Label-Free and Homogenous Detection of Caspase-3-Like Proteases

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Label-Free and Homogenous Detection of Caspase-3-Like Proteases by Disrupting Homodimerization-Directed Bipartite Tetracysteine Display Yong Yang, Yan Liang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04771 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Analytical Chemistry

Label-Free and Homogenous Detection of Caspase-3-Like Proteases by Disrupting Homodimerization-Directed Bipartite Tetracysteine Display

Yong Yang, § Yan Liang, § and Chun-yang Zhang*† §

Laboratory for Food Safety and Environmental Technology, Shenzhen Institutes of Advanced Technology,

Chinese Academy of Sciences, Shenzhen 518055, China †

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of

Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Caspase-3-like proteases (e.g., caspase-3 and caspase-7) constitute the core players of cell apoptosis, and their dysregulation has been linked to a number of human diseases, such as cancer and neurodegenerative disorders. However, the methods for measuring caspase-3-like proteases are often complex and time-consuming. Herein, we develop a label-free method to homogenously detect caspase-3-like proteases in vitro and in complex cell lysate. This assay uses a modular peptide that contains a dimerization domain, a caspase 3/7 cleavage sequence, and a di-cysteine motif as the activity sensor to detect caspase-3-like proteases. In the absence of caspase-3-like proteases, the homodimerization of modular peptide brings the di-cysteine motif into close proximity and forms a particular configuration suitable for the binding to bis-arsenical dye FlAsH-EDT2. The coordination of FlAsH-EDT2 to dimeric peptide forms a highly fluorescent FlAsH-peptide complex. In contrast, the cleavage of the modular peptide by caspase-3-like proteases removes the di-cysteine motif from the peptide and abrogates the bipartite tetracysteine display, leading to the disappearance of fluorescence. As a result, the caspase-3-like proteases can be quantitatively evaluated by measuring the change in fluorescence. This assay may be carried out in a “mix-and-read” manner, and is thus quite simple and convenient. Moreover, this assay is extremely specific and can measure caspase-3 protein down to 1.28 × 10-4 µg/mL. Importantly, the assay is compatible with complex biological samples and can measure both the activation and inhibition of intracellular caspase-3, thereby providing a new approach for the screening of caspase-targeted drugs and the diagnosis of apoptosis-associated diseases.

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Introduction Proteases, also known as peptidases or proteinases, are a large group of enzymes that selectively hydrolyze peptide bonds within proteins and polypeptides 1. The human genomes encode approximately 600 different proteases which regulate virtually all biological pathways and networks 2. One important class of human protease is caspases, a family of cysteine proteases that demonstrate primary specificity for aspartic acid and may cleave the substrates at the C-terminal of specific aspartate residue. It is well documented that caspases play essential roles in apoptosis, inflammation, cell differentiation, innate immune, cell proliferation, and cell migration

3-5

.

Excessive or insufficient activation of caspases may lead to a broad range of human diseases, including cancers, cardiovascular diseases, immunodeficiency, autoimmune diseases, and neurological disorders3,6-9. Among the apoptotic proteases, the caspase-3-like proteases, such as caspase-3 and caspase-7, are key executioners of cell apoptosis and may cleave hundreds of substrate proteins. The caspase-3 like protease is initially expressed as inactive zymogen in cells and often requires initiator caspase-mediated cleavage for activation. The active form of caspase-3 like protease can specifically recognize a tetrapeptide sequence of Asp-Glu-Val-Asp (DEVD) and hydrolyze peptide bonds after the C-terminal Asp residue. By using the DEVD as the cleavable substrate, a series of analytical methods including fluorometric, electrochemical, electrochemiluminescent, bioluminescent, surface plasmon resonance (SPR), colorimetric, Förster resonance energy transfer (FRET)-based methods have been developed to measure caspase-3/7 activity.10-15 The integration of DEVD-containing peptides with nanoparticles (e.g., quantum dots

16,17

, gold nanoparticles

18

and grapheme oxide

15

) has also been used for the study of

caspase-3/7 activation and inhibition. In addition, a variety of genetically encoded FRET-based reporters

15

and

switch-on fluorescent biosensors19 have been constructed to track caspase-3/7 dynamics in living cells. However, some of these methods require labor-intensive procedures or high levels of technical expertise. Therefore, a facile and versatile method with the capability of measuring both the activity and inhibition of caspase-3 like protease is highly desirable. Fluorescent proteins have revolutionized cell biology by enabling what was formerly invisible to be seen clearly. The genetic fusion of fluorescent proteins to target protein allows for the visualization of the localization, dynamics and interaction of specific proteins in living cells and even in whole organism. Nevertheless, an issue associated with fluorescent proteins is their relatively large size (~27 kDa) and the potential risk of perturbing the 3



