Genetically Encoded Bioluminescent Indicators for Stress Hormones

Anal. Chem. 2009, 81, 3760–3768

Genetically Encoded Bioluminescent Indicators for Stress Hormones Sung Bae Kim,† Moritoshi Sato,‡ 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 Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of corepressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP. Nuclear receptors are one of the most abundant classes of transcriptional regulators in living subjects.1 They are known to regulate diverse functions such as homeostasis, reproduction, development, and metabolism. The molecular mechanisms of ligand-activated nuclear receptors (NRs) have been utilized in designing new fusion protein probes emitting fluorescence or bioluminescence; for instance, single-molecular conformation change of NR was indexed for determining a ligand activity.2 The interactions between an R-helical peptide and ligand binding domain of NR (NR LBD) were utilized in both fluorescent resonance energy transfer (FRET) 3,4 and single-chain probes.5,6 * To whom correspondence should be addressed. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ The University of Tokyo. (1) Lee, H. J.; Chang, C. Cell. Mol. Life Sci. 2003, 60, 1613–1622. (2) Paulmurugan, R.; Gambhir, S. S. Proc. Natl. Acad. Sci. U.S A. 2006, 103, 15883–15888. (3) Awais, M.; Sato, M.; Lee, X. F.; Umezawa, Y. Angew. Chem., Int. Ed. 2006, 45, 2707–2712. (4) Awais, M.; Sato, M.; Sasaki, K.; Umezawa, Y. Anal. Chem. 2004, 76, 2181– 2186. (5) Kim, S. B.; Awais, M.; Sato, M.; Umezawa, Y.; Tao, H. Anal. Chem. 2007, 79, 1874–1880. (6) Kim, S. B.; Umezawa, Y.; Kanno, K. A.; Tao, H. ACS Chem. Biol. 2008, 3, 359–372.

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Nuclear trafficking of NRs was also monitored with GFP-tagging7 and a protein splicing technique.8 Dimerization of NRs was examined with FRET to evaluate the contribution of a ligand to NR actions.9 These precedent studies suggest great potential for NRs to explore ligand property in the complex context of living subjects. In NR actions, coactivators and corepressors play a pivotal role in transcriptional functions through an association with NR LBD.10,11 The corepressors of NRs were originally isolated with yeast two-hybrid screenings, which included the nuclear receptor corepressor (N-CoR)12 and the silencing mediator for retinoic acid and thyroid hormone receptors (SMRT).13 These corepressors share the consensus LXXIIXXXL motif for binding with NR LBDs in the absence of ligand.10 As previously expected from the results using LXXLL coactivator helical motifs,14 we assumed that residues flanking the corepressor motif were of quantitative importance. Recent advances in the knowledge of corepressor actions inspired us to design new types of fusion protein probes. Corepressors generally stabilize the host molecules and repress the transcriptional activity before agonist activation. Thus, we hypothesized that corepressors may exert bioanalytical importance for ligand-sensing properties of NRs upon linkage to a host singlechain indicator. On the basis of this speculation, we designed new single-chain probes comprising a LXXIIXXXL motif of corepressors at the N- and C-termini to mimic the physiological circumstance of unliganded NRs (Figure 1). The ligand binding domain of the glucocorticoid receptor (GR LBD) was initially fused to an R-helical coactivator motif (LXXLL) with a GS linker. The fusion protein was then sandwiched between the N- and C-terminal fragments of split-Gaussia luciferase (GLuc), which was dissected at Q105. (7) Maruvada, P.; Baumann, C. T.; Hager, G. L.; Yen, P. M. J. Biol. Chem. 2003, 278, 12425–12432. (8) Kim, S. B.; Ozawa, T.; Watanabe, S.; Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11542–11547. (9) Tamrazi, A.; Carlson, K. E.; Daniels, J. R.; Hurth, K. M.; Katzenellenbogen, J. A. Mol. Endocrinol. 2002, 16, 2706–2719. (10) Perissi, V.; Staszewski, L. M.; McInerney, E. M.; Kurokawa, R.; Krones, A.; Rose, D. W.; Lambert, M. H.; Milburn, M. V.; Glass, C. K.; Rosenfeld, M. G. Genes Dev. 1999, 13, 3198–3208. (11) Hu, X.; Lazar, M. A. Nature 1999, 402, 93–96. (12) Horlein, A. J.; Naar, A. M.; Heinzel, T.; Torchia, J.; Gloss, B.; Kurokawa, R.; Ryan, A.; Kamei, Y.; Soderstrom, M.; Glass, C. K. Nature 1995, 377, 397–404. (13) Chen, J. D.; Evans, R. M. Nature 1995, 377, 454–457. (14) Darimont, B. D.; Wagner, R. L.; Apriletti, J. W.; Stallcup, M. R.; Kushner, P. J.; Baxter, J. D.; Fletterick, R. J.; Yamamoto, K. R. Genes Dev. 1998, 12, 3343–3356. 10.1021/ac802674w CCC: $40.75  2009 American Chemical Society Published on Web 04/23/2009

Figure 1. (A) Schematic structures of cDNA constructs encoding the present bioluminescent indicators. Abbreviations: GLuc-N, N-terminal fragment of Gaussia luciferase; GLuc-C, C-terminal fragment of Gaussia luciferase; GR LBD, ligand binding domain of glucocorticoid receptor. (B) Cartoon structures of the synthesized indicators. (C) Basic strategy for illuminating ligand-induced conformational change of cPRESSO-C1. In the absence of agonist, the probe is stabilized with corepressor. Agonist activates an intramolecular complementation between the fragments, resulting in recovery of the luciferase activity.

