Bioluminescent Indicator for Determining ProteinProtein Interactions

Protein-Protein Interactions Using Intramolecular. Complementation of Split Click Beetle Luciferase. Sung Bae Kim,† Yosuke Otani,‡ Yoshio Umezawa,...
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Anal. Chem. 2007, 79, 4820-4826

Bioluminescent Indicator for Determining Protein-Protein Interactions Using Intramolecular Complementation of Split Click Beetle Luciferase Sung Bae Kim,† Yosuke Otani,‡ Yoshio Umezawa,*,‡ and Hiroaki Tao*,†

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan, and Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan

Click beetle luciferase (CBLuc) is insensitive to pH, temperature, and heavy metals, and emits a stable, highly tissue-transparent red light with luciferin in physiological circumstances. Thus, the luminescence signal is optimal for a bioanalytical index reporting the magnitude of a signal transduction of interest. Here, we validated a singlemolecule-format complementation system of split CBLuc to study signal-controlled protein-protein (peptide) interactions. First, we generated 10 pairs of N- and Cterminal fragments of CBLuc to examine respectively whether a significant recovery of the activity occurs through the intramolecular complementation. The ligand binding domain of androgen receptor (AR LBD) was connected to a functional peptide sequence through a flexible linker. The fusion protein was then sandwiched between the dissected N- and C-terminal fragments of CBLuc. Androgen induces the association between AR LBD and a functional peptide and the subsequent complementation of N- and C-terminal fragments of split CBLuc inside the single-molecule-format probe, which restores the activities of CBLuc. The examination about the dissection sites of CBLuc revealed that the dissection positions next to the amino acids D412 and I439 admit a stable recovery of CBLuc activity through an intramolecular complementation. The ligand sensitivity and kinetics of the single molecular probe with split CBLuc were discussed in various cell lines and in different proteinpeptide binding models. The probe is applicable to developing biotherapeutic agents on the AR signaling and for screening adverse chemicals that possibly influence the signal transduction of proteins in living cells or animals. Luciferase is a family of photoproteins that can be isolated from a large variety of insects, marine organisms, and prokaryotes.1-4 * Corresponding authors. E-mail: [email protected]; [email protected]. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ The University of Tokyo. (1) Kim, S. B.; Ozawa, T.; Watanabe, S.; Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11542-11547. (2) Kim, S. B.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2005, 77, 6588-6593. (3) Viviani, V. R.; Uchida, A.; Viviani, W.; Ohmiya, Y. Photochem. Photobiol. 2002, 76, 538-544.

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The emission spectrum ranges between 400 and 620 nm. Firefly luciferase (FLuc) and Renilla luciferase (RLuc) are sensitive luciferases for luminescence development and are widely used by researchers to identify different biological events of cells in culture and in living subjects. However, they are sensitive to pH, temperature, and heavy metals in physiological conditions and emit comparably short-wavelengh reporter lights (λmax ) 560 nm for FLuc and λmax ) 480 nm for RLuc). Most of the emitted light is thus unfortunately absorbed in tissues before determined by external light detectors upon the luciferases utilized in living animals.5 Beetle luciferases produce bioluminescence of different colors ranging from green to red using the same luciferin substrate.3,6,7 They are categorized into two groups according to their bioluminescence spectral sensitivity to pH, i.e., pH-sensitive and -insensitive luciferase. pH-sensitive luciferases such as FLuc undergo a red shift with decreasing pH or increasing temperature or heavy metal cation concentration, whereas pH-insensitive luciferases like click beetle- and railroad worms-derived ones are insensitive to those conditions.3 Click beetle luciferase (CBLuc) emits a stable light in various physiological circumstances inside cells or tissues, due to the insensitivity to pH, temperature, and heavy metals. In addition, an engineered CBLuc for mammalian expression emits red light (λmax ) 615 nm), which is a highly red-shifted wavelength compared to those from other insect and marine organism luciferases. The red light, close to near-infrared, is highly tissuetransparent, thus especially attractive for the molecular imaging of a signal transduction of interest in living animals.5 Therefore, the luminescence light by CBLuc is optimal for a bioanalytical index reporting the extent of a signal transduction of interest inside living cells or animals. Monomeric photoproteins such as beetle luciferases can be split into two portions with resulting functionally inactive fragments. These split reporters have been used for measuring realtime protein-protein interactions in living cells or animals. This combination of two protein fragments to restore activity has been termed protein fragment complementation. Paulmurugan et al. split FLuc and RLuc, and the resulting self-association of the split (4) Hastings, J. W. Gene 1996, 173, 5-11. (5) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123-128. (6) Kaihara, A.; Kawai, Y.; Sato, M.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2003, 75, 4176-4181. (7) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2005, 77, 1295-1302. 10.1021/ac0621571 CCC: $37.00

