyme-linked immunosorbent assay (ELISA). Recently, methods in relative high-throughput platforms have been developed for studying protein-DNA interactions based on fluorescence resonance energy transfer (FRET) (12–15). For instance, Lu and his colleagues have described an assay that combines exonuclease III (ExoIII) protection strategy and FRET detection or SYBR ®  Green I staining method (14,15). In their assay, a double-stranded DNA (dsDNA) probe is designed to contain a pair of FRET fluorophores in the middle and two identical protein binding sites on each side of the FRET fluorophores. The NF- κ  B protein can protect the probe from ExoIII digestion, which results in a high FRET signal. This binding configuration, however, does not neces-sarily reflect endogenous DNA binding. Further, protein binding on one side may interfere the binding on the other side, because of the steric effect from complexation with other cotranscription factors likely present in cell extracts.In this study, we developed a method employing both restriction endonu-clease digestion and FRET detection strategy to study NF- κ  B-DNA inter-action. The DNA-FRET probe used is a dsDNA that contains a pair of FRET fluorophores at the same end of the probe and an endonuclease recog-nition site of the κ  B DNA consensus sequence. When the transcription factor binds to the dsDNA, it prevents the DNA probe from binding and subsequently being cleaved by the endonuclease, which then results in a high FRET signal. We compared this assay with the commonly used EMSA using purified recombinant NF- κ  B p50, nuclear extracts, and whole cell lysates. We examined the suitability of this FRET-based assay for high-throughput screening of NF κ  B activation. MATERIALS AND METHODSFRET Probes Oligonucleotides were synthe-sized and high-performance liquid chromatography (HPLC)-purified from Invitrogen (Carlsbad, CA, Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor κ  B Hua-Jun He 1,4 , Rick Pires 2 , Tie-Nian Zhu 3 , Anhong Zhou 4 , Adolfas K. Gaigalas 1 , Sige Zou 3 , and Lili Wang 1 1 National Institute of Standards and Technology, Gaithersburg, MD, 2 Montgomery College, Germantown, MD, 3 National Institute on Aging, Baltimore, MD, and 4 Utah State University, Logan, UT, USA  BioTechniques 43:93-98 (July 2007) doi 10.2144/000112475 The DNA binding protein nuclear factor κ    B (NF- κ    B) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including in-nate and adaptive immune responses, oxidative stress response, and aging. NF- κ    B also plays a key role in diseases, for example, cancer. A simple, convenient, and high-throughput detec-tion of NF- κ    B activation is therefore important for systematically studying signaling path-ways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF- κ    B. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET  fluorophores at the same end of the probe and an endonuclease binding site within the NF- κ    B consensus sequence. The activated NF- κ    B was detected by FRET following the restriction en- zyme digestion. Using three different analyte materials—( i ) purified recombinant NF- κ    B p50, ( ii ) nuclear extracts, and ( iii ) whole cell lysates—we demonstrated that this assay is as sensi-tive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF- κ    B DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF- κ    B activation.  