Additionally, the N- or C-termini of the prototypical single-chain indicator was modified with a corepressor motif. These series of bioluminescent indicators may be called “PRESSO” from corepressors. The first impact of cofusion of a corepressor motif to singlechain probes was a biphasic response of the probe to agonists. In addition, modification of the host indicators was conducted by a circular permutation (CP). A CP alone dramatically enhanced the detection limit of the host probe to 10-8 M cortisol, while the addition of a corepressor motif extended the dynamic range in the calibration curve. In addition, we report that a CP and a corepressor motif synergistically contributed to (i) the enhanced absolute luminescence intensity, (ii) the low background luminescence intensity, (iii) the improved signal-tonoise ratio, and (iv) a better ligand selectivity. EXPERIMENTS Plasmid Construction. A cDNA template encoding full-length Gaussia luciferase (GLuc) was purchased from Nanolight. The cDNAs of N- and C-terminal fragments (GLuc-N [18-105 aa]; GLuc-C [106-185 aa]) were generated by PCR to introduce unique restriction sites, HindIII/ KpnI (GLuc-N) and BamHI/ XhoI (GLucC), at both ends of the fragments using the template and adequate primers. The cDNAs encoding the ligand binding domain of the glucocorticoid receptor (GR LBD; 527-777 aa) was synthesized by PCR to introduce adequate restriction sites, KpnI/ NotI, at both the ends. The cDNA oligomers encoding the following peptides and the restriction sites, NotI/ BamHI, at both ends were ordersynthesized by Exigen (Tokyo, Japan): (i) an R-helical LXXLL motif of glucocorticoid receptor interacting protein 1 interaction domain 3 (GRIP1 ID3: NALLRYLLDKD); (ii) an LXXLL motif of GRIP1 ID2 (GRIP1 ID2: HKILHRLLQDS); (iii) an alanine-mutated form of LXXLL motif of GRIP1 ID3 (GRIP1 ID3m: NALARYALDKD). The upper cDNA fragments were ligated in the construct backbone as shown in Figure 1 and subcloned into the pcDNA 3.1(+) vector.

The plasmid comprising cDNAs encoding GRIP1 ID3, a mutated form of GRIP1 ID3, and GRIP1 ID2 were respectively named pSimgr3, pSimgr3m, and pSimgr2, meaning a single molecule-format bioluminescent probe for the glucocorticoid receptor. The corresponding fusion protein probes after expression may be called SIMGR3, SIMGR3M, and SIMGR2, respectively. The cDNA construct in pSimgr3 was additionally modified to add a repressor peptide at the N- or C-terminal ends: The N-terminal end was flanked with a LXXIIXXXL motif that originated from the silencing mediator for retinoic acid and thyroid hormone receptors interaction domain 1 (SMRT ID1; ASTNMGLEAIIRKALMG) or nuclear receptor corepressor interaction domain 1 (NcoR ID1; ASNLGLEDIIRKALMG). In addition, the reversed LXXIIXXXL motifs were attached at the N-terminal end to examine the binding property with GR LBD, that is, the revised form of SMRT ID1 (GMLAKRIIAELGMNTSA) and the revised form of NcoR ID1 (GMLAKRIIDELGLNSA). The C-terminal end was similarly flanked with the peptides derived from SMRT ID1 or NcoR ID1. The structures are shown in Figure 1. These probes were named an integrated-molecule-format bioluminescent probe carrying a corepressor motif (PRESSO). Probes with a corepressor motif at the N- and C-terminal ends may be called PRESSO-N and -C series indicators, respectively. A circularly permutated version of PRESSO was made on the basis of the expectation of (i) enhanced ligand sensitivity and (ii) an improved signal-to-background ratio. The cDNA of GLuc was first fragmented into two parts at the point encoding Q105 for making artificial 5′ and 3′ terminals in the middle of GLuc. The original 5′ and 3′ ends of the fragments were genetically fused with an oligomer encoding three GS linkers, whereas the new 5′ and 3′ ends were respectively fused with cDNAs encoding a LXXLL motif and GR LBD (527-777 aa). This plasmid was named pcPresso, where “c” means a circular permutation. The 3′ end of the construct in pcPresso was extensively linked with a corepressor peptide. These types of plasmids were named pcPresso-C. Each probe expressed from the plasmids may be called cPRESSO and cPRESSO-C, respectively. Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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Table 1. List of Probes Synthesized for the Present Study

plasmid name probe name (expression ordera)

coactivator motif (LXXLL motif)

corepressor motif (LXXIIXXXL motif)

pSimgr3 pSimgr3m pSimgr2

SIMGR3 (Gn-GL-LL-Gc) SIMGR3M (Gn-GL-LL-Gc) SIMGR2 (Gn-GL-LL-Gc)

pPresso-N1

PRESSO-N1 (II-Gn-GL-LL-Gc)

NALLRYLLDKD (GRIP1 ID3) HKILHRLLQDS (GRIP1 ID2) NALARYALDKD (mutated GRIP1 ID3) NALLRYLLDKD (GRIP1 ID3)

pPresso-N2

PRESSO-N2 (II-Gn-GL-LL-Gc)

NALLRYLLDKD (GRIP1 ID3)

pPresso-C1

PRESSO-C1 (Gn-GL-LL-Gc-II)


pPresso-C2



pPresso-C3



pPresso-C4



pcPresso pcPresso-C1

cPRESSO (LL-Gc-Gn-GL) cPRESSO-C1 (LL-Gc-Gn-GL-II)

