Genetically Encoded Molecular Tension Probe for Tracing Protein

Aug 31, 2015 - Abstract Image. Optical imaging of protein–protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecul...
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Genetically Encoded Molecular Tension Probe for Tracing Protein− Protein Interactions in Mammalian Cells Sung Bae Kim,*,† Ryo Nishihara,‡ Daniel Citterio,‡ and Koji Suzuki‡ †

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan ‡ Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan S Supporting Information *

ABSTRACT: Optical imaging of protein−protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named “a combinational probe”. The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

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INTRODUCTION Proteins fill most of the mass of a cell and play a predominant part in most biological processes.1 Optical imaging of the protein−protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events.2 To date, PPIs have been visualized with various strategies using fluorescent proteins and luciferases, which include fluorescence resonance energy transfer (FRET),3 bioluminescence resonance energy transfer (BRET),4 and protein complementation assay (PCA).5,6 PCA is an emerging technology for determining the occurrence of PPIs in mammalian cell lines, where a monomeric luciferase is split into two fragments for a temporal loss and conditional reconstitution of the activities only upon PPI.7,8 To date, many luciferases including firefly luciferase (FLuc),5,6 Renilla reniformis luciferase (RLuc),9 and artificial luciferase (ALuc)10 have been utilized for PCA. However, this methodology requires a complex probe design and a tedious optimization process for seeking a suitable dissection site and restores merely 0.5−5% of the original optical intensity after complementation.11,12 The sophisticated molecular design limits the general applicability of an optimized PPI model to other bioassays. We previously introduced a unique strategy for illuminating PPIs called “molecular strain probes”, where a full-length RLuc8 was sandwiched between two proteins of interest.13 The full-length luciferases in the strain probe allow greater absolute bioluminescence than split-luciferases in PCAs. Although the © XXXX American Chemical Society

basic concept of a molecular strain probe is unique and broadly applicable for bioluminescence imaging (BLI) and PPI, no follow-up studies have been elucidated to date. Further, it is unclear whether the RLuc8 is the only luciferase that enables us to fabricate a molecular strain probe. Whether or not this is the case, we should know the reason. In the present study, we hypothesized that a full-length luciferase basically has the ability to vary its enzymatic activity even by molecular tension physically induced by PPIs, besides conventionally recited factors such as ions, temperature, and pH. We excluded beetle luciferases in this evaluation because they are said to be generally darker than copepod luciferases and comprise a long flexible region, naturally easing molecular tension.13,14 As ALucs are brighter and more stable than any other luciferases,10,15 we first fabricated a prototypical bioluminescent probe comprising full-length ALucs sandwiched between the FK506-binding protein (FKBP) and the FKBPrapamycin-binding domain of mTOR (FRB) as a model protein pair (Figure 1). The model luciferase, ALuc, is originally established through extraction of frequently occurring amino acids from multiple sequence alignment of highly conserved copepod luciferases from zooplankton, which were Special Issue: Molecular Imaging Probe Chemistry Received: July 28, 2015 Revised: August 28, 2015

A

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Figure 1. (A) Basic working mechanism of molecular tension probes in mammalian cells. Proteins A and B in the tension probe are approximated in response to a ligand. The protein binding causes molecular tension to the sandwiched ALuc. The molecular distortion dramatically varies bioluminescence. (B) Schematic structures of cDNA constructs of bioluminescent template candidates for sensing molecular tension. Tension probe series 1 and 2 differ in the order of proteins A and B of interest. Abbreviations: TP1 and 2, Tension probe series 1 and 2; Kz, kozak sequence. (C) Optical intensity variance of 16 kinds of tension probe candidates before and after ligand stimulation (n = 3). The gray and black bars indicate TP1 and 2 series templates, respectively. The signs “+” and “−” at the X-axis represent the presence or absence of rapamycin, respectively. The red bars highlight the dramatic elevation of bioluminescence from a tension probe named “TP2.4”. The inset a shows the bioluminescence spectra of TP2.4 before and after rapamycin stimulation. The maximal optical intensity is found at 530 nm. Abbreviations: TP1.1, Tension probe version 1.1; TP2.1, Tension probe version 2.1. The detailed components were listed in Table 1.

Table 1. Detailed Composition of the Molecular Tension Probes Fabricated in This Studya probe name TP0.1 TP0.2 TP1.1 TP1.2 TP1.3 TP1.4 TP1.5 TP1.6 TP1.7 TP1.8 TP2.1 TP2.2 TP2.3 TP2.4 TP2.5 TP2.6 TP2.7 TP2.8 TP3.1 TP3.2 TP3.3 TP4.1 TP4.2

protein Ab FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP FRB FRB FRB FRB FRB FRB FRB FRB FRB FRB FRB ER LBD FRB

inserted luciferase

length of luciferasec

internal secretion peptide (SP)d

protein Bb

ALuc23 ALuc23 ALuc16 ALuc16 ALuc23 ALuc23 ALuc24 ALuc24 ALuc30 ALuc30 ALuc16 ALuc16 ALuc23 ALuc23 ALuc24 ALuc24 ALuc30 ALuc30 ALuc23 ALuc23 ALuc23 ALuc23 RLuc8

19−212 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 1−212 (full) 19−212 19−209 19−207 19−198 19−212 1−311

+ + + + + + + + -

FKBP FRB FRB FRB FRB FRB FRB FRB FRB FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP FKBP SH2 FKBP

figure numbers in use Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure

2; SI Figure 2; SI Figure 1 1 1 1 1 1 1 1 1 1 1 1, 2, 3, 4, 5 1 1 1 1 3 3 3 5; SI Figure 5; SI Figure