© 2007 American Chemical Society Published on Web 06/01/2007

fragments was used for studying known positive interacting proteins such as Id and myoD.7-9 In our previous studies, we split various functional proteins including RLuc and GFP for the purpose of developing methods for determining the extent of activities of signaling factors in intracellular signaling network.1,2,6,10-12 These split reporter proteins have been used for measuring biologically meaningful signal transductions inside cells, where the activity of the reporter proteins is recovered by a complementation or splicing of the protein fragments. The methods were constructed on the premise that the two component proteins of the analysis system should be equally expressed beforehand. However, biased expression efficiencies of the two component proteins may result in an inefficiency of the systems. Very recently, we developed a single-molecule-format bioluminescent probe for determining ligand-induced protein-protein (peptide) interactions inside a single molecular backbone.13 The probe contains the N- and C-terminal fragments of a split luciferase, between which two proteins (peptides) of interest were inserted. Upon stimulation of a signal, an intramolecular protein complementation occurs to restore the luciferase activities.13 The probe is characterized as a bioluminescent indicator that comprises all the component proteins (peptides) of separate entities, e.g., more than two proteins (peptides), for ligand sensing and signal development in the single molecular backbone. Here, we validated a new single-molecule-format bioluminescent system using an intramolecular complementation of split CBLuc to study ligand-controlled protein-protein interactions (Figure 1A). We made use of a CBLuc as a functional reporter protein, which is engineered for red light emission and mammalian expression, originally cloned from Pyrophorus plagiophthalamus, a large click beetle indigenous to the Caribbean. We identified suitable dissection sites of the CBLuc to generate rational fragments of the monomeric reporter protein that can recover bioluminescence through an intramolecular complementation after the temporal loss of the activity by dissection. Ten dissection points in CBLuc were selected inside/near the hinge region of the two subdomains based on the molecular structural knowledge. The ligand binding domain of androgen receptor (AR LBD) was fused to a peptide sequence such as a conserved LXXLL motif of coactivators through a flexible linker. This fusion protein was sandwiched between the dissected fragments of CBLuc. Thus, we tested 10 pairs of fragments of split CBLuc by flanking them respectively at both ends of the single molecular backbone (Figure 1B). The present probe with the fragments of split CBLuc at an optimum dissection point provides high agonist selectivity and reproducible luminescence to a repeated androgen addition and withdrawal. We demonstrate that (1) the present single-moleculeformat indicator with split CBLuc enables the host cells to emit highly androgen-selective, red bioluminescence upon transfected and (2) AR LBD prefers to bind the conserved LXXLL motif of (8) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2003, 75, 1584-1589. (9) Paulmurugan, R.; Umezawa, Y.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15608-15613. (10) Ozawa, T.; Takeuchi, T. M.; Kaihara, A.; Sato, M.; Umezawa, Y. Anal. Chem. 2001, 73, 5866-5874. (11) Kim, S. B.; Ozawa, T.; Umezawa, Y. Anal. Biochem. 2005, 347, 213-220. (12) Kim, S. B.; Takao, R.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2005, 77, 69286934. (13) Kim, S. B.; Awais, M.; Sato, M.; Umezawa, Y.; Tao, H. Anal. Chem. 2006, 79, 1874-1880.