NALLRYLLDKD (GRIP1 ID3) NALLRYLLDKD (GRIP1 ID3)

pcPresso-C2

cPRESSO-C2 (LL-Gc-Gn-GL-II)


pcPresso-C3



pcPresso-C4



pCP-ctrl1 pCP-ctrl2

CP-ctrl1 (Gc-Gn-GL) CP-ctrl2 (Gc-Gn-GL-II)

signal-to-background ratio at 10-5 M cortisol (absolute luminescenceb) 5.6(4.7 × 105 RLU) 4.0(3.3 × 105 RLU) 5.9(1.5 × 105 RLU)

ASNLGLEDIIRKALMG (NcoR ID1; fused to N-termini) GMLAKRIIDELGLNSA (revised NcoR ID1; fused to N-termini) ASNLGLEDIIRKALMG (NcoR ID1; fused to C-termini) GMLAKRIIDELGLNSA (revised NcoR ID1; fused to C-termini) GMLAKRIIAELGMNTSA (SMRT ID1; fused to C-termini) ASTNMGLEAIIRKALMG (revised SMRT ID1; fused to C-termini) ASNLGLEDIIRKALMG (NcoR ID1; fused to C-termini) GMLAKRIIDELGLNSA (revised NcoR ID1; fused to C-termini) GMLAKRIIAELGMNTSA (SMRT ID1; fused to C-termini) ASTNMGLEAIIRKALMG (revised SMRT ID1; fused to C-termini)

5.0 2.0 5.6 5.5 7.8 33(1.9 × 106 RLU) 24(3.6 × 106 RLU) 20(1.4 × 106 RLU)

ASNLGLEDIIRKALMG (NcoR ID1; fused to C-termini)

a The “c” character in the probe names means circular permutation. Parentheses shows the expression order of each probe. Abbreviations: Gn, GLuc-N; Gc, GLuc-C; GL, GLuc; LL, LXXLL motif; II, LXXIIXXXL motif. b Parentheses shows the absolute luminescence intensity.

As control probes for cPRESSO-C1, CP-ctrl1 and CP-ctrl2 were constructed. CP-ctrl2 was made by eliminating the LXXLL motif from cPRESSO-C1, while CP-ctrl1 was constructed by deleting both LXXLL and LXXIIXXXL motifs from cPRESSO-C1. The plasmids carrying cDNAs expressing CP-ctrl1 and -ctrl2 were named pCP-ctrl1 and pCP-ctrl2, respectively. The plasmids were sequenced to ensure fidelity with a BigDye Terminator Cycle sequencing kit and a genetic analyzer ABI Prism310 (PE Biosystems). The constructs synthesized in this study are illustrated in Figure 1, and the components of the constructs are listed in Table 1. For the reader’s quick reference, the domain sequences in the expression orders were appended to the next of the probe names (Table 1), where GLuc-N and -C were respectively symbolized by “Gn” and “Gc”, while LXXLL and LXXIIXXXL motifs were simplified as “LL” and “II”, respectively. GLuc was represented as “GL”. Ligand Sensitivity of SIMGR Series Probes in Response to 10-6 M Cortisol. COS-7 cells were cultured in a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37 °C in a 5% CO2 incubator. COS-7 cells in the plate were transiently transfected with pSimgr3, pSimgr2, or pSimgr3m (0.2 µg per each well) using a transfection reagent, TransITLT1 (Mirus). The cells were extensively incubated for 16 h for experiments. The cells in the plate were stimulated with vehicle (0.1% DMSO) or with 10-6 M cortisol (final concentration) for 20 min 3762


(Supporting Information, Suppl. Figure 1). The recovered luciferase activities were examined with the specific substrate kit (Promega) according to the manufacturer’s instruction, that is, the cells were briefly washed once with PBS, and lysed with a lysis buffer supplied from the kit for 10 min. The lysates were transferred into a test tube, and mixed with 50 µL of the substrate solution provided from the kit. Finally, the luminescence intensities were read for the first 15 s with a luminometer (Minilumat LB9506; Berthold). The luminescence intensities were normalized by two methods: (i) one was by the amount of proteins in cell lysates. The unit of absolute luminescence is subsequently RLU/ µg of protein; (ii) the other was expressed by a relative luminescence unit (RLU) ratio (±), where RLU (+) and RLU (-) represent the luminescence intensity from a 1 µg protein of cell lysate after COS-7 cells were stimulated with and without a ligand, respectively. Western Blot Analysis. The amounts of the total proteins and expression levels of the probes in Supporting Information, Suppl. Figure 1 were examined with a Western blot analysis (Supporting Information, Suppl. Figure 2). COS-7 cells raised in a 6-well plate were transiently transfected with either pSimgr3, pSimgr2, or pSimgr3m, and incubated for 16 h. The cells were washed once in PBS and lysed in 150 µL of lysis buffer (1% SDS/10% glycerol/ 10% 2-mercaptoethanol/0.001% bromophenol blue/50 mM TrisHCl, pH 6.8). An equal amount of the samples (5 µL) was electrophoresed in a precasted 10% acrylamide gel (TEFCO), transferred to a nitrocellulose membrane, and blotted with a rabbit

Figure 2. Dose-Response curves of the PRESSO series indicators for cortisol based on bioluminescence intensities of the complemented GLuc. (A) Bifacial curves of the PRESSO-C3 and -C4 in response to varying concentrations of cortisol. (B) Bifacial curves of the PRESSO-N2 and -C2 in response to cortisol. Insets show the expression order of the probes.