1 1

1 1

Abbreviations: ER LBD, the ligand-binding domain of human estrogen receptor; SH2, the the src homology domain of ν-Src. bProteins A and B refer to the proteins linked at the N- and C-terminal ends of the luciferase, respectively. c“Length of Luciferase” indicates the amino acid numbers of the inserted luciferase. dThe signs “+” and “-” represent the presence or absence of secretion peptide (SP). a

N-terminal end of FRBP, which was named a “combinational probe”. The practical utility of TPs was elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and other PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

collected at the southern deep sea of Hokkaido, Japan (13 species) by our colleagues in AIST and two other conventional copepod luciferases (2 species).16,17 Rapamycin triggers a FKBP−FRB interaction, which induces molecular tension to the sandwiched full-length ALuc. The tensed ALuc enhances or weakens the optical intensities in a ligand dependent manner (Figure 1C). This unique tension probe was named “molecular tension probe” (abbreviated TP for short). Further, a series of combinational probes were generated, where the C-terminal end of ALuc in the probe is eliminated and designed to be compensated by the homological

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RESULTS AND DISCUSSION Luciferases under Tension by Protein−Protein Interactions (PPIs) Enhance Their Optical Intensities. A series B

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Figure 2. (A) Optical intensities of TP2.4 in response to varying concentrations of rapamycin (n = 4). Inset a indicates the absolute optical intensities adjusted by protein amount (μg), integration time (s), and light-emitting area (mm2). (B) Relative optical intensities of TP2.4 and its negative control probes (tension-free; TP0.1 and/or TP0.2) before and after rapamycin stimulation (n = 3). Inset a illustrates the molecular binding models of the tension probes. Upon stimulation with 10−6 M of rapamycin, no tension is applied to ALuc23 in the probes, (i) TP0.1, (ii) TP0.2, or (iii) cotransfection of TP0.1 and TP0.2, whereas tension is induced to ALuc23 in the case of TP2.4. An intermolecular binding between TP0.1 and TP0.2 do not vary the optical intensity. Inset b shows the optical images. (C) Western blot analysis to show protein amounts of lysates of TP2.4. The lysates were electrophoresed and blotted with rabbit anti-FKBP antibody (abcam) or anti-β-actin antibody (Sigma). Both β-actin and TP2.4 are found at approximately 45 kDa.

of new bioluminescent probes were designed for illuminating molecular tension induced by intramolecular PPIs (Figure 1C). Some of the 16 tension probe candidates showed significantly enhanced or reduced optical intensities in response to rapamycin: TP2.4 exhibited 6.7-fold enhanced bioluminescence intensity in the presence of 10−6 M of rapamycin compared to the presence of vehicle alone (0.1% ethanol dissolved in the culture medium), whereas TP1.3 and TP1.7 showed optical intensities reduced by one-third in response to 10−6 M of rapamycin. TP2.7 and TP2.8 result in approximately 2-fold stronger bioluminescence upon stimulation with the same ligand. The corresponding optical spectra were obtained with African green monkey kidney-derived COS-7 cells carrying TP2.4, which were stimulated by vehicle (0.1% ethanol) or 10−6 M of rapamycin. The optical intensity in the spectra was greatly enhanced by rapamycin and the maximum optical intensity (λmax) was found at ca. 530 nm. About 13% of the overall light emission was located in the red and near-infrared region at a wavelength longer than 600 nm, which is highly tissue-permeable and commonly referred to as “optical window” (Figure 1C, inset a). It is interesting to compare TP2.4 and TP1.4, whose only difference is the consecutive order of the domains, i.e., FRBALuc23 (19−212)-FKBP vs FKBP-ALuc23 (19−212)-FRB, respectively. Considering all the other ingredients to be equivalent with each other, it is rational that (i) intramolecular steric hindrance between the moieties or (ii) spatial mismatch works in TP1.4. Further study on the structural information in future should reveal the details. Secretion Peptide (SP)-Embedding and Deficient Probes Showed Distinctive Ligand Sensitivity. It has been previously predicted that copepod luciferases consist of two repeated mirror image-like catalytic domains, besides a highly variable domain at the N-terminal region according to the multiple alignment.15,18 The variable domain comprises a unique secretion peptide (SP) at the N-terminal end.17

It is interesting to compare the ligand sensitivities of SPembedding and -deficient probes among the 16 molecular designs (Figure 1C). It was observed that (i) all of the molecular designs with SP failed to elevate the optical intensities, and (ii) a significant reduction of optical intensities was found only in the SP-embedding group of probes (TP1.1, TP1.3, TP1.7, and TP2.1). The SP-bearing probes in Figure 1C are potentially secreted into the extracellular compartment. To examine the secreted proportion, we determined the intra- and extracellular fractions of the probes, TP2.3 and TP2.4, whose only difference is the presence or absence of the secretion peptide (SP) at the Nterminal end of ALuc23 as shown in Table 1. The results show that 86 ± 6% of the TP2.3 bearing SP is secreted into the extracellular compartment, whereas only 14 ± 1% of TP2.3 retained in the cellular compartment. On the other hand, in case of TP2.4 without SP, the majority (96 ± 7%) is retained in the cellular compartment (Supporting Information Figure 3(A)). We further determined whether TP2.3 in the supernatant exerts rapamycin sensitivity (Supporting Information Figure 3(B)). The result shows that no optical variance is observed between the supernatants treated with the vehicle (0.1% ethanol) or rapamycin (10−6 M). Both TP2.3s in the intracellular and extracellular compartments have no rapamycin sensitivity. It indicates that (i) the SP even located at the middle of TP2.3 can exert secretion of the host probe, and (ii) considering that TP2.3 has no rapamycin sensitivity even in the intracellular compartment, we speculate that the majority of intracellular TP2.3 is in the secretion process, i.e., inside the endoplasmic reticulum (ER) with a fragmented form at the SP region. Alternatively, the insensitivity of TP2.3 to rapamycin may be interpreted as follows: the SPs may act as natural flexible linkers inside the probes and, thus, may ease the intramolecular tension raised by the FKBP−FRB interaction, considering that the SP is located at the flexible N-terminal boundary. A corresponding C