coactivators with an antiparallel orientation. The probe is applicable to developing biotherapeutic agents active in a protein signaling network and for screening adverse chemicals that possibly induce malfunctions of signaling proteins. Many of the molecular signaling events in living cells can be determined with a similar single-molecule-based scheme. EXPERIMENTAL SECTION Plasmid Construction. As a template, pCBR-control vector containing the full-length cDNA of an engineered click beetle luciferase was purchased from Promega. The cDNAs of N-terminal (CBLuc-N) and C-terminal (CBLuc-C) fragments were generated by PCR to introduce unique restriction sites, HindIII, KpnI, BamHI, or XhoI, at both ends of the fragments using adequate primers (Supporting Information Table 1) and the template vector. The cDNAs encoding ligand binding domains of AR (672-910 AA) and glucocorticoid receptor (GR) (527-777 AA) were synthesized by PCR to introduce adequate restriction sites, KpnI and NotI, at the both ends. cDNA oligomers encoding the following peptides were purchased from Exigen (Tokyo, Japan) (Supporting Information Table 1): (1) an AR N-terminal motif (11 AA; 20RGAFQNLFQSV30), (2) a normal LXXLL motif of TIF2 (13 AA; 686KHKILHRLLQDSS698), (3) a reversed LXXLL motif of TIF2 (13 AA; 698SSDQLLRHLIKHK686), or (4) mutated LXXLL motifs of TIF2 (13 AA; 686KHKIRHRLLQDSS698 for pSimbe-LXM1; 686KHKILHRRLQDSS698 for pSimbe-LXM2; and 686KHKILHRLRQDSS698 for pSimbe-LXM3). The cDNA oligomers commonly comprise unique restriction sites, NotI and BamHI, at their both ends. The amplified fragments of each site were cloned in the corresponding restriction enzyme-digested pcDNA 3.1 (+) vector backbone (Invitrogen). The constructed plasmids were sequenced to ensure fidelity with a BigDye Terminator Cycle Sequencing kit and a genetic analyzer ABI Prism310 (PE Biosystems). Decision of Suitable Dissection Points in CBLuc for a Single-Molecule-Format Bioluminescent Probe. Dissection points in CBLuc suitable for constructing a single-molecule-format probe were explored based on the recovery of the luminescence intensities in the presence of 10-5 M 5R-dihyoxytestosterone (DHT). Ten dissection points were illustrated in Figure 1C. Based on the dissection points, 10 pairs of N- and C-terminal fragments of CBLuc were flanked in a single-molecule backbone consisting of AR LBD and a FQNLF motif, 20RGAFQNLFQSV30, as shown in Figure 1B. The plasmids were numbered from 1 to 10 according to the dissection points. The N- and C-terminal fragments consisting of each plasmid are respectively indicated as follows: plasmid 1, 1-387 and 388-542; plasmid 2, 1-430 and 431-542; plasmid 3, 1-439 and 440-542; plasmid 4, 1-463 and 464-542; plasmid 5, 1-480 and 481-542; plasmid 6, 1-412 and 413-542; plasmid 7, 1-442 and 443-542; plasmid 8, 1-439 and 413-542; plasmid 9, 1-439 and 443-542; plasmid 10, 1-439 and 437-542. The luminescence intensities by each probe expressed in human cervical carcinoma-derived HeLa cells were estimated using a Bright-Glo substrate kit (Promega) in the presence or absence of 10-5 M DHT for 20 min. A brief procedure for the Bright-Glo substrate kit is as follows: The cells on the 12-well plates were transiently transfected with a plasmid and washed once with PBS. An 80-µL aliquot of substrate solution was added to each well of the plates. After a 3-min incubation at 37 °C, the developed luminescence intensities Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

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Figure 1. (A) Schematic diagram showing the detection scheme of the single-molecule-format bioluminescent indicator based on an intramolecular complementation strategy of split CBLuc to monitor bioactive small molecules. An agonist induces the conformational change in the ligand binding domain of a nuclear receptor (NR LBD). It subsequently activates the association of NR LBD with the specific recognition peptide sequence such as “FQNLF” or “LXXLL” motif. The association triggers the recovery of the CBLuc activities by an intramolecular complementation of split CBLuc. The recovered luciferase activities were taken as a measure of the androgenicity of ligands. On the other hand, removal of the agonist dissociates the complementation between NR LBD and the motif and cancels the developed CBLuc activities. Agonist was animated with a key, while the split CBLuc was drawn as a half-segmented ball. Abbreviations: CBLuc-N, N-terminal fragment of click beetle luciferase; CBLuc-C, C-terminal fragment of click beetle luciferase; NR LBD, ligand binding domain of nuclear receptor; N-term, a peptide sequence such as ‘FQNLF’ or ‘LXXLL’ motif; GS linker, a flexible amino acid sequence consisting of glycines and serines. (B) Schematic structures of cDNA constructs. The plasmids constructed in the present study were named “pSimbe”, which means a plasmid encoding a SIngle Molecule-format probe using click BEetle luciferase. The plasmids modified from the pSimbe plasmid backbone were named as follows according to the cofused peptide sequences: plasmids 1-10 (with a FQNLF motif; 20RGAFQNLFQSV30), pSimbe-LXA (with a normal LXXLL motif; 686KHKILHRLLQDSS698), and pSimbe-LXP (with a reversed LXXLL motif; 698SSDQLLRHLIKHK686). The constructs were respectively subcloned inside a pcDNA 3.1(+) backbone. Abbreviations: AR LBD, ligand binding domain of androgen receptor; peptide, a specific peptide sequence for binding AR LBD such as ‘FQNLF’ or ‘LXXLL’ motif. (C). Determination of appropriate dissection points in CBLuc applicable for a singlemolecule-format probe based on the recovered luminescence intensities by 10-5 M DHT. The numbers 1-10 in bracket show respective plasmid numbers with different N- and C-terminal fragments of split-CBLuc (see Experimental Section for the specific domains). Luminescence intensities by the respective single-molecule-format probes expressed with the plasmids from 1 to 10 were determined in the presence or absence of 10-5 M DHT. The plasmids 8-10 contain overlapping fragments or shortened fragments. The plasmids 3, 6, 9, and 10 showed significant signal-to-background ratios upon exposure to 10-5 M DHT. The error bar is the standard error of the mean for three samples (n ) 3).