Figure 3. (A) Determination of the contribution of LXXLL and LXXIIXXXL motifs in the probe performance of cPRESSO-C1. The molecular structures were specified in the inset. (B) Dose-Response curves of cPRESSO-C1 and SIMGR3 to varying concentrations of cortisol or DHT (n ) 3). (C) Determination of the biochemical accuracy of cPRESSO-C1 in response to varying concentrations of cortisol. The recovered bioluminescence was determined 20, 40, 80 min after stimulation of cPRESSO-C1 with cortisol and normalized in percentage.

anti-GR antibody (Santa Cruz) or a mouse anti-β-Actin antibody (Sigma). The blots were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody and visualized by chemiluminescence (GE healthcare). The Western blot analysis in Supporting Information, Suppl. Figure 2B was examined using the same procedure. Dose-Response Curves of the PRESSO Series Probes to Cortisol. Dose-response curves of the present PRESSO series probes to cortisol were examined (Figure 2). COS-7 cells carrying PRESSO-C1, -C2, -C3, -C4 (expression order: Gn-GL-LL-Gc-II), or SIMGR3 (order: Gn-GL-LL-Gc) were respectively stimulated with varying concentrations of cortisol for 20 min. Similarly, COS-7 cells carrying PRESSO-N1, -N2 (order: II-Gn-GL-LL-Gc), -C1, -C2 (order: Gn-GL-LL-Gc-II), or SIMGR3 (order: Gn-GL-LL-Gc) were treated with varying doses of cortisol for 20 min. The recovered luciferase activities were monitored using the same procedure as Supporting Information, Suppl. Figure 1. Figure 2 shows the representative curves. Synergistic Effects of CP and a Corepressor Motif. The synergistic effects of CP and a corepressor motif in ligand sensitivity of the present probe were examined (Supporting Information, Suppl. Figure 2A). COS-7 cells raised in a 24-well plate were transiently transfected with pcPresso-c1 (order: LL-

Gc-Gn-GL-II), pcPresso (order: LL-Gc-Gn-GL), pcPresso-c2 (order: LL-Gc-Gn-GL-II), or pSimgr3 (order: Gn-GL-II-Gc). The cells were extensively incubated for another 16 h and stimulated with vehicle (0.1% DMSO) or 10-5 M cortisol for 20 min. The luminescence intensities were estimated from the cell lysate prepared using the method described in Supporting Information, Suppl. Figure 1. The absolute bioluminescence intensities were specified in Supporting Information, Suppl. Figure 2A. In addition, the contribution of LXXLL and LXXIIXXXL motifs to the recovery of the bioluminescence was examined with a series of control probes, CP-ctrl1 and CP-ctrl2 (Figure 3A). COS-7 cells raised in 24-well plates were transfected with pCP-ctrl1, pCP-ctrl2, or pcPresso-c1, and incubated for 16 h. The cells were stimulated with vehicle (0.1% DMSO) or 10-6 M cortisol for 20 min. The recovered bioluminescence intensities were estimated in the presence of coelenterazine. Dose-Response Curves of cPRESSO-C1 to Cortisol. Doseresponse curves of cPRESSO-C1 to cortisol and DHT were compared with that of SIMGR3 (Figure 3B). COS-7 cells raised in 24-well plates were transfected with pcPresso-c1 or pSimgr3, and incubated for 16 h before ligand stimulation. The cells were Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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Figure 4. (A) Comparison of the ligand selectivities of cPRESSO-C1, PRESSO-C1, and SIMGR3. Inset shows the absolute luminescence intensities of cPRESSO-C1 in response to 10-6 M cortisol. Abbreviations: vehicle, 0.1% dimethyl sulfoxide; DHT, 5R-dihydrotestosterone; T, testosterone; E2, 17β-estradiol; OHT, 4-hydroxytamoxifen; genis, genistein; ICI, ICI-182780. (B) Reversibility of the ligand sensitivity of cPRESSOC1. Stimulation and withdrawal of 10-6 M cortisol were repeated as specified in inset A.

Figure 5. (A) Ligand-dependent kinetics in the luminescence intensities from cPRESSO-C1 (n ) 3). (B) Determination of a ligand activity on a bioluminescent strip as a cell-free condition. (C) Determination of stress hormone levels in physiological samples. COS-7 cells carrying cPRESSOC1 were exposed to ten urine samples, and the recovered bioluminescence was fitted for quantifying the cortisol levels in the urine samples. The cortisol levels in same urine samples were simultaneously determined with an ELISA kit (Cayman). The correlation was analyzed.

treated with varying concentrations of cortisol or DHT for 20 min. The subsequent luminescence intensities were estimated using the method described for Figure 2. In addition, variances in the dose-response curves of cPRESSOC1 according to the incubation time were similarly monitored (Figure 3C). COS-7 cells raised in 24-well plates were transfected with pcPresso-c1 and incubated for 16 h. After incubation with cortisol for 20, 40, 80 min, the cells were harvested to determine the bioluminescence intensities in the presence of coelenterazine. Ligand Selectivity of COS-7 Cells Carrying pcPresso-c1. Ligand selectivity of cPRESSO-C1 (order: LL-Gc-Gn-GL-II) was compared with that of SIMGR3 (order: Gn-GL-LL-Gc) (Figure 4A). COS-7 cells raised in 24-well plates were transfected with pcPressoc1, pPresso-c1, or pSimgr3. The cells were incubated with 10-6 M steroids or chemicals for 20 min. The corresponding luminescence intensities were developed using the method described for Supporting Information, Suppl. Figure 1. Reversibility of cPRESSO-C1 in Sensing Cortisol. The reversibility of cPRESSO-C1 (order: LL-Gc-Gn-GL-II) upon sensing cortisol was estimated with a repeated treatment of 10-6 M cortisol and the withdrawal (Figure 4B). COS-7 cells raised in 24-well plates were transiently transfected with pcPresso-c1. 3764