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Figure 3. (A) Predicted super 2-dimensional structure of ALuc30.15 The inset a highlights the C-terminal end of ALuc, which shares high sequential homology with the N-terminal end of FKBP. (B) Optical intensities of TP3 series in the presence or absence of rapamycin (n = 4). TP3 series templates contain a shortened C-terminal end of ALuc23 between FRB and FKBP, compared with the TP2 series. The white dotted box in the optical image indicates extremely suppressed background intensity. The corresponding amino acid length of the ALuc23 is specified in the parentheses. The asterisk indicates the greatly diminished optical intensity. The inset a shows the corresponding optical image.

Figure 4. Substrate selectivity of the tension probe TP2.4: (A) Absolute optical intensities of TP2.4 in response to rapamycin (n = 3). The numbers on the bars show fold intensities before and after the stimulation with rapamycin, 10−6 M. The upper and lower insets show an optical image of the bioluminescence and the chemical structure of native coelenterazine (nCTZ), respectively. Abbreviations: 6-pi-OH, 6-pi-OH-coelenterazine. The white and black bars highlight optical intensities with nCTZ and 6-pi-OH-CTZ, respectively. (B) Time-course of the optical intensities after substrate injection (n = 3). The optical intensities were monitored every 5 min and are expressed in relative percentages to the initial optical intensities.

at 10−4 M resulted in only a basic level of optical intensities, close to those observed upon treatment with vehicle alone (0.1% ethanol). The poor optical intensity is interpreted as being caused by cell death induced by the excess amount of rapamycin. Optical Intensities of TP2.4 Are Solely Enhanced by Intramolecular Tension Induced by Ligand-Activated FRB−FKBP Interactions. The presence of 10−6 M of rapamycin failed to increase or decrease the optical intensities of the cells carrying (i) TP0.1 alone, (ii) TP0.2 alone, or (iii) both TP0.1 and TP0.2, whereas the same stimulation enhanced the optical intensities up to 5.4-fold in the case of cells expressing TP2.4 (Figure 2B). An apparent variance in the expression levels of TP2.4 was examined with Western blot analysis (Figure 2C). Anti-FKBP and anti-β-actin antibodies recognized specific proteins at 45 kDa, which are the same as the expected molecular weight of TP2.4 and β-actin (housekeeping protein) after expression. Apparently biased variance in the protein amounts at 45 kDa was not observed by stimulation of rapamycin.

view of the linker length, where a minimal length of the linkers was adapted between the probe domains to efficiently induce intramolecular tension to the sandwiched luciferase, was previously discussed.13 All of the above results show that (i) luciferases may have the intrinsic nature to modulate their enzymatic activity in response to molecular tension induced by PPIs although the extent of optical variation might be trivial, and (ii) the sensitivity of the probes to molecular tension is dominated by the molecular designs including the flexible region of the luciferase, the length of the flexible linkers, and the presence of the SP. TP2.4 Dramatically Elevates the Optical Intensities in a Quantitative Manner. The ligand-driven feature of the optical intensities of TP2.4 was determined with varying concentrations of rapamycin (Figure 2). Stimulation with vehicle (0.1% ethanol) resulted in a basic optical intensity, whereas the presence of rapamycin increased the optical intensities even at low concentrations, with the signal reaching a plateau at 10−5 M. The detectable concentration of rapamycin was as low as 10−9 M. In contrast to our expectation, rapamycin D

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Figure 5. (A) Rapamycin-driven optical intensity of living COS-7 cells on a microslide. TP2.4 emits rapamycin-dependent bioluminescence. The inset a shows a profile of the optical image of the optical slide. (B) Relative optical intensities of TP4.1 and TP4.2 in response to various ligands (n = 3). The inset illustrates the molecular structures of TP4.1 and TP4.2. Abbreviations: ER LBD, the ligand-binding domain of human estrogen receptor; SH2, the Src homology domain of v-Src; RLuc8, an 8-mutation-bearing variant of Renilla reniformis luciferase; E2, 17β-estradiol; OHT, 4hydroxytamoxifen.