from the cell lysates were recorded with a luminometer (Minilumat LB9506; Berthold). The amount of proteins was sequentially determined using a Bradford reagent for the following normalization. The click beetle luminescence normalized against the determined protein amount was expressed as “RLU/µg of protein (Bright Glo)”, which means the click beetle luminescence intensity from the 1 µg of cell lysate. “RLU ratio (()” means the luminescence ratio, RLU (+)/RLU (-), where RLU (+) is the RLU/µg of protein (Bright-Glo) of the cell lysate upon stimulation with a ligand, and RLU (-) is that of the lysate upon stimulation with a vehicle (0.1% DMSO). Association of AR LBD with Several Motifs in a Single Molecule. The study on the dissection points showed that plasmid 3 exhibits the highest signal-to-background ratio to 10-5 M DHT (Figure 1C). The extent of recovered activity of the fragmented CBLuc by 10-5 M DHT was estimated to be 0.97 ( 0.09% of the 4822 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

full CBLuc activity. The extent of this recovered activity is consistent with the previously reported case of split FLuc, i.e., from 0.01 to 4%,7 which was enough to be applied for small animals. Based on the plasmid 3 backbone, two plasmids with a normal or a reverse LXXLL motif were newly constructed for exploring the association of AR LBD with a conserved LXXLL motif of nuclear receptor coactivators (Figure 2A and Supporting Information Figure 1). The specific amino acid sequences of the motifs were as follows: (1) a normal FXXLF motif of human AR, 20RGAFQNLFQSV30, (2) a normal LXXLL motif of African clawed frog transcription intermediary factor 2 (TIF2), 686KHKILHRLLQDSS698, and (3) a reversed LXXLL motif, 698SSDQLLRHLIKHK686. The reversed LXXLL motif is an artificial amino acid sequence made by reversing the codons of the normal LXXLL motif to explore the orientation upon the association of AR LBD with a LXXLL motif. The newly constructed plasmids with a

reversed LXXLL motif and a normal LXXLL motif were named pSimbe-LXP and pSimbe-LXA, respectively. In the case of the probe from pSimbe-LXA, AR LBD binds a normal LXXLL motif with an antiparallel orientation (see Figure 1A). That is why the plasmid is named pSimbe-LXA. HeLa cells carrying plasmid 3, pSimbe-LXP, or pSimbe-LXA, were stimulated with 10-5 M DHT or vehicle (0.1% DMSO) for 20 min. The luminescence intensities were developed with the Bright-Glo substrate kit and determined with the luminometer. Association of AR LBD or GR LBD with the Recognition Peptide in a Single Molecule. Androgen-induced association of AR LBD or GR LBD with LXXLL motifs was studied on the basis of the developed luminescence intensities (Figure 2A). Two new plasmids with GR LBD were constructed as a control for the androgen-induced association of AR LBD with a LXXLL motif in human breast cancer-derived MCF-7 cells. The plasmids with GR LBD were named pSimbe-GRA and pSimbe-GRP, respectively, according to the normal or reversed orientation of the linked LXXLL motif (Figure 1B). The MCF-7 cells carrying pSimbe-LXA, pSimbe-LXP, pSimbe-GRA, or pSimbe-GRP were stimulated with 0.1% DMSO or 10-5 M DHT for 20 min. The luminescence intensities were developed with the Bright-Glo substrate kit and determined with the luminometer. Furthermore, the recognition peptide for AR LBD in pSimbeLXA was replaced with a mutated peptide of the LXXLL motif of TIF2 for an additional negative control study (Supporting Information Figure 2). Each leucine in the LXXLL motif was one-point mutated with arginine for constructing the mutated peptides, i.e., RXXLL, LXXRL, and LXXLR. The plasmids with the three mutated peptides were respectively named pSimbe-LXM1, pSimbe-LXM2, and pSimbe-LXM3. The respective plasmids were transiently transfected into MCF-7 cells. The luminescence intensities were then determined 20 min after the addition of 10-5 M DHT. Ligand Selectivity of the Probe Expressed with pSimbeLXA in Different Cell Lines. The variations of the ligand selectivity of the probe from pSimbe-LXA were estimated in four cell lines: (1) human breast cancer-derived MCF-7, (2) Chinese hamster ovary-derived CHO, (3) human cervical carcinomaderived HeLa, and (4) mouse fibroblast-derived NIH 3T3 cells (Figure 2B). pSimbe-LXA was transiently transfected in the four cell lines using a transfection reagent (TransIT-LT, Mirus). The cells were stimulated with a 10-5 M of 17β-estradiol (E2), testosterone (T), or DHT for 20 min to initiate the association between AR LBD and the reversed LXXLL motif. The luminescence intensities were developed with the Bright-Glo substrate kit, and determined with the luminometer. Androgenicities of various steroids and synthetic chemicals were determined with the probe expressed from pSimbe-LXA (Supporting Information Figure 3). The MCF-7 cells carrying pSimbe-LXA were stimulated with 10-5 M of each ligand for 20 min. The relative luminescence intensities from the MCF-7 cells with pSimbe-LXA in response to ligands were taken as their androgenic activities. Dose-Response Curves of the Probe Expressed with pSimbe-LXA to Ligands. Dose-response curves of pSimbe-LXA expressed in MCF-7 cells to various concentrations of ligands were determined and compared with that of plasmid 3 (Figure 3A). MCF-7 cells were transfected with pSimbe-LXA or plasmid 3 using