Sixteen hours after additional incubation, the cells were first stimulated with vehicle (0.1% DMSO) or 10-6 M cortisol for 20 min. The culture medium was replaced with a fresh, steroidfree one for temporally removing the cortisol. The cells were repeatedly stimulated with 10-6 M cortisol as shown in the inset of Figure 4B. The luminescence intensities were monitored at every stimulation step with the specific substrate kit (Promega) according to the procedure of Supporting Information, Suppl. Figure 1. Time-Course of the Luminescence Intensities by cPRESSOC1 to Ligands. The time-course of the luminescence intensities generated by cPRESSO-C1 (order: LL-Gc-Gn-GL-II) in response to ligands was monitored for 1 h after stimulation (Figure 5A). COS-7 cells cultured in a 24-well plate were transiently transfected with pcPresso-c1, using a transfection reagent (TransIT-LT1, Mirus). The cells were incubated in a CO2 incubator for 16 h. The cells were then stimulated with 10-6 M cortisol or 5R-hydrotestosterone (DHT). At 5, 10, 15, 20, 30, 40, and 60 min after ligand addition, the luminescence intensities from the cells were developed with a specific substrate solution (Promega) according to the procedure described for Supporting Information, Suppl. Figure 1.

Bioluminescence Strip for Imaging Steroid Activity in Vitro. A bioluminescence strip was constructed on a glass slide (2 × 7 cm) for examining a potential advantage of the present probe in a cell-free condition (Figure 5B). A “Y”-shaped paper strip (1.1 × 1.1 cm) was clipped from a sheet of nitrocellulose paper and carefully fixed onto the glass slide. In addition, a cell crude or a cell lysate carrying cPRESSO-C1 (order: LL-Gc-Gn-GL-II) was prepared with the method for Supporting Information, Suppl. Figure 1. A total of 12 µL of cell lysates (protein amount: 21.8 µg) were respectively spotted on each end circle (area: ca. 6.8 mm2) of the “Y”-shaped strip. The spots were carefully dried not to spread on the whole slide. A 10 µL substrate solution supplemented with vehicle (0.1% DMSO) or 10-6 M cortisol was dropped in the middle of the strip (cross-section), and the subsequent luminescence intensities on the strip were integrated with a luminescence scanner (RAS-3000, FujiFilm) for 10 min. Determination of Cortisol Levels in Urine. The cortisol levels in a physiological sample, urine, were estimated with both COS-7 cells carrying cPRESSO-C1 and an enzyme-linked immunosorbent assay (ELISA) kit (Cayman) (Figure 5C). COS-7 cells raised in 24-well plates were transfected with pcPresso-c1 and incubated for 16 h. The cells were washed once with PBS and directly exposed for 20 min to 200 µL of urine samples donated from ten volunteers affiliated in AIST. Some of the cells were stimulated with var.ying concentrations of controlled cortisol for 20 min for calibration curves. The recovered bioluminescence intensities from the cells were finally estimated in the presence of coelenterazine as shown in the procedure described for Supporting Information, Suppl. Figure 1. An ELISA assay (Cayman) was conducted in parallel to the same urine samples. The assay was proceeded according to manufacturer’s instruction. RESULTS Intramolecular GR LBDsLXXLL Motif Interaction. An interaction between GR LBD and a LXXLL motif was initially examined to show whether the binding is indeed ligand-dependent inside the single-chain probe (Supporting Information, Suppl. Figure 1). The results exhibited that the luminescence intensities were influenced by the cofused motifs. Both SIMGR3 and SIMGR2 reported 4 to 5 times enhanced bioluminescence intensities (per microgram protein lysate) compared with the background in response to 10-6 M cortisol. However, SIMGR3M comprising an alanine-mutated LXXLL motif (Table 1) showed a decreased ligand sensitivity to cortisol. The apparent expression levels of the three probes did not significantly vary according to a Western blot analysis (Supporting Information, Suppl. Figure 1B). The results suggest that (i) an intramolecular GR LBDsLXXLL motif interaction indeed occurs ligand-dependently, as expected with the crystal structure of GR (Protein Data Bank accession no. 1m2z) and (ii) the component amino acids in a LXXLL motif is a determining factor for binding GR LBD, as assured with the weakened interaction between a mutated LXXLL motif and GR LBD. Contribution of a Corepressor Motif to the Sensorial Performance of Single-Chain Probes. Roles of a corepressor motif in the sensorial performance of single-chain probes were examined by cofusing the motif to the N- or C-termini of the