The strongest optical intensities for TP2.4 were found with nCTZ, CTZ h, and CTZ f (Figure 4A). The absolute optical intensities were about 4- to 5-fold stronger than those with CTZ i and 6-pi-OH-CTZ. In contrast, poor optical intensities close to the background were observed with CTZs hcp, fcp, and ip. A detailed comparison of the chemical structure−intensity relationship (CSIR) reveals that the intensity is dominated by the size of the substituent at the C-2 position of coelenterazine: i.e., the size of the side chains decreases in the order of CTZ n, CTZ i, CTZ f, and CTZ h, whereas the optical intensities approximately increase in the same order. The poor intensities with CTZs cp, hcp, fcp, and ip can be explained by modification of the side chain at C-8 of the coelenterazine backbone. The corresponding feature was observed in our previous study on substrate selectivity of artificial luciferases,15 where the optical intensities of the substrates with ALuc34 were elevated in the same order of CTZ n, CTZ i, CTZ f, and CTZ h. Prolonged optical emission compared with native CTZ was observed with CTZ n, CTZ i, and 6-pi-OH-CTZ (Figure 4B). 6-pi-OH-CTZ and CTZ i maintained a half-maximal optical intensity after about 10 and 15 min, respectively. CTZ n surprisingly maintained about 60% of the initial optical intensity even 20 min after substrate injection. The overall results are interpreted as (i) the optical intensity and stability of substrates are basically dominated by the size effects of the functional groups at C-2, C-6, and C-8 positions of coelenterazine, (ii) the C-2 position of CTZ analogs is the most important functional group to determine both of the optical intensity and stability of TPs, (iii) the C-8 position of CTZ analogs is a conservative site for recognizing TPs and any modification of the C-8 position generally hampers its recognition of TPs, and (iv) considering that the molecular tension modulates the active site of ALucs in TPs, the substrates emitting poor bioluminescence with TP2.4 in Figure 4A may have potential to enhance bioluminescence with other TPs. TP2.4 Induces a Ligand-Driven Elevation of Bioluminescence in Live Cells. A bioluminescence imaging in small animal models is an attractive subject in medical and pharmaceutical sciences. The feasibility was examined with

This negative control study revealed that (i) molecular tension induced by rapamycin-driven FRB−FKBP interaction is the only factor contributing to the enhancement of ALuc activities, (ii) intermolecular PPIs do not enhance the ALuc activities, (iii) the presence of 10−6 M of rapamycin itself does not boost or inhibit the ALuc activities, and (iv) the Western blot analysis showed that TP2.4 is properly expressed at the expected size and the expression levels are not greatly affected by stimulation of rapamycin for 4 h. Combinational Probes Showed Improved Signal-toBackground (S/B) Ratios. A series of combinational probes were fabricated, which comprise both features of molecular TPs and protein-fragment complementation assays (PCAs) (Figure 3). Consecutive removal of the amino acids at the C-terminal end of ALuc23 gradually decreased the overall optical intensities of the probes (Figure 3B). The maximal S/B ratio of 9.2 was found with TP3.3 embedding the shortest ALuc fragment (18−198 AA). The improved S/B ratio is achieved by the greatly diminished “background” intensity, rather than the “signal” intensity developed by rapamycin. The basic optical signal intensity of TP3.3 induced by the vehicle was as low as that of the optical bottom plate itself (Figure 3B, inset a; dotted line). The dramatic decline in the basal bioluminescence of TP3.3 is interpreted as follow: as predicted from the super 2Dstructure of ALuc (Figure 3A), the C-terminal end of ALuc is very close to the substrate and constitutes part of the active site. We consider that the elimination of the C-terminal end resulted in suppression of the basal optical intensities. The overall results show that (i) a combinational single-chain probe can be made by combining the concept of a molecular TP with that of PCA and (ii) by an optimization process, combinational probes potentially allow both strong bioluminescence and high S/N ratios, considered as the merits of TP and PCA, respectively. TP2.4 Prefers nCTZ, CTZ h, and CTZ f for Strong Bioluminescence. The substrate selectivity and the kinetic characteristics of TP2.4 were estimated with various coelenterazine analogues (Figure 4). E

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plateau comprise all the periods for (i) plasma membrane (PM) permeation of rapamycin, (ii) rapamycin-induced intramolecular binding between FRB and FKBP, and (iii) the tensed ALuc−substrate interaction and corresponding emission of bioluminescence. Steps (ii) and (iii) are unlikely to be a ratedetermining step because molecular interaction between FRB and FKBP or between FKBP and rapamycin generally reaches a plateau within 2 min, according to surface plasmon resonance (SPR) studies.22,23 Thus, the remaining step (i) should be the rate-determining step. The PM permeability of chemicals greatly depends on the hydrophobicity. Variance of the stimulation times between steroid and rapamycin, i.e., 20 min vs 4 h, is considered to reflect the PM permeability of the chemicals. Taken together, the molecular TPs provide unique utility for determining PPI in cells with high bioanalytical performance comparable to other conventional tools, e.g., PCA and BLI. TPs are characteristic in point that they make use of a full-length luciferase and molecular tension and thus allow strong bioluminescence signal, compared to PCA. We found that the S/B ratios can be modified by fusing the concept of PCA to the TP design. We previously reported that a molecular strain of RLuc8 may be indexed for bioassays, but it was unclear whether the RLuc8 is the only luciferase that allows a molecular tension probe. The present study confirms with RLuc8 and ALuc23 that any luciferases basically have the ability to vary its enzymatic activity more or less by molecular tension physically appended by PPIs. It is still unclear how molecular tension works in the active site of ALuc23 of TPs. Further study on structural information before and after rapamycin addition in the future will help to rationally explain the working mechanism. We found that TPs exhibited unique substrate selectivity and bioluminescence kinetics. Considering that luciferases are broadly used as an optical readout for bioassays, TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.