94ıBioTechniquesı Vol. 43 ı No. 1 ı 2007 Short Technical Reports USA). The sequences of two pairs of NF- κ  B FRET probes are: (probe 1) 5 ′ -(FAM)AAGTG GGAAATTCC TCT G-3 ′  5 ′ -CAGA GGAATTTCC CAC TT(TAMRA)-3 ′ ; and (probe 2, a mutant probe) 5 ′ -(FAM)AAGTG TTAAATTC C TCTG-3 ′ , 5 ′ -CAGA GGAATTTAA CACTT(TAMRA)-3 ′  (16). The donor [carboxyfluorescein (FAM)] and acceptor [tetramethyl-6-carboxy-rhodamine (TAMRA)] fluorophores are attached to 5 ′  (dA) and 3 ′  (dT) via a C6 linker, respectively. The bold   sequences represent the NF- κ  B binding sites, and the underlined sequences are the recognition sites for restriction enzyme  Apo I, respectively. To obtain dsDNA-FRET probes, complementary oligonucleotide pairs were mixed at the same molar concentrations of 20 μ M in a 100- μ L Tris buffer solution (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). The mixture was heated for 4 min at 92 ° C and cooled down slowly to 25 ° C. The formed dsDNA probes were then purified by using native polyacrylamide gel electropho-resis (PAGE). DNA Binding Proteins, Cell Culture, and Preparation of Cell Extracts Purified recombinant NF- κ  B p50 (rhNF- κ  B p50) was purchased from Promega (Madison, WI, USA). The restriction endonuclease,  Apo I, was obtained from New England Biolabs (Ipswich, MA, USA). A control protein, glutathione S-transferase, was purified from  Escherichia coli  in house. To obtain NF- κ  B protein mixtures, HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin in a humid-ified chamber containing 5% CO 2  at 37 ° C, and were then treated with or without 20 ng/mL tumor necrosis factor- α  (TNF- α ) from R&D Systems (Minneapolis, MN, USA) for 30 min. Nuclear and cytoplasmic fractions were extracted with the NE-PER Nuclear and Cytoplasmic Extraction kits from Pierce Biotechnology (Rockford, IL, USA). The nuclear extracts were transferred to the binding reaction buffer using Microcon ®  YM-10 centrifugal filters (Millipore, Billerica, MA, USA) prior to the analysis. To collect the whole cell lysates, cell pellets were suspended in the lysis buffer [20 mM HEPES, pH 7.5, 0.35 M NaCl, 20% glycerol, 1% Nonidet ®  P40 (NP40), 0.5 mM EDTA] with Protease Inhibitor Cocktail Set I from Calbiochem (La Jolla, CA, USA). After setting on ice for 10 min, the lysates were centrifuged at 14,000 ×   g  for 20 min at 4 ° C, and the supernatant was collected and stored at -70 ° C until analysis. The protein concentration was determined using a BCA™ kit from Pierce Biotechnology. Electrophoretic Mobility Shift Assay EMSA was performed at room temperature as follows: recombinant NF- κ  B p50 or cell extracts were incubated with a DNA probe in the DNA binding buffer [10 mM HEPES, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 2.5 mM dithiothreitol (DTT), 0.05% NP40, 0.01 U poly(dI-dC) (Pierce Biotechnology), 0.5 mg/mL bovine serum albumin (BSA), and 10% (v/v) glycerol] for 10 min. To measure the dissociation constants in a compet-itive EMSA, restriction endonuclease  Apo I was mixed with NF- κ  B p50 in the binding buffer. After addition of dsDNA-FRET probes, the mixture was incubated for additional 20 min. Two microliters of the gel-loading buffer (250 mM Tris-HCl, pH 7.5, 40% glycerol) were then added to the reaction mixture, and the mixture was loaded on a pre-run 5% polyacryl-amide gel and electrophoresed at 100 V in 0.5 ×  Tris-borate-EDTA (TBE) buffer. DNA detection was performed using an FMBIO III Multi-View scanner. The band intensity was analyzed using FMBIO Image Analysis 3.0 software (both from MiraiBio, Alameda, CA, USA). � �  �  Figure 1.   