probes (Figure 2A and 2B). The first notable appearance by cofusing a corepressor motif to the probes was the biphasal calibration curves in response to cortisol. Compared with the control (i.e., without a corepressor; open circles), PRESSO-C1 and -C2 (order: Gn-GL-LL-Gc-II) weakly enhanced the luminescence intensities in response to low levels of cortisol ranging from 10-9 to 10-7 M (Figure 2A). Similarly, PRESSO-N2 (order: II-Gn-GLLL-Gc) increased the luminescence intensities in the range from 10-11 to 10-9 M cortisol (Figure 2B). In addition, the contribution of LXXLL and LXXIIXXXL motifs in cPRESSO-C1 was examined with a series of control probes, CP-ctrl1 and CP-ctrl2 (Figure 3A). cPRESSO-C1 carrying both LXXLL and LXXIIXXXL motifs, exhibited 13-times stronger luminescence intensities than the background in response to 10-6 M cortisol, whereas CP-ctrl1 and CP-ctrl2 respectively showed 2.5 and 1.5-times stronger luminescence in the same stimulation. This result indicates that (i) the LXXLL motif is an active component for recruiting GR LBD and (ii) intramolecular LXXLL motif-GR LBD binding efficiently triggers the reconstitution of split-GLuc fragments. These conclusions are also supported by multiple references: The selective LXXLL motifGR LBD binding was well investigated with conventional methods.10,12,13,14 Their X-ray crystalographic structures are accessible in Protein Data Bank (e.g., 1m2z for GR LBD-LXXLL motif binding; 2jfa for ER LBD-LXXIIXXXL motif binding). In addition, it is interesting to consider the reason that CPctrl1 and -ctrl2 weakly respond to cortisol. GR LBD exposes a hydrophobic region to the cytosol upon agonist binding.15 This may aggregate nonspecifically with the remaining components, GLuc-N and -C, increasing the encounter chance between the splitGLuc fragments. Synergistic Effects of a Corepressor Motif and a CP to Ligand Sensitivity of Single-Chain Probes. Synergistic effects of a corepressor motif and a CP to ligand sensitivity of singlechain probes were examined in COS-7 cells (Supporting Information, Suppl. Figure 2A). The luminescence intensities from COS-7 cells carrying SIMGR3 (non-CP; a control) (order: Gn-GL-LL-Gc), cPRESSO (order: LL-Gc-Gn-GL), cPRESSO-C1, or cPRESSO-C2 (order: LL-Gc-Gn-GL-II) were compared after stimulation with varying concentrations of cortisol. cPRESSO-C1 (order: LL-GcGn-GL-II) expressed the strongest luminescence intensities and the longest dynamic range to cortisol upon comparison with the others. On the other hand, cPRESSO exhibited an excellent signalto-background ratio among the tested probes. The detection limits of CP probes were improved about 50-100 times better than the non-CP control, SIMGR3. The absolute bioluminescence intensities from cPRESSO in response to 10-5 M cortisol were approximately 33 times better than the background. The detectable concentration window of cPRESSO-C1 was broadened to the range from 10-8 to 10-5 M cortisol (Figure 3B, Supporting Information, Suppl. Figure 2B). The apparent effective concentration 50% (EC50) was about 2.5 × 10-7 M. An EC50 value between full-length GR and cortisol is approximately 1 × 10-7 M according to a previous MMTV promoter-β-galactosidase assay.16 Considering that GR LBD alone reduces its ligand (15) Doweyko, A. M. Drug Dev. Res. 2007, 68, 95–106. (16) Tanaka, H.; Hirano, F.; Nomura, Y.; Miura, T.; Makino, Y.; Fukawa, E.; Makino, I. Rheumatol. Int. 1994, 14, 9–12.


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sensitivity,17 the present value, 2.5 × 10-7 M, is considered reasonable. The background luminescence of CP probes was generally less than that of the non-CP controls. In addition, biochemical accuracy in the dose-response curves of cPRESSO-C1 according to the incubation time were examined (Figure 3C). COS-7 cells were stimulated with varying concentrations of cortisol for 20, 40, and 80 min. The EC50 values in calibration curves did not shift considerably to the right with incubation times, and the drift of the baseline with time was not observed. Ligand Selectivity of cPRESSO-C1 in COS-7 Cells. The ligand selectivity of cPRESSO-C1 (order: LL-Gc-Gn-GL-II) was compared with those of SIMGR3 (order: Gn-GL-LL-Gc) and PRESSO-C1 in COS-7 cells (Figure 4A). cPRESSO-C1 was superior to SIMGR3 in the (i) absolute luminescence intensities, (ii) signalto-noise ratio, and (iii) ligand selectivity. cPRESSO-C1 emitted 17 times stronger luminescence intensities than the background in response to 10-6 M cortisol. Overall, a corepressor peptide and a CP synergistically contributed to both (i) a decrease in the background luminescence and (ii) enhancement of the signal luminescence. SIMGR3 as a low-efficiency probe showed a discrepancy in Figures 2, 3, and 4. It may be caused by uncontrollable factors among batches such as variances in (i) probe amounts caused by overexpressing CMV promoter in pcDNA 3.1 (+) and (ii) transfection efficiency caused by cell population and aging. Reversibility of cPRESSO-C1 in Sensing Cortisol. The reversible sensitivity of cPRESSO-C1 (order: LL-Gc-Gn-GL-II) to an agonist was examined by a cortisol treatment and its withdrawal (Figure 4B). The result shows that cPRESSO-C1 preserves the ligand sensitivity even after repeated treatment and withdrawal of 10-6 M cortisol. The results show that (i) the interactions between GR LBD and a LXXLL motif are reversible and (ii) cPRESSO-C1 is robust enough to repeatedly determine cortisol actions, which is correspondent with the previous study in that liganded GR LBD is recycled after transcription activation.18,19 Time-Course of the Luminescence Intensities from cPRESSO-C1. The time-course of the luminescence intensities from cPRESSO-C1 (order: LL-Gc-Gn-GL-II) after ligand addition was monitored for 60 min (Figure 5A). COS-7 cells carrying cPRESSOC1 were first stimulated by 10-6 M cortisol (final concentration). The luminescence intensities from cPRESSO-C1 were quickly enhanced in response to the stimulation and reached a plateau in about 20 min, whereas the intensities in response to 10-6 M DHT remained in the basal line for 60 min. The time-course shows that (i) 20 min are required for cPRESSO-C1 to completely bind with cortisol, (ii) the time may be taken for the following steps, that is, (a) membrane penetration of cortisol, (b) cortisol-cPRESSO-C1 binding, and (c) the subsequent intramolecular conformation change and complementation of cPRESSO-C1. The response time is correspondent with that of a FRET study based on a dexamethasone-activated interaction between GR LBD and a LXXLL motif.20 (17) Keightley, M. C.; Fuller, P. J. Steroids 1995, 60, 87–92. (18) Galigniana, M. D.; Housley, P. R.; DeFranco, D. B.; Pratt, W. B. J. Biol. Chem. 1999, 274, 16222–16227. (19) DeFranco, D. B. Mol. Endocrinol. 2002, 16, 1449–1455. (20) Awais, M.; Sato, M.; Umezawa, Y. Steroids 2007, 72, 949–954.