Figure 2. (A) Negative control study for examining NR LBD-peptide association. Comparison of the binding affinity of AR LBD or GR LBD with a LXXLL motif (n ) 3). For the present study, four different plasmids were constructed based on the combinations of the component domains, i.e., AR LBD, GR LBD, a normal LXXLL motif, and a reversed LXXLL motif. The luminescence intensities by the probe expressed with plasmids in MCF-7 cells were compared in the presence or absence of 10-5 M DHT. (B). Determination of ligand selectivity of the probe expressed with pSimbe-LXA in four different cell lines, MCF-7, CHO, HeLa, and NIH 3T3 cells, utilizing transient transfection studies (n ) 3). The cell lines carrying pSimbe-LXA were stimulated with a 10-5 M E2, T, or DHT for 20 min to initiate the association between AR LBD and a LXXLL motif and the emission of bioluminescence light (λmax ) 615 nm). The specific amino acid sequence of the LXXLL motif was as follows: 686KHKILHRLLQDSS698, which is the sequence from 686 to 698 AA of African clawed frog TIF2 (transcription intermediary factor 2). Abbreviations: CHO, Chinese hamster ovary cell; HeLa, human cervical carcinoma cell; MCF-7, human breast cancer cell; NIH 3T3, mouse fibroblast cell.

the transfection reagent. The cells were stimulated with E2, T, or DHT, with the concentrations ranging from 10-8 to 10-4 M, for 20 min for initiating the association of AR LBD and the respective motifs. The luminescence intensities were developed with the Bright-Glo substrate kit and determined with the luminometer. Inhibitory Effects of Androgen Antagonists. An inhibitory effect of an androgen antagonist on the luminescence intensities developed by DHT was tested with the MCF-7 cells carrying pSimbe-LXA (Figure 3B). MCF-7 cells were transfected with pSimbe-LXA using the transfection reagent and extensively incubated for 16 h. The MCF-7 cells in each well were prestimuAnalytical Chemistry, Vol. 79, No. 13, July 1, 2007

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Figure 4. Time course of the bioluminescence intensities from the probe expressed with pSimbe-LXA in MCF-7 cells by the deprivation of DHT after the stimulation of 10-5 M DHT for 20 min (n ) 3). The changes of luminescence intensities were monitored for 2 h after the DHT deprivation by medium change. The fluctuation of the luminescence intensities by the repeated stimulation and deprivation of DHT was monitored for another 2 h. The other experiments respectively exhibited 30 times and 8 times higher luminescence intensities with the cells carrying pSimbe-LXA than the background. In the present experiments, the method of sample preparation highly influenced the signal-to-noise (S/N) ratios. Supernatants of the cell lysates after adding the substrate solution caused 8 times higher luminescence intensities than the background, whereas the homogenized mixture of cell lysates with the substrate solution exhibited 30 times higher luminescence intensities compared to the background. The homogenization of the sample highly decreased the background luminescence intensities, which resulted in the high S/N ratios.