living COS-7 cells expressing TP2.4, grown in a 6-channel microslide (Figure 5A). The right three channels stimulated with 10−6 M rapamycin exhibited about 6-fold stronger optical intensities (i.e., 6.1 ± 0.7), compared to the left three channels stimulated with the vehicle (0.1% ethanol). The high S/B ratio in the model study indicates that living mammalian cells carrying TP2.4 can be implanted into a target organ of living animals, where it reports on rapamycin activities by elevated bioluminescence intensities. Basic Concept of Molecular TPs is Generally Applicable to Other Protein−Protein Binding Models. The general applicability of the concept of the present molecular TPs was examined with the following two PPI models (Figure 5B): i.e., (i) TP4.1 (ER LBD-ALuc23-SH2), in which 17βestradiol (E2) activates ER LBD−SH2 binding; (ii) TP4.2 (FRB-RLuc8-FKBP), in which rapamycin triggers FRB−FKBP interaction. The binding model between ER LBD and SH2 shown in TP4.1 was originally demonstrated by Kim et al. for constructing a multicolor imaging probe set.8 Tyr537 of ligand-activated ER LBD is phosphorylated, which is recognized by the adjacent SH2 domain in the probe. TP4.1 and TP4.2 enhanced the optical intensities up to 1.4and 4-fold by E2 and rapamycin, respectively, compared with those by the vehicles. 4-Hydroxytamoxifen (OHT), known as anti-estrogen, weakly increased the optical intensities. These results show that the basic concept of “molecular tension” probes indexing molecular tension induced by PPIs is generally applicable to other PPI models including homo- and hetrodimerization of FRB and FKBP12, and thus useful as a bioluminescent template. We have no direct evidence why the S/B ratio by the ER LBD-SH2 pair is much lower than that by the FRB−FKBP pair. The molecular tension in TPs is variable by many possible reasons including steric hindrance among the internal components, spatial mismatch, the linker length, and directions of N- and C-terminal ends of the components. The poor S/B ratio with the ER LBD−SH2 pair should be improved by an optimization process including truncation of the ER LBD. Molecular TPs Facilitate a Rapid Determination of Protein−Protein Interactions. Kim et al. previously suggested that conventional molecular imaging probes are categorized into two major groups: i.e., genetic, transcriptional assay (GTA) and nontranscriptional assay (NTA).19 The present assay is categorized into NTA accompanying intramolecular distortion of the sandwiched luciferase. The probe is expressed beforehand and located in an adequate intracellular compartment of interest. The probe is ready to emit bioluminescence once the cell is stimulated by a ligand. This method thus largely shortens the assay time, compared with GTA, which consumes 12−24 h for accumulating a reporter protein after ligand stimulation. Membrane Permeability of Ligands Is a RateDetermining Step in the Molecular TPs. A high sample throughput generally provides great potential for an early stage screening of drug candidates in medical and pharmaceutical sciences. The present TPs require about 4 h of incubation time to reach the optical plateau according to our evaluation (data not shown). Although this is a greatly shortened assay time compared with conventional reporter-gene assays, it is still longer than the assay time (20 min) of our single-chain probes illuminating the activities of steroid hormones.19−21 It is considered that the 4 h of incubation time to reach the optical

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EXPERIMENTAL PROCEDURES Construction of Plasmids. We generated a series of DNA constructs encoding 23 kinds of different molecular designs for estimating their potential as molecular tension probes able to sense PPIs (Table 1). The basic molecular structures of TP1 and TP2 series probes differ in the consecutive order of their component proteins from the N-terminal end. The schematic diagrams of the cDNA constructs are illustrated in Figure 1B and SI Figure 1. As a template for polymerase chain reaction (PCR), cDNAs encoding the following components were obtained from the corresponding providers: Renilla luciferase 8 (RLuc8) was kindly presented by Prof. Gambhir (Stanford University); ALucs 16, 23, 24, 30 were from our previous studies;10,15 the human FKBP (12 kDa, Genbank access number: AAP36774.1) and FRB (11 kDa, PDB access number: 1AUE_A) were custom-synthesized by Eurofins Genomics (Tokyo) on the basis of the sequence information on a public database (NCBI), and the ligand binding domain of human estrogen receptor (ER LBD, 305−550 AA) and the SH2 domain of v-Src were from our previous study.13 A series of cDNA segments encoding the components shown in Table 1 were generated by PCR using corresponding primers to introduce unique restriction sites, HindIII/BamHI, BamHI/ KpnI, or KpnI/XhoI at the 5′ and 3′ ends, respectively. The F