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function of target protein

20

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. Alternatively, small genetically encoded tags are introduced for the labeling of

cellular proteins. A typical example is the tetracysteine tag that was first reported by Tsien and colleagues 21. The tetracysteine tag is a short peptide with the sequence of CCPGCC (Cys-Cys-Pro-Gly-Cys-Cys)

22

or

FLNCCPGCCMEP (Phe-Leu-Asn-Cys-Cys-Pro-Gly-Cys-Cys-Met-Glu-Pro) 23, which can be selectively labeled by biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2 with each arsenic atom binding to a pair of adjacent cysteines. These dyes are nonfluorescent in ethanedithiol (EDT)-bound form but become fluorescent upon binding to the tetracysteine motif. In addition to the linear tetracysteine tags, the biarsenical dyes may bind to split tetracysteine motifs in which the central di-peptide Pro-Gly is replaced by one or more folded protein domains whilst the two Cys-Cys pairs remain spatially close. The biarsenical dyes can interact with the split tetracysteine motifs without significant loss in affinity and brightness. This approach, termed as bipartite tetracysteine display

24,25

, has been

successfully employed to monitor the protein conformational change and dynamics26, and to characterize specific protein-protein interactions

27

. Herein, we take advantage of the binding capability of biarsenical dyes toward

split tetracysteine motif, and develop a label-free method to homogenously measure caspase-3-like proteases. Our strategy relies on homodimerization-directed bipartite tetracysteine display and the subsequent abrogation by specific protease cleavage. This assay is compatible with complex biological environments and can measure both the activation and inhibition of intracellular caspase 3/7, providing a convenient way for the detection of caspase activity and the screening of caspase-targeted drugs.

Experimental Section Materials The TC-FlAsH™ II in-cell tetracysteine tag detection kit, the trypsin protein, and the reagents used for cell culture were purchased from Invitrogen (USA). The recombinant human caspase-3 protein was obtained from R&D Systems (Minneapolis, USA). The apoptosis inducer (staurosporine, STS) and caspase-3 inhibitor (Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO) was purchased from Cell Signaling Technology (USA) and Enzo Life Sciences Inc. (USA), respectively. Recombinant human SENP1 protein was purchased from Enzo Life Sciences Inc. (USA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). The peptides listed in table 1 were synthesized by SciLight Biotechnology LLC (Beijing, China) with purity greater than 95%. All 4


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peptides were analyzed by MALDI-TOF mass spectrometry (MS) to determine their molecular weight. The MS result of Jun-CCC and Jun-TCC are provided in the Supporting Information (see Supporting Information, Figures S1 and S4, respectively). All solutions were prepared using ultra-pure water (Millipore, > 18.2 MΩ). Caspase-3-mediated cleavage of Jun-CCC and fluorescence measurement The peptides were dissolved in DMSO as 1 mM stock solution, and the stock solution was immediately frozen stored in aliquots at -20°C. For the cleavage assay, 0.1 µL of peptide solution (1 mM) and 0.2 µL of diluted protease solution were added into 10 µL of caspase assay buffer (25 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10% sucrose, 0.1% CHAPS, 1 mM DTT, pH = 7.4) and incubated at 37°C for 60 minutes. Then the reaction mixture was added to 40 µL of bipartite tetracysteine display buffer (100 mM Tris·Cl, 75 mM NaCl, 1 mM EDTA, 1 mM DTT, pH = 7.4) followed by incubation with 1 µM FlAsH-EDT2 and 0.05 mM BAL at room temperature for 30 minutes. The fluorescence signal of the mixture was measured by an F-4600 spectrometer (Hitachi, Japan) with an excitation wavelength of 510 nm. To verify the specificity of Jun-CCC toward caspase-3, the Jun-CCC was incubated with 0.05 µg of casapse-3, 0.05 µg of thrombin, and 0.05 µg of trypsin in caspase assay buffer at 37°C for 60 minutes, respectively. The fluorescence measurement was performed as described above. To determine the limit of detection (LOD), the assay was performed with a constant concentration of Jun-CCC and varying concentrations of caspase-3. Meanwhile, the fluorescence of Jun-CCC without caspase-3 treatment was also measured. The change in fluorescence intensity is calculated according to equation 1: ∆F = Fnegative — Fcaspase

(1)

where ∆F represents the decrease of fluorescence intensity, Fnegative is the measured fluorescence intensity in the absence of capase-3, and Fcaspase is the measured fluorescence intensity in the presence of caspase-3. The calculated ∆F is plotted against the concentration of caspase-3 to determine the LOD.

Table 1.