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Stress Hormone Activity Visualized with a Bioluminescence Strip on a Glass Slide. To explicitly demonstrate the advantages of the present single-chain probes in a cell-free circumstance, we constructed a bioluminescence strip on a glass slide carrying a cell crude and cPRESSO-C1 (order: LL-Gc-GnGL-II) (Figure 5B). After the inlet of a substrate solution supplemented with the vehicle (0.1% DMSO) or 10-6 M cortisol in the middle of the cross-section, the enhanced bioluminescence intensities at the end circles of the strip were scanned. As shown in the intensity profile, the bioluminescence strip selectively enhanced the luminescence intensity in response to cortisol. The signal-to-noise ratios in response to 10-6 M cortisol in Figure 5B were not as large as those in Figure 3 and 4. On the strip, the probe is once dried and dampened with a sample solution. This drying procedure may exert an aggregation and decomposition of the probe hampering the sensitive ligand determination. Overall, the results exhibited that (i) stress hormone activity can be imaged at the end circle of the strip, (ii) cPRESSO-C1 is robust enough to sense cortisol even after drying on a paper strip, and (iii) the developed luminescence intensity was strong enough to be detected on a glass plate using a conventional luminescence scanner. Determination of Stress Hormone Levels in Urine. We additionally determined cortisol levels in physiological samples and compared with those from an ELISA assay (Figure 5C). COS-7 cells carrying cPRESSO-C1 were utilized in the determination of cortisol levels in urine samples donated by 10 volunteers. The cortisol levels in urine samples were crosschecked with an ELISA kit (Cayman). Figure 5C shows that the cortisol sensitivity of cPRESSO-C1 is correspondent with that of ELISA (correl. coeff. ) 0.62; Slope by Least-squares analysis ) 0.75). Normal clinical range of cortisol in urine is from 0.6 to 7.5 × 10-7 M. Among the tested urine samples, one sample was ranked in a higher cortisol level than the normal clinical range, whereas some samples were near the lowest limit of the normal clinical range. The high cortisol level in the sample suggests a probability of Cushing’s syndrome, which causes truncal obesity, hypertension, hypokalemic metabolic alkalosis, carbohydrate intolerance, disturbance of reproductive function, and neuropsychiatric symptoms. DISCUSSION Clinical levels of “stress” hormones such as cortisol in physiological samples direct the signs and symptoms of diseases such as Cushing’s Syndrome and Addison’s Disease.21 The representative levels of total cortisol in physiological samples range from 0.8 to 6.4 × 10-7 M for serum, from 0.6-7.5 × 10-7 M for urine, and from 0.2 to 2.8 × 10-8 M for saliva. These clinical ranges demand a sensitive measure for determining low levels of endocrinal cortisol in physiological samples without concentration. The conventional determinations of stress hormones have heavily depended on either isotopic or nonisotopic immunoassays. Instead of the conventional isotopic and/or immunological approaches, we synthesized single-chainbased bioluminescent indicators for sensitively determining low (21) Burtis, C. A.; Ashwood, E. R.; Tietz, N. W. Tietz textbook of clinical chemistry, 3rd ed.; W. B. Saunders: Philadelphia, 1999.

levels of cortisol. This may be characterized as a nonisotopic, nonimmunological, colorimetric approach for determining endocrinal steroids. A biphasic calibration curve of the probes in Figure 2 may be taken as a characteristic effect of a corepressor motif to the host probes. These biophysical calibration curves generally suggest that two equilibriums exist in the system. Considering the ingredients of the probe, one equilibrium may be the GR LBDcorepressor motif binding and the other would be the GR LBDcoactivator motif interaction. With the calibration curves, we speculate that GR releases corepressors and recruits coactivators respectively in different concentration ranges of agonist. A gray zone may exist between the two ranges like a buffer region. Our initial approach with GR LBD sandwiched between the split-GLuc fragments generally exhibited a poor detection limit (SIMGR series; Supporting Information, Suppl. Figure 1). The poor detection limit was largely relieved by a CP of split-GLuc. The detection limits of cPRESSO series probes were surprisingly decreased to about 50-100 times lower than that of SIMGR3 (order: Gn-GL-LL-Gc). A temporal loss of GLuc activity inside cPRESSO may be caused by the dissection of the putative active site of GLuc. A CP places a dissected active site of GLuc on the opposite side of the other dissected site in the single-chain probe (Figure 1A). In this single molecular conformation, the basal encountering chance between the fragmented active sites should be extremely rare. This structural reason may contribute to (i) the dramatic decrease of the basal luminescence by the indicator and (ii) the improved signal-to-background ratios in this study. The synergistic contribution of both a CP and a corepressor motif to the single-chain probe was carefully examined (Supporting Information, Suppl. Figure 2A). First, comparison between SIMGR3 (order: Gn-GL-LL-Gc) and cPRESSO-C1 revealed that a CP improves the detection limit through decreasing the background luminescence as discussed in the previous paragraphs. In addition, cofusion of a corepressor motif (derived from NcoR) to cPRESSO unexpectedly exerted (i) a wider dynamic range in case of cPRESSO-C1 and (ii) an enhancement of the absolute luminescence intensity (Supporting Information, Suppl. Figure 2A). The probe was fabricated with a corepressor motif at the terminals of fusion protein probes. Here, we propose a probable explanation of the effects of the corepressor motif. It was already known that (i) the corepressor motif binds GR LBD in the absence of ligands like a typical corepressor of NR 11,22 and (ii) the corepressor motifs block the access of a LXXLL motif to the hydrophobic coactivator interaction pocket of the GR LBD.23 We suppose that this basal interaction conducts two different roles, that is, (i) the repressor motif sterically hinders an access of the cofused LXXLL motif to GR LBD in the basal condition, which contributes to a decrease in the detection limit as shown in Figure 2, and (ii) the repressor motif may exert rigidity of the whole probe backbone. The rigidity of the reconstituted active site may positively contribute to the efficiency of the enzyme reaction of reconstituted GLuc and the enhanced absolute luminescence intensities. We briefly examined the heat stability, detergent resistance, and luminescence sustainability of cPRESSO-C1. Unfortunately, we could not observe their considerable influence (22) McKenna, N. J.; O’Malley, B. W. Cell 2002, 108, 465–474. (23) Stevens, A.; Garside, H.; Berry, A.; Waters, C.; White, A.; Ray, D. Mol. Endocrinol. 2003, 17, 845–859.