The fluorescence resonance energy transfer (FRET) and restriction enzyme-based assay. (A) Representation of the assay. FRET-double-stranded DNA (dsDNA) probe 1 is used as an example. The italicized sequence refers to the nuclear factor κ  B (NF- κ  B) p50 binding site, and the underlined sequence represents the restriction enzyme recognition site, respectively. (B) Emission spectra of the FRET-dsDNA probe alone (curve 1), the FRET probe digested for 60 min by a 10 U  Apo I (curve 2), and the FRET probe digested for 60 min by 10 U  Apo I in the presence of 20 pmol recombinant NF- κ  B p50 (curve 3). Fam, carboxyfluorescein; TAMRA, tetramethyl-6-carboxyrhodamine; A.U., arbitrary units. AB  Vol. 43 ı No. 1 ı 2007 ı BioTechniques ı 95 Short Technical Reports Fluorescence Measurement and FRET Analysis For FRET analysis, the dsDNA-FRET probe and the DNA binding protein were incubated in the binding buffer for 20 min, followed by addition of MgCl 2  and restriction endonuclease  Apo I to a final concentration of 2 mM. The reaction mixture was kept at 25 ° C for different time periods. EDTA was then added to a final concentration of 5 mM in a total volume of 20 μ L to terminate the restriction reaction. The mixture was further diluted with 480 μ L buffer solution containing 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA and was immediately subjected to fluorescence measure-ments at 30 ° –32 ° C. Emission spectra were collected by using a Starna ®  Semi-Micro cuvette, type 9F, and a spectrofluorimeter with a 488-nm excitation source (17). Approximately 0.5 mL reaction mixture was used for the measurements while stirred with a small magnetic bar.The FRET efficiency,  R , is calculated as  R  = 1 - [( F  ( t  ) - F  (0)]/[ F  ( max  ) - F  (0)], where F(t)  refers to the fluorescence intensity of a reaction mixture under the endonuclease treatment for a given time t   at 518 nm, and F  ( 0 ) represents the fluorescence intensity at 518 nm at zero-time point following endonuclease addition in the absence of DNA binding proteins (14). The parameter F  ( max  ) is the fluorescence emission intensity at 518 nm under endonuclease treatment in the absence of DNA binding proteins for a maximal time point of an experiment. A relative FRET efficiency, Δ  R  =  R ( t  ) -  R ( c ), where  R ( t  ) is the transfer efficiency of an analyte protein sample and  R ( c ) is the value of the corresponding control sample (purified GST protein, nuclear extracts, or whole cell lysates without TNF- α  treatment), respectively. RESULTS AND DISCUSSIONExperimental Design Figure 1A describes the scheme for the detection of the DNA binding activities of NF- κ  B using dsDNA-FRET probe 1 as an example. The dsDNA-FRET probe consists of two complementary oligonucleotides, one labeled with a FAM fluorophore at the 5 ′  end serving as the energy donor, and the other with a TAMRA fluorophore at the 3 ′  end as the acceptor. After annealing of the two oligonucleotides, the donor and acceptor fluorophores are brought adjacently for efficient energy transfer (18,19). The oligonucleotide sequences are chosen based on the NF- κ  B binding sequences, also known as κ  B elements (5 ′ -GGGRNYYYCC-3 ′; underlined sequences define the inner variables). The outer GG cores in these elements are separated by variable inner base pairs, whereas  R  is a purine, Y   is a pyrimidine, and  N   can be any nucleotide. Four flanking nucleotides were added to both ends of the sequences for enhancing the stability of dsDNA. Within the conserved GG cores is the recognition site for the restriction enzyme  Apo I.In the absence of NF- κ  B and a endonuclease, the free probe contains two fluorophores close to each other, which results in very low donor fluorescence at 518 nm and high FRET signal at 588 nm as shown in Figure 1B (curve 1). With the presence of the endouclease, the free probe was cleaved into