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containing nCTZ in a 200 μL microtube. The corresponding optical intensities were integrated for 30 s with a high-precision spectrophotometer (AB-1850, ATTO) equipped with a cooled charge-coupled device (CCD) camera that enables one-shot capture of the entire light. To examine the secreted proportion, we determined the intra- and extracellular fractions of the probes, TP2.3 and TP2.4 to examine the secreted proportion as shown in Table 1 (SI Figure 3(A)). COS-7 cells raised in a 12-well plate were transiently transfected with pcDNA3.1 encoding TP2.3 or TP2.4, as described in Figure 1C. The cells were incubated 16 h and then the supernatants were transferred into fresh microtubes. The remaining cells were washed once with a PBS buffer and lysed with a lysis buffer (Promega). The optical intensities from 50 μL of the supernatants and 5 μL of the lysates were determined for 5 s with a luminometer (GloMax 20/20n, Promega) after injection of 110 μL of the assay solution carrying nCTZ (Promega). We further determined whether TP2.3 in the supernatant exerts rapamycin sensitivity (SI Figure 3(B)). The supernatants were first treated with vehicle (0.1% ethanol) or 10−6 M of rapamycin (final concentration). The supernatants (50 μL) on a 96-well optical bottom plate were then simultaneously mixed with 150 μL of the assay solution carrying nCTZ (Promega) using a multichannel pipet, and transferred into the chamber of LAS-4000 (FujiFilm). The optical image was determined for 5 min. The experiments were conducted in quadruplicate for an error bar. Ligand-Dependent Elevation of Optical Intensities of TP2.4. The ligand-driven feature of the optical intensities of TP2.4 was determined with varying concentrations of rapamycin (Figure 2A). The COS-7 cells carrying TP2.4 were prepared as described above. The cells were stimulated with vehicle (0.1% ethanol) or varying concentrations of rapamycin from 10−9 to 10−4 M for 4 h. The cells on each well, where every three wells on the plate were grouped for error bars, were lysed with a lysis buffer (Promega) and an aliquot (10 μL) of the lysates from the wells was transferred into each well of a fresh 96-well optical-bottom plate (Thermo Scientific). The lysates in the plate were simultaneously mixed with 50 μL of assay solution (Promega) containing a nCTZ analog, 6-pi-OHCTZ, (final concentration = 0.01 mg/mL) with a multichannel pipet (Gilson).24 All samples were prepared in quadruplicate (n = 4). The plate was immediately transferred into the chamber of the image analyzer and the optical intensities were recorded. The optical intensities (relative luminescence unit; RLU) were normalized to protein amount (μg), integration time (second), and area (mm2), i.e., the unit is expressed in RLU/μg/s/mm2. Negative Control Study for Proving the Molecular Tension−Optical Intensity Correlation of TP2.4. We further examined whether the enhanced optical intensities are solely caused by intramolecular tension induced by ligandactivated PPIs, and not by any other driving force including any unexpected intermolecular PPIs (Figure 2B and SI Figure 1). To exclude the probability of intermolecular interaction, we first fabricated TP0.1 and TP0.2, which are FKBP- and FRBdeficient probes, respectively (Table 1 and SI Figure 1). The negative control probes were made by replacing the cDNA segments encoding FRB and FKBP in TP2.4 with small cDNA fragments embedding a start codon (ATG) and a stop codon (TGA), respectively. The constructs were subcloned into a

linkers connecting the probe components were minimized to efficiently develop intramolecular tension inside the probe. The cDNA segments were restricted by the corresponding restriction enzymes (NEB), ligated with a ligation kit (Takara Bio), and finally subcloned into a pcDNA 3.1(+) mammalian expression vector (Invitrogen) using the HindIII and XhoI sites. The probes are categorized into 5 groups (TP0, TP1, TP2, TP3, and TP4 series) according to the molecular designs (Table 1). The cDNA constructs encoding TP1 and TP2 series probes differ in the consecutive order of the segments from the 5′ end (Figure 1B). The constructs encoding TP3 series probes are characteristic in that they carry cDNA segments encoding ALuc23 with a shortened C-terminal end, compared with the others. The constructs encoding TP0 series probes were designed for conducting negative control studies, while the constructs encoding TP4 series were fabricated for examining the general applicability of the present probe design to other PPI models. The cDNA sequences of all the constructs were confirmed with a genetic analysis system (GenomeLab GeXP, Beckman Coulter). Evaluation of an Optimal Molecular Design for Molecular TPs. Sixteen kinds of molecular designs were examined for evaluating efficient molecular TPs (Figure 1C). COS-7 cells were cultured in a 96-well plate (Nunc) in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% penicillin/ streptomycin (P/S; Gibco) at 37 °C in a cell incubator (5% CO2; Sanyo). The cells on the plate were transiently transfected with an aliquot (0.2 μg per well) of the pcDNA 3.1(+) vector encoding one of the TP1 and TP2 series probes as specified in Figure 1C using a lipofection reagent (TransIT-LT1; Mirus), and incubated for 16 h at 37 °C in 5% CO2 before the following experiments. The cells on the plate wells, where every three wells were grouped for error bars, were stimulated with vehicle (0.1% ethanol dissolved in the culture medium) or 10−6 M of rapamycin for 4 h and lysed with a lysis buffer (Promega). An aliquot (10 μL) of the lysate from the wells was transferred into each well of a fresh 96-well optical bottom plate (Thermo Scientific) and simultaneously mixed with 50 μL of assay solution (Promega) containing native coelenterazine (nCTZ) with a multichannel pipet (Gilson). The plate was immediately placed into the chamber of an image analyzer (LAS-4000, FujiFilm) equipped with a cooled CCD camera system (−25 °C). The optical intensities were determined with the image acquisition software (Image Reader v 2.0) and analyzed with the specific image analysis software (Multi Gauge v 3.1). The error bars hereafter show the standard deviation (SD) of each mean in single sigma. The luminescence intensities are expressed as a fold intensity of relative luminescence units (RLU), i.e., RLU ratios (+/−), where RLU (+) and RLU (−) represent the luminescence intensities with 1 μg of cell lysate after the cells were incubated with and without rapamycin, respectively; the RLU is an amplified value of photon counts generated from the image analyzer (arbitrary unit). The corresponding optical spectra were determined in the presence or absence of rapamycin (Figure 1C, inset a). COS-7 cells expressing TP2.4 were stimulated with 10−6 M of rapamycin for 4 h and lysed with the lysis buffer. 5 μL of the lysate was mixed with 35 μL of the assay solution (Promega) G