Sequences of peptides and their binding affinity to FlAsH-EDT2

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Name

Peptide sequences (N- to C-terminal)a

Kd (µM) b

Jun-CCC

Ac-asaaeleervktlkaeiyelrskanmlreqiaqlgapDEVD↓GCC-NH2c

3.17 ± 0.39

JunP-CCC

Ac-asaaepeervktpkaeiyeprskanmpreqiaqpgapDEVDGCC-NH2c

> 1000

CCC

Ac-GCC-NH2

566.7 ± 160.9

Positive control

Ac-FLNCCPGCCMEP-NH2

4.55 ± 0.36

Negative control

Ac-FLNCCPPPPPPPPPCCMEP-NH2

122.1 ± 7.86

a

The cleavage site by caspase-3 is indicated by downward arrow.

b

The Kd is determined by adding increasing amount of peptides to 0.1 µM FlAsH-EDT2 in 50 µL of

bipartite tetracysteine display buffer. Note: 0.05 mM BAL was added to the mixture to eliminate the nonspecific binding. c

Lowercase characters denote D-amino acid. The underlined characters indicate the capping motif.

Western Blotting Assay The western blotting assay was carried out according to the general protocol. Briefly, the crude cell lysates were resolved in 15% SDS-PAGE gel before transferring to a nitrocellulose membrane. The membrane was then blocked with non-fat powdered milk at room temperature for 1 hour. After extensive washing, the blot was incubated with anti-cleaved caspase-3 antibody (Cell Signaling Technology, USA) and anti-tubulin antibody (Invitrogen, USA) for 60 minutes, respectively. Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch, USA) were used as the secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence with ECL detection kit (Amersham Biosciences, USA) according to the instruction of the manufacturer. The images were acquired by Kodak 4000 MM (Kodak, Japan). Kd Measurement 6


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The equilibrium dissociation constants (Kd) of biarsenical-peptide complexes were determined by monitoring the increase of fluorescence intensity as a function of peptide concentration (see Supporting Information, Figure S2). Specifically, the titrations were performed in bipartite tetracysteine display buffer, and the apparent Kd values was obtained by fitting the data to equation 2 as previously described 24: F = F +

( )

x (dye + peptide +

!) −

#(dye + peptide +

!)

− 4xdyexpeptide % （2）

where Fmin and Fmax represent the minimum and maximum fluorescence values, respectively. The [dye] is the concentration of FlAsH-EDT2, and the F is the measured fluorescence intensity at any total peptide concentration（i.e., [peptide]）. Cell Culture and Induction of Apoptosis RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in a humidified chamber containing 5% CO2 at 37 °C. After the cells being cultured for 12 hours, 1 µM staurosporine (STS, a nonspecific kinase inhibitor) was added to the culture medium to induce cell apoptosis. The cells treated with equal volume of DMSO were used as the control. At the indicated time points, the culture medium was discarded and the cells were washed with PBS buffer (pH 7.4) for three times. The cells were then lysated in caspase assay buffer by sonication, followed by centrifugation to remove the cell debris. The obtained supernatant was then incubated with Jun-CCC, and the mixtures were subsequently subjected to western blotting assay and fluorescence measurement. For Ac-DEVD-CHO-mediated inhibition assay, the supernatant was pre-incubated with 10 µM Ac-DEVD-CHO on ice for 30 minutes before incubation with Jun-CCC. 7



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RESULTS AND DISCUSSION Principle of the assay Based on the unique specificity of caspase-3 toward tetrapeptide DEVD, a number of DEVD-based

fluorogenic

peptides

(e.g.,

Ac-DEVD-AMC,

Ac-DEVD-AFC,

Ac-DEVD-AMAC, (Z-DEVD)2-R110, Z-DEVD-ProRed 620)28-30 have been developed to detect caspase-3 activity. However, the conjugation of non-natural fluorogenic groups to DEVD may cause some steric hindrance for enzymatic digestion, and in some cases may abolish the cleavage

31

. In addition, the labeling of tetrapeptide by a specific fluorogenic

group implies that the measurement is limited to certain excitation and emission spectra, making the assay less versatile. In contrast, the tetracysteine-biarsenical system possesses small peptide-tag and a set of different biarsenical dyes20,24,32, and may provide a flexible approach to measure caspase-3 activity. In the tetracysteine-biarsenical system, the fluorogenicity of bis-arsenical dye requires the tetracysteine motif forming a special configuration to accommodate the fluorophore. A minor change in the conformation will lead to drastic changes in the stability (by up to 23,000-fold) and quantum yield (by up to 500%) of resulting biarsenical complexes

24

. Since protease-mediated cleavage may cause notable

change in the structure / conformation of substrate proteins, this unique structure-activity relationship of tetracysteine motif toward biarsenical dye may be used as a reporter to sense protease activity. Herein, we designed a modular peptide with three different domains as an activity probe to measure caspase-3-like protease (Scheme 1). The modular peptide contains an N-terminal dimerization domain, an intervening caspase-3 cleavage sequence, and a C-terminal di-Cys motif (Figure 1A). The dimerization domain functions as a scaffold to 8