small fragments. Since the melting temperatures (T m ) of these fragments were < 20 ° C, which were lower than the reaction and detection temperatures ( ≥ 25 ° C), dissociation of these fragments to single strands was expected. This resulted in higher donor fluorescence at 518 nm and a lower FRET signal at 588 nm as shown in Figure 1B (curve 2). When NF- κ  B was added into the probe solution, NF- κ  B was bound to the endonuclease recog-nition site, which protected the probe from cleavage by the endonuclease and resulted in a high FRET signal (Figure 1B, curve 3). The amount of the FRET signal, therefore, reflects the DNA binding activities of NF- κ  B. Competitive EMSA Showing the Feasibility of the Experimental Design Figure 2 shows the results of compet-itive EMSA with either a fixed amount of 1.2 pmol  Apo I (A) or a fixed amount of 3-pmol recombinant NF- κ  B p50 (B). In both assays, the amount of FRET probe was fixed at 0.28 pmol. Assuming that both DNA binding proteins (  Apo I represented by  E   and NF- κ  B p50 by T  ) exist in two states only—either bound to dsDNA probe ( S  ) or free in solution—the protein distributions are governed by the following equilibria: where k  1 [  E  ][ S  ] = k  -1 [  ES  ] and k  2 [ T  ][ S  ] = k  -2 [ TS  ]. The dissociation constant of  E    Figure 2.   Competitive electrophoretic mobility shift assay (EMSA) for evaluating the feasibil-ity of the experimental design.  In the assays, the amount of the fluorescence resonance energy transfer (FRET)-double-stranded DNA (dsDNA) probe was fixed at 0.28 pmol in a total volume of 20 μ L. (A) Competitive binding of the DNA probe by various amount nuclear factor κ  B (NF- κ  B) p50 in presence of the fixed amount of 1.2 pmol  Apo I. Lane 1, 10 pmol; lane 2, 5 pmol; lane 3, 2 pmol; lane 4, 1 pmol; lane 5, 0.5 pmol; and lane 6, 0.2 pmol. The only FRET probe is pre-sented in lane 7. (B) Competitive binding of the DNA probe by various amount of  Apo I at the fixed amount of 3 pmol NF- κ  B p50: lane 2, 0 pmol; 3, 0.12 pmol; 4, 0.24 pmol; 5, 0.6 pmol; 6, 1.2 pmol; 7, 2.4 pmol; 8, 4.8 pmol. The only FRET probe is presented in lane 1. Arrows indi-cate the positions for free FRET-dsDNA probe (lower),  Apo I-DNA probe complex (middle), and NF- κ  B p50-DNA probe complex (upper), respectively. AB S + EESk  1 k  -1 +TTSk  2 k  -2 NF- κ  B p50 Probe  Apo I ProbeFree ProbeNF- κ  B p50 Probe  Apo I ProbeFree Probe  96ıBioTechniquesı Vol. 43 ı No. 1 ı 2007 Short Technical Reports and S   binding is thus defined as K  d1  = k  -1  /  k  1  = [  E  ][ S  ]/[  ES  ], and the dissociation constant of T   and S   binding is as K  d2  = k  -2  /  k  2  = [ T  ][ S  ]/[ TS  ]. Hence, K  d2  /  K  d1  = ([  ES  ] [ T  ]) / ([ TS  ] [  E  ]) = ([  ES  ] ([ T  ] 0  - [ TS  ])) / ([ TS  ] ([  E  ] 0  - [  ES  ])), whereas [  E  ] 0  is the concentration of the endonuclease added and [T] 0  is the concentration of NF- κ  B added, respectively. Based on the band intensities shown in Figure 2, the dissociation constants of K  d1  and K  d2  were calculated to be 1.7 ±  0.3 nM and 1.3 ±  0.3 nM, respectively. These constants suggest that the dsDNA probe binds more strongly to NF- κ  B p50 than  Apo I, considering the fact that not all of the recombinant NF- κ  B p50 is in the active state. The competitive EMSA demonstrated the feasibility of the experimental design described above. Optimization of NF- κ  B-DNA Binding and Endonuclease Reaction To optimize the parameters for NF- κ  B-DNA binding and endonuclease reaction, we examined incubation time, temperature, and buffer composition. First, we investigated the effect of