Figure 3. (A). Dose-response curves for steroid hormones based on the bioluminescence intensity of the MCF-7 cells with plasmid 3 or pSimbe-LXA (n ) 3). (B). An inhibitory effect of CPA on the bioluminescence intensities developed by 10-6 M DHT in MCF-7 cells carrying pSimbe-LXA. The 10 and 100 times higher concentrations of CPA than DHT antagonized the association of AR LBD with a LXXLL motif in MCF-7 cells (n ) 3).

lated with 10-5 M cyproterone acetate (CPA), 10-4 M CPA, or vehicle (0.1% DMSO) for 5 min. All the cells except for the control were then additionally stimulated with 10-6 M DHT (final concentration) for 20 min. The luminescence intensities of each well were determined with the Bright-Glo substrate kit (Promega). Time Course of the Luminescence Intensities by the Probe to Ligands. The time course of the bioluminescence intensities from the MCF-7 cells with pSimbe-LXA in response to DHT was determined (Supporting Information Figure 4). MCF-7 cells cultured in a 24-well plate were transfected with pSimbeLXA, using the transfection reagent, and were incubated in 37 °C for 16 h. The cells in each well were stimulated with DHT ranging from 10-7 to 10-5 M for 0, 5, 10, 20, or 30 min. The luminescence intensities of the cells were then estimated with the Bright-Glo substrate kit (Promega). Reversibility of the Probe Expressed with pSimbe-LXA in Response to Androgen. Reversibility of the luminescence intensities of the present luminescent probe was estimated by a DHT treatment and its withdrawal (Figure 4). The MCF-7 cells cultured in a 24-well plate were transfected with pSimbe-LXA using the transfection reagent. After the subsequent incubation for 16 h, all the cells in the plate wells were first stimulated with 10-5 M DHT for 20 min. The medium in the plate wells was then 4824 Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

replaced with a fresh MEM supplemented with 10% FBS and 1% P/S. At 0.33, 1, and 2 h after the medium change, the luminescence intensities from the cells were respectively determined with the Bright-Glo substrate kit. The remaining cells in the 24-well plate were stimulated once again with 10-5 M DHT for 20 min. The medium filled in the plate wells was replaced repeatedly with a fresh MEM supplemented with 10% FBS and 1% P/S for removing DHT. At 0.33, 1, and 2 h after the second medium change, the luminescence intensities were respectively determined with the Bright-Glo substrate kit. RESULTS AND DISCUSSION Appropriate Dissection Point in CBLuc for a SingleMolecule-Format Probe. Beetle luciferases are the members of a superfamily of acyl-adenylate-forming enzymes, which include acyl-coenzyme A ligases and peptide synthetases.14 We considered the following points for determining the dissection points of CBLuc: (1) the predicted secondary structure of CBLuc (1-542 AA) and (2) the hydrophobicity analysis of the amino acids based on the scale of Kyte and Doolittle.15 The hydrophobicity analysis showed that a long hydrophilic region comprising the flexible loop exists at around 400-450 AA of CBLuc. On the basis of the knowledge for the predicted protein structure of CBLuc, we concluded that the effective dissection points of CBLuc for constructing a single-molecule-format bioluminescent probe should be inside or near the flexible loop region between 380 and 480 AA of CBLuc. Here, we tested 10 differing potential dissection points near the flexible loop region of CBLuc. To examine the utility of the dissected fragments of CBLuc, a tandem protein of the AR LBD (14) Conti, E.; Franks, N. P.; Brick, P. Structure 1996, 4, 287-298. (15) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132.