to the probe compared to the control (data not shown). Precise thermodynamic consideration should be followed for more discussion. Reversibility of the present on-off systems needs to be considered for convincing quantitative ligand sensing. It was previously proven that the GR LBD-LXXLL motif binding itself is a naturally occurring, reversible interaction in live cells.14 However, the modification of GR LBD with GLuc fragments and a LXXLL motif could affect the intrinsic ligand sensitivity and reversibility. As to the complementation mechanism, Michnick et al. previously proposed the molecular mechanisms of the protein complementation assay (PCA) on the basis of split-GLuc.24,25 Their study suggested two models to explain how PCA with split-GLuc would be reversible using the possible equilibrium distribution states and the free-energy landscape among the states changes. The present GLuc fragments in the cPRESSO series probes did not exhibit a spontaneous folding but did make a robust calibration curve to varying concentrations of ligands. Also of another consideration is whether the present singlechain probes afford a sufficient gap for exerting the on-off system through an intramolecular complementation. As to the necessary gap, Michnick et al. previously demonstrated a study with a molecular ruler that the valid distance between two fragments of a split-reporter protein for PCA should be less than about 8 Å.26 The small gap claims that protein complementation should occur only when the fragments are in very close proximity. This distance hurdle appears too restricted to exert a nonspecific, basal complementation between the CP fragments even inside a single fusion molecule. In addition, a 3-dimensional orientation of the fragments may be another factor in preventing the nonspecific complementation. The present probes can be characterized as single-chain-based bioluminescent measures for determining stress hormones, where all the components for ligand-sensing and bioluminescence emission were integrated into a single molecule. This advantage of the present probe was explicitly demonstrated with a bioluminescence strip mounted on a glass slide and a real sample (urine) analysis (Figure 5B and 5C). This technique to illuminate stress hormone activity with a bioluminescence strip can largely expand the scope of usage by replacing the component proteins. In addition, these kinds of bioluminescence strips should be a cost-effective alternative for ELISAs, pregnancy test kits, and ovulation prediction kits using antibodies. The potential hurdles for real use in a cell-free condition may be summarized as follows: (i) the photon emission should be strong enough to be determined with a conventional bioluminescence scanner, (ii) the probe should not decompose even in a dried, cell-free condition, (iii) the conformation change of GR LBD and the subsequent protein complementation inside the probe should not require any energy source or live-cell circumstance for ligand sensing. The results conclude that (i) this sensor strip enables an on-site determination of ligand activities and (ii) a small amount of the probe emitting bioluminescence (24) Remy, I.; Michnick, S. W. Nat. Methods 2006, 3, 977–979. (25) Michnick, S. W.; Ear, P. H.; Manderson, E. N.; Remy, I.; Stefan, E. Nat. Rev. Drug Discovery 2007, 6, 569–582. (26) Michnick, S. W. Curr. Opin. Struct. Biol. 2001, 11, 472–477.


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on a strip is enough for evaluating ligand activities with a conventional scanner. We additionally determined cortisol levels in urine samples donated by 10 volunteers. Urine is not harmful to cell lines and reflects the cortisol levels in blood. Thus, urine is an excellent physiological sample for accessing the stress hormone levels. The cortisol levels in urine samples were crosschecked with an ELISA kit (Cayman). Figure 5C shows that the cortisol sensitivity of cPRESSO-C1 is correspondent with that of ELISA. The normal clinical range of cortisol in urine is from 0.6 to 7.5 × 10-7 M, which is approximately 105-times higher levels than other steroids such as testosterone and 17β-estradiol. In addition, cPRESSOC1 was highly selective to cortisol and not interfered by other steroids and chemicals as shown in Figure 4A.

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The present probe reconstituted the bioluminescence intensities in response to physiological cortisol in 20 min, whereas ELISA consumed approximately 4 h for the outcome. This comparison concludes that (i) the present probe is a cost-effective and laboreffective method compared to a ELISA assay, and (ii) mammalian cells carrying bioluminescent probes can be utilized in the determination of steroid levels in real samples like urine. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 18, 2008. Accepted April 3, 2009. AC802674W

Genetically Encoded Bioluminescent Indicators for Stress Hormones

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