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in 25 μL of ethanol and diluted 10-fold (final concentration: 0.1 μg/μL) with an assay buffer (Promega). An aliquot (50 μL) of the prepared substrate solutions was simultaneously mixed with the lysates on the wells of the plate using a multichannel pipet and immediately transferred into the image analyzer. The optical intensities were measured every 5 min from 0 to 20 min after substrate injection in a high resolution and 30 s integration mode, where four wells were treated with the same substrate and grouped for error bars. The examined coelenterazine analogs in the present study include nine analogs from a coelenterazine sampler kit (Promokine): native coelenterazine (nCTZ), coelenterazine h (CTZ h), coelenterazine f (CTZ f), coelenterazine i (CTZ i), coelenterazine n (CTZ n), coelenterazine cp (CTZ-cp), coelenterazine hcp (CTZ hcp), coelenterazine fcp (CTZ fcp), coelenterazine ip (CTZ ip), in addition to one synthesized coelenterazine analog from our previous study 6-pi-OH-CTZ (6-pi-OH).24 Live-Cell Image of COS-7 Cells Carrying TP2.4. A bioluminescence imaging was conducted with living COS-7 cells carrying TP2.4 in a 6-channel microslide (μ-slide VI0.4, ibidi) (Figure 5). COS-7 cells grown on the 6-channel microslide were transiently transfected with the pcDNA3.1(+) vector encoding TP2.4 and incubated for 2 days. The cells on the left three channels and right three channels were then stimulated with vehicle (0.1% ethanol) or 10−6 M rapamycin for 4 h, respectively. The culture media in the slide channels was replaced with an HBSS buffer containing nCTZ. The slide was immediately transferred into the image analyzer and the consequent optical image was taken every 5 min in a high precision and 30 s integration mode. Evaluation of the General Applicability of the Probe Scheme to Other Protein−Protein Binding Models. TP2.4 was further modified to examine whether the concept is generally applicable to other protein−protein binding models (Figure 5B). The cDNA segments encoding FRB and FKBP in TP2.4 were replaced with those encoding ER LBD and SH2 domain and the consequent probe was named TP4.1. The binding between the phosphorylated ER LBD and SH2 represents a typical nongenomic signaling pathway of ER in mammalian cells. Separately, the cDNA segment encoding ALuc23 (18− 212 AA) in TP2.4 was replaced with that encoding RLuc8 and the consequent probe was named TP4.2 (Table 1, Figure 5B, SI Figure 1). Bioluminescence intensities were recorded by the image analyzer using nCTZ as the substrate in analogy to the procedures described above, where three wells were treated with a same stimulator and grouped for error bars.

pcDNA3.1(+) vector and the fidelity of the sequences was confirmed with the DNA sequencer. COS-7 cells grown in a 96-well plate (Nunc) were transiently transfected with pcDNA3.1(+) vectors encoding (i) TP0.1, (ii) TP0.2, (iii) TP0.1 plus TP0.2, or (iv) TP2.4. The cells on each well, where every three wells on the plate were grouped for error bars, were incubated for 16 h in a CO2 incubator (Sanyo), and stimulated with 10−6 M of rapamycin for 4 h. The cells were lysed and an aliquot (20 μL) of the lysates was transferred in a 96-well optical bottom plate. Finally, the lysates were simultaneously mixed with an aliquot (50 μL) of assay buffer (Promega) containing nCTZ, immediately set into the chamber of the image analyzer, and the corresponding optical intensities determined. Apparent variance in the expression levels of TP2.4 was examined with a Western blot analysis (Figure 2C). COS-7 cells transiently expressing TP2.4 were stimulated with 10−6 M rapamycin for 4 h and lysed with an aliquot of sample buffer (Wako). The lysates were electrophoresed and transferred to a nitrocellulose paper. The specific proteins were blotted with rabbit anti-FKBP antibody (abcam) or mouse anti-β-actin antibody (Sigma). The proteins were subjected to anti-rabbit and anti-mouse secondary antibodies, respectively. The stains were finally blotted with a horseradish peroxidase (HRP) substrate solution (Immunostar, Wako). Fabrication of a Combinational Probe for Illuminating Protein−Protein Interactions. On the basis of the success of TP2.4, TP2.4 was further modified to fabricate a combinational probe with the synergistic property of the molecular TP and a conventional protein complementation assay (PCA) (Figure 3; SI Figure 2). We paid attention to the C-terminal end of ALuc23, which according to an alignment of the sequences (alignment score: 26, ClustalW v 2.1), shares high sequential homology with the adjacent N-terminal end of FKBP inside TP2.4 (Figure 3A, Inset a). Based on this information, a series of new bioluminescent probes were designed by consecutively eliminating several amino acids at the C-terminal end of ALuc23 in TP2.4, and were named TP3.1 to 3.3 (Table 1, SI Figure 2B). They were generated by replacing cDNA of the full-length ALuc23 with the corresponding shortened cDNA fragments of ALuc23. COS-7 cells expressing one of the above-described TP3 probes (i.e., TP3.1, TP3.2, and TP3.3) besides TP2.4 were prepared in a 96-well optical bottom plate with the same method as described above (Figure 3B). The cells on each well of the plate were stimulated with vehicle (0.1% ethanol) or 10−6 M of rapamycin for 4 h, and the optical intensities from the cell lysates were determined with the image analyzer as described above, where four wells were treated with a same stimulator and grouped for error bars. Substrate-Driven Feature of the Optical Intensity and Kinetics of TP2.4. The substrate-driven feature of the optical intensity and kinetics of TP2.4 was determined both in the presence or absence of rapamycin (Figure 4). COS-7 cells grown in each well of a 96-well plate were transiently transfected with pcDNA3.1(+) vector encoding TP2.4 and incubated for 2 days. Four hours after the stimulation with 10−6 M of rapamycin, the cells were lysed for 20 min with 50 μL of a lysis buffer (Promega). An aliquot (10 μL) of the lysates was transferred to each well of a 96-well optical bottom plate. Furthermore, 25 μg of ten different kinds of coelenterazine analogs (see below) were separately dissolved