glycerol concentrations on NF- κ  B-DNA binding and restriction digestion and found that a 10% glycerol was optimal for  Apo I activity (see Supplementary Figure S1A available online at In general, high concentrations of glycerol enhanced NF- κ  B-DNA binding (data not shown). Using  Apo I, we further tested the temper-atures for reactions ranging from room temperature to 50 ° C (the temperature for optimal  Apo I enzymatic activity). We found that 25 ° C was suitable for both NF- κ  B-DNA binding and  Apo I cleavage reaction. For FRET detection, temperatures from 30 ° –32 ° C were shown to be optimal (Supplementary Figure S1B), which were below the T m  of FRET dsDNA probe (approxi-mately 40 ° C). In addition, to minimize the background signal partially from nonspecific binding, the concentra-tions of BSA, poly(dI-dC), and salmon sperm DNA were examined. We found that 0.01 U poly(dI-dC) plus 0.5 mg/ mL BSA showed the best result, while salmon sperm DNA was not critical. Lastly, we observed that 5 mM EDTA was sufficient to terminate the reaction (data not shown).Based on these tests, we concluded that an optimal assay would be performed in the binding buffer containing 10% glycerol, 0.01 U poly(dI-dC), and 0.5 mg/mL BSA at 25 ° C, and terminated by EDTA at a final concentration of 5 mM. The fluorescence reading would be carried out at 30 ° C. Detection of DNA Binding Activities of Recombinant NF- κ  B p50 We applied the optimized assay condition to detect DNA binding activities of recombinant NF- κ  B p50 and compared the results with EMSA. For EMSA, with increased concentra-tions of NF- κ  B p50, the intensity of the band for NF- κ  B p50-dsDNA complex increased until reaching saturation (Figure 3, A and B). A protein concen- Figure 3.   Comparison of electrophoretic mobility shift assay (EMSA), fluorescence resonance energy transfer (FRET), and restriction enzyme-based assays.  (A) EMSA was performed with vari-ous amounts of recombinant nuclear factor κ  B (NF- κ  B) p50. Lane 1, 0 pmol; lane 2, 0.1 pmol; lane 3, 0.2 pmol; lane 4, 0.5 pmol; lane 5, 1.0 pmol; lane 6, 2 pmol; lane 7, 5 pmol; and lane 8, 10 pmol. (B) The relative FRET efficiency was measured by FRET assays ( Δ  R , left axis) and compared with the intensities of shifted bands in the EMSA assays (right axis) using various concentrations of the recombinant NF- κ  B p50. (C) EMSA was performed with different amounts of tumor necrosis factor- α  (TNF- α )-treated nuclear extracts. Lane 1, 0 μ g; lane 2, 0.2 μ g; lane 3, 0.5 μ g; lane 4, 1 μ g; lane 5, 2 μ g; lane 6, 5 μ g; lane 7, 10 μ g; lane 8, 20 μ g; lane 9, 5 μ g with a 20 ×  nonfluorescent probe 1; and lane 10, 5 μ g with a 100 ×  nonfluorescent probe 1. (D) The relative FRET efficiency ( Δ  R ) was measured by FRET assays ( Δ  R , left axis) and compared with the intensities of shifted bands in the EMSA assays (right axis) using various concentration of the TNF- α -treated nuclear extracts. (E) EMSA was performed with different amounts of TNF- α -treated whole cell lysates: lane 1, 0 μ g; lane 2, 0.5 μ g; lane 3, 1 μ g; lane 4, 2 μ g; lane 5, 5 μ g; lane 6, 10 μ g; lane 7, 20 μ g; lane 8, 40 μ g; lane 9, 10 μ g with the mutant probe 4; lane 10, 10 μ g with 200 ×  nonfluorescent probe. (F) The relative FRET efficiency ( Δ  R ) was measured by FRET assays ( Δ  R , left axis) and compared with the intensities of shifted bands in the EMSA assays (right axis) using vari-ous concentrations of the TNF- α -treated whole cell lysates. Less than 10% of standard deviations were observed for EMSA. ACEBDF κ κ κ  NF- κ  B
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