linked with a FQNLF motif was sandwiched between the N- and C-terminal fragments of split CBLuc generated from 10 point dissections. Among 10 potential probes, plasmids 3, 6, 9, and 10 showed significant signal-to-background ratios upon exposure to 10-5 M DHT. Especially, plasmid 3 exhibited a luminescence intensity with 10-5 M DHT 5 times larger than that with a vehicle (0.1% DMSO) in HeLa cells (Figure 1C). Previously, Paulmurugan et al. studied the various combination of overlapping N- and C-terminal fragments of FLuc for complementation.7 Here, we also tested two overlapping pairs of fragments by constructing single-molecule-format probes, plasmids 8 and 10, with the fragments. However, we finally concluded that the overlapping fragments of split CBLuc are not a correct choice for the present single-molecule format probes. Rather, we concluded that nonoverlapping fragments are suitable for the present single-molecule format. As a representative example, the plasmid 3 with nonoverlapping fragments exhibited the largest signal-tobackground ratio and quasi-reversible androgen sensitivity. Orientation in the Association of NR LBD with a LXXLL Motif. Most of coactivators contain conserved leucine-rich LXXLL motifs (L ) leucine, X ) any amino acid) that are responsible for interaction with agonist-bound AR LBD and other NR LBDs.16-19 However, it was unclear whether AR LBD binds the LXXLL motif with a parallel or antiparallel conformation. To extensively explore the binding chemistry between NR LBD and a peptide sequence, four additional single-molecule-format indicators were constructed based on the backbone of plasmid 3 (Figure 1B). Four indicators contain NR LBD with a normal or a reversed LXXLL motif, which were named pSimbe-LXA, pSimbeLXP, pSimbe-GRA, and pSimbe-GRP, respectively (Figure 2A and Supporting Information Figure 1). Relative androgen sensitivities were evaluated with the two cell lines, HeLa and MCF-7, carrying plasmids 3, pSimbe-LXA, or pSimbe-LXP (Figure 2A with MCF-7 cells; Supporting Information Figure 1 with HeLa cells). In both cases with HeLa and MCF-7 cells, pSimbe-LXA exhibited much higher luminescence intensities than pSimbe-LXP. The results can be explained that androgenactivated AR LBD prefers to bind the LXXLL motif with an antiparallel orientation, but it is still possible to bind the LXXLL motif with a parallel orientation. Two affinities are considered to motivate the binding of AR LBD with the LXXLL motif: one is their affinity between their side chains based on hydrogen bond, and the other is their physical hydrophobic affinity.17-19 The affinity between the two hydrophobic regions may be the reason for the luminescence intensities shown in the cells with pSimbeLXP. In addition, a component domain of pSimbe-LXA was replaced from AR LBD to GR LBD (pSimbe-GRP and pSimbe-GRA) (Figure 2A). pSimbe-GRP and pSimbe-GRA expressed in MCF-7 cells did not show any sensitivity to androgen. It is explained that androgen (16) Awais, M.; Sato, M.; Lee, X. F.; Umezawa, Y. Angew. Chem., Int. Ed. 2006, 45, 2707-2712. (17) He, B.; Gampe, R. T., Jr.; Kole, A. J.; Hnat, A. T.; Stanley, T. B.; An, G.; Stewart, E. L.; Kalman, R. I.; Minges, J. T.; Wilson, E. M. Mol. Cell 2004, 16, 425-438. (18) He, B.; Bowen, N. T.; Minges, J. T.; Wilson, E. M. J. Biol. Chem. 2001, 276, 42293-42301. (19) Dubbink, H. J.; Hersmus, R.; Verma, C. S.; van der Korput, H. A.; Berrevoets, C. A.; van Tol, J.; Ziel-van der Made, A. C.; Brinkmann, A. O.; Pike, A. C.; Trapman, J. Mol. Endocrinol. 2004, 18, 2132-2150.

is the only driving factor for the conformation change of AR LBD and consequent binding with a normal or a reversed LXXLL motif. Determination of the Ligand Sensitivity of the SingleMolecule-Format Probe. The sensitivities of the probe from pSimbe-LXA to ligands were explored in four different cell lines (Figure 2B). First, pSimbe-LXA expressed in MCF-7, CHO, HeLa, or NIH 3T3 cells was stimulated with ligands, 10-5 M DHT, T, or E2. All the cells carrying pSimbe-LXA had sensitivity to androgens, but not to E2. The relative signal-to-background ratios were as follows in decreasing order: MCF-7 > NIH 3T3 > HeLa > CHO. The results show that pSimbe-LXA is generally applicable to developing biotherapeutic agents and for screening adverse chemicals in various contexts of living cell lines or animals. An inhibitory effect of CPA on the luminescence intensities by the probe from pSimbe-LXA was estimated (Figure 3B). In the absence of CPA, the luminescence intensities by 10-6 M DHT were developed up to 7 times higher than those by the vehicle (0.1% DMSO). However, the luminescence intensities were inhibited by increasing concentrations of CPA: i.e., 28% by 10-5 M CPA and 79% by 10-4 M CPA. The results are interpreted as follows: The luminescence intensities are indeed developed by the intramolecular association of androgen-activated AR LBD with the LXXLL motif. The present probe can be used for a highthroughput screening for potential prostate cancer drugs (androgen antagonist). The time course of the luminescence intensities by the probe from pSimbe-LXA was monitored in response to 10-7, 10-6, or 10-5 M DHT (Supporting Information Figure 4). The present singlemolecule-format probe expressed with pSimbe-LXA in MCF-7 cells clearly differentiated the concentrations of DHT. The luminescence intensities by 10-5 M DHT reached a plateau in 5 min, whereas those by 10-6 M DHT were gradually increased up to 30 min after the stimulation. The 10-7 M DHT weakly increased the luminescence up to 1.7 times over background. The result shows that, although the response times differ according to concentrations of DHT, the probe requires roughly 20 min to make a sensitive response to DHT. Dose-Response Curves of Various Ligands Based on the Luminescence by the Single-Molecule-Format Probe. Doseresponse curves of ligands, DHT, T, and E2, were determined with plasmid 3 or with pSimbe-LXA expressed in MCF-7 cells (Figure 3A). Both plasmid 3 and pSimbe-LXA expressed in MCF-7 cells quickly induced the biolumimescence in response to DHT ranging from 10-7 to 10-5 M. pSimbe-LXA induced 6 times higher luminescence intensities with 10-5 M DHT than that with the vehicle (0.1% DMSO), while plasmid 3 increased the luminescence up to 8 times in response to 10-5 M DHT compared to the case stimulated with the vehicle. pSimbe-LXA induced basal luminescence intensities in response to E2 compared to DHT. The luminescence intensities by 10-4 M T were barely detectable with the cells carrying pSimbe-LXA. The detection limits of the plasmid 3 and pSimbe-LXA were both at ∼10-7 M DHT and the luminescence intensities reached to a plateau at 10-5 M DHT. The MCF-7 cells carrying pSimbe-LXA exhibited 100 times weaker sensitivity to T than to DHT. The detection limit to T was merely at ∼10-5 M. E2 did not induce any luminescence in the tested range from 10-8 to 10-4 M. Although both DHT and T may have an ability to bind AR LBD, they will induce different conformational changes Analytical Chemistry, Vol. 79, No. 13, July 1, 2007