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00421. Schematic structures of cDNA constructs encoding the molecular tension probes (Figure S1); illustration showing the working mechanism and cDNA constructs of combinational bioluminescent probes (Figure S2); the intra- and extracellular fractions of the probes, TP2.3 and TP2.4 (Figure S3) (PDF) H

DOI: 10.1021/acs.bioconjchem.5b00421 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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(18) Inouye, S., and Sahara, Y. (2008) Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps. Biochem. Biophys. Res. Commun. 365, 96−101. (19) Kim, S. B., Takenaka, Y., and Torimura, M. (2011) A bioluminescent probe for salivary cortisol. Bioconjugate Chem. 22, 1835−1841. (20) Kim, S. B., Sato, M., and Tao, H. (2009) Split Gaussia luciferasebased bioluminescence template for tracing protein dynamics in living cells. Anal. Chem. 81, 67−74. (21) Kim, S. B., Umezawa, Y., and Tao, H. (2009) Determination of the androgenicity of ligands using a single-chain probe carrying androgen receptor N-terminal peptides. Anal. Sci. 25, 1415−1420. (22) Banaszynski, L. A., Liu, C. W., and Wandless, T. J. (2005) Characterization of the FKBP-Rapamycin-FRB ternary complex. J. Am. Chem. Soc. 127, 4715−4721. (23) Wear, M. A., and Walkinshaw, M. D. (2007) Determination of the rate constants for the FK506 binding protein/rapamycin interaction using surface plasmon resonance: An alternative sensor surface for Ni2+-nitrilotriacetic acid immobilization of His-tagged proteins. Anal. Biochem. 371, 250−252. (24) Nishihara, R., Suzuki, H., Hoshino, E., Suganuma, S., Sato, M., Saitoh, T., Nishiyama, S., Iwasawa, N., Citterioa, D., and Suzuki, K. (2015) Bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution. Chem. Commun. 51, 391−394.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by JSPS KAKENHI Grants: Numbers 26288088 and 24225001.

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REFERENCES

(1) Alberts, B. (1994) Molecular biology of the cell, 3rd ed., Garland Pub., New York. (2) Ozawa, T., Yoshimura, H., and Kim, S. B. (2013) Advances in Fluorescence and Bioluminescence Imaging. Anal. Chem. 85, 590−609. (3) Vogel, S. S., Thaler, C., and Koushik, S. V. (2006) Fanciful FRET. Sci. Signaling 2006, re2. (4) Hoshino, H., Nakajima, Y., and Ohmiya, Y. (2007) LuciferaseYFP fusion tag with enhanced emission for single-cell luminescence imaging. Nat. Methods 4, 637−639. (5) Luker, K. E., Smith, M. C., Luker, G. D., Gammon, S. T., PiwnicaWorms, H., and Piwnica-Worms, D. (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. U. S. A. 101, 12288−12293. (6) Kim, S. B., Awais, M., Sato, M., Umezawa, Y., and Tao, H. (2007) Integrated molecule-format bioluminescent probe for visualizing androgenicity of ligands based on the intramolecular association of androgen receptor with its recognition peptide. Anal. Chem. 79, 1874− 1880. (7) Tarassov, K., Messier, V., Landry, C. R., Radinovic, S., Serna Molina, M. M., Shames, I., Malitskaya, Y., Vogel, J., Bussey, H., and Michnick, S. W. (2008) An in vivo map of the yeast protein interactome. Science 320, 1465−1470. (8) Kim, S. B., Umezawa, Y., Kanno, K. A., and Tao, H. (2008) An integrated-molecule-format multicolor probe for monitoring multiple activities of a bioactive small molecule. ACS Chem. Biol. 3, 359−372. (9) Kaihara, A., and Umezawa, Y. (2008) Genetically encoded bioluminescent indicator for ERK2 dimer in living cells. Chem. - Asian J. 3, 38−45. (10) Kim, S. B., Torimura, M., and Tao, H. (2013) Creation of artificial luciferases for bioassays. Bioconjugate Chem. 24, 2067−2075. (11) Paulmurugan, R., and Gambhir, S. S. (2005) Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal. Chem. 77, 1295−1302. (12) Kim, S. B., Otani, Y., Umezawa, Y., and Tao, H. (2007) Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase. Anal. Chem. 79, 4820−4826. (13) Kim, S. B., Sato, M., and Tao, H. (2009) Molecular tension indexed bioluminescent probe for determining protein-protein interactions. Bioconjugate Chem. 20, 2324−2330. (14) Tannous, B. A., Kim, D. E., Fernandez, J. L., Weissleder, R., and Breakefield, X. O. (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11, 435−443. (15) Kim, S. B., and Izumi, H. (2014) Functional artificial luciferases as an optical readout for bioassays. Biochem. Biophys. Res. Commun. 448, 418−423. (16) Takenaka, Y., Masuda, H., Yamaguchi, A., Nishikawa, S., Shigeri, Y., Yoshida, Y., and Mizuno, H. (2008) Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica. Gene 425, 28−35. (17) Takenaka, Y., Yamaguchi, A., Tsuruoka, N., Torimura, M., Gojobori, T., and Shigeri, Y. (2012) Evolution of bioluminescence in marine planktonic copepods. Mol. Biol. Evol. 29, 1669−1681. I

DOI: 10.1021/acs.bioconjchem.5b00421 Bioconjugate Chem. XXXX, XXX, XXX−XXX