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of AR LBD inside the probe. The differences in the conformation of AR LBD may result in the selectivity variance of the probe to androgens. The results are also interpreted as follows: Upon binding androgen, AR experiences complex signal transduction pathways from dimerization to transcription activation. Many useful methods may be made based on such a specific step of the pathways for determining androgen activities. Nevertheless, AR itself has an intrinsic ability to discriminate androgen activities even in the simplest model, androgen-AR binding, which is the first step to the long journey to transcription activation. Relative ligand sensitivity of the present probe expressed in MCF-7 cells to various ligands was estimated (Supporting Information Figure 3). The luminescence intensities by steroids were decreased as follows in order: DHT > 19T > T > E2 > progesterone ) cortisol ) mifepristone ) CPA. The sensitivity variance of the present probe to steroids is correspondent with that of other methods using trafficking of GFP-fused AR or expression of reporter proteins.20-22 The probe did not exhibit any increase of luminescence in response to synthetic chemicals including known endocrine disrupting chemicals. The results show that the present probe has a high sensitivity to androgens and selectivity against synthetic chemicals. Reversibility of the Present Single-Molecule-Format Probe in Response to Androgen. The reversibility of the present probe in response to DHT was explored with repeated stimulation and (20) Roy, A. K.; Tyagi, R. K.; Song, C. S.; Lavrovsky, Y.; Ahn, S. C.; Oh, T. S.; Chatterjee, B. Ann. N. Y. Acad. Sci. 2001, 949, 44-57. (21) Georget, V.; Lobaccaro, J. M.; Terouanne, B.; Mangeat, P.; Nicolas, J. C.; Sultan, C. Mol. Cell. Endocrinol. 1997, 129, 17-26. (22) Mak, P.; Cruz, F. D.; Chen, S. Environ. Health Perspect. 1999, 107, 855860.

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withdrawal of 10-5 M DHT (Figure 4). First, 20-min stimulation of the MCF-7 cells carrying pSimbe-LXA with 10-5 M DHT produced luminescence intensities 29 times higher than a control. After the withdrawal of DHT by medium change, the luminescence intensities were quickly decreased in 20 min. The luminescence intensities from the MCF-7 cells carrying pSimbe-LXA were decreased to half of the original intensities 1 h after the medium change and returned to background after 2 h. Additional stimulation of the cells with 10-5 M DHT repeatedly increased the luminescence intensities up to 27 times compared to the case with the vehicle. The luminescence intensities from the MCF-7 cells were slowly decreased again by the medium change. The luminescence was completely back to background 2 h after the medium change. The result shows that the intramolecular complementation of the present probe is reversible in androgen sensing and reusable 2 h after the medium change and that the sensitivity of the present probe to DHT is weakly decreased by the repeated use. ACKNOWLEDGMENT This work was supported by grants from Japan Science and Technology Agency (JST), Japan Society for the Promotion of Science (JSPS). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 16, 2006. Accepted April 17, 2007. AC0621571