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bring two Cys-Cys pairs into close proximity upon homodimerization, and the specific recognition sequence for caspase-3/7 is DEVD, and the di-cysteine motif may function as the readout of protease activity. In order to facilitate the binding of biarsenical dyes to di-cysteine motifs, a glycine residue is placed between the di-Cys motif and the DEVD sequence to increase the flexibility of the probe. In the absence of caspase-3, the homodimerization of modular peptide will bring the di-Cys motifs into close proximity and forms a specific configuration for FlAsH binding. The coordination of bis-arsenical dye to bipartite tetracysteine motif may yield a strong fluorescence signal. In contrast, the cleavage of modular peptide by caspase-3 will dissociate the di-Cys motif from the modular peptide and abrogates the bipartite tetracysteine display, leaving the biarsenical dye in dark state. As a result, the caspase-3/7 activity may be simply measured by monitoring the change in fluorescence intensity. To the best of our knowledge, this is the first report that exploits bipartite tetracysteine display as the sensing system to measure protease activity.

Scheme 1. Schematic illustration of homodimerization-directed bipartite tetracysteine display for caspase-3 assay. A modular peptide consisting of a dimerization domain (red), a caspase-3 cleavage sequence (light sea green), and a di-cysteine motif (orange) serves as the sensing probe to measure caspase-3-like proteases. In its intact state, the homodimerization of the peptide brings two Cys-Cys pairs into close proximity with the geometry suitable for bisarsenical dye binding. 9



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The coordination of bisarsenical dye (e.g., FlAsH-EDT2) to dimeric peptide forms a biarsenical-peptide complex with strong fluorescence. In contrast, the cleavage of modular peptide by caspase-3 disrupts the appropriate geometry by dissociating the di-cysteine motif from the modular peptide, resulting in the disappearance of fluorescence. By monitoring the decrease of fluorescence intensity, the caspase-3-like proteases can be quantitatively determined.

Characterization and validation of homodimerization-directed bipartite tetracysteine display The complete amino acid sequence of Jun-CCC is shown in table 1. In this multifunctional peptide, the dimerization domain is derived from the leucine zipper region of c-Jun with additional capping motifs (underlined) for the improvement of helix stability and solubility 33,34

. To avoid unnecessary proteolysis by proteases, the dimerization domain is synthesized

using D-amino acids. In addition, the dimerization domain does not include cysteine residue so that the dye can bind di-Cys motifs specifically. To verify whether the modular peptide is eligible for bipartite tetracysteine display, we monitored the emission fluorescence of FlAsH-EDT2 after mixing with Jun-CCC. As shown in Figure 1B, a distinct fluorescence signal is obtained for the mixture. In contrast, only an extremely weak fluorescence is observed for an analogue of Jun-CCC (i.e., JunP-CCC), in which all the leucine residues are mutated to prolines to disable its dimerization capability

24

. Similarly, the removal of the

dimerization domain from the modular peptide (i.e., CCC) results in only negligible background signal (Figure 1B). These results suggest that the proposed assay relies on the successful homodimerization of the probe. Notably, the dimerized peptide binds to the dye 10


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rapidly and reaches a plateau in less than 10 minutes, whereas the peptides lacking the ability to form dimers generate only low background signal even after a long incubation time (Figure 1C). The homodimeric peptide may form a stable complex with biarsenical dye, and the apparent dissociation constant (Kd) for Jun-CCC is comparable to that of the optimized linear tetracysteine sequence (Table 1). The above data clearly demonstrate that the proposed bipartite tetracysteine display is homodimerization-dependent and highly sensitive to the precise arrangement of two Cys-Cys pairs.

Figure 1. Characterization of homodimerization-directed bipartite tetracysteine display. (A)

Domain organization of Jun-CCC. The Jun-CCC contains three functional domains: a dimerization domain (red), a caspase-3 recognition / cleavage sequence (light sea green), and a di-cysteine motif (orange); (B) Fluorescence spectra of FlAsH–EDT2 upon binding with Jun-CCC, JunP-CCC, and CCC, respectively. The peptides of FLNCCPGCCMEP and FLNCCPPPPPPPPPC CMEP were used as the positive control and the negative control, respectively. (C) 11



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Time-dependent fluorescence of FIAsH-EDT2 upon binding to Jun-CCC (red trace), JunP-CCC (green trace), and CCC (blue trace), respectively. The fluorescence was measured in a time-scan mode (λex = 510 nm, λem = 532 nm).

Measurement of caspase-3 activity in vitro using homodimerization-directed bipartite tetracysteine display We further used Jun-CCC as an active probe to measure caspase-3. As shown in Figure S1 and Figure 2A, caspase-3-mediated cleavage results in the generation of expected cleavage product (see Supporting Information, Figure S1) and substantial reduction of fluorescence intensity (Figure 2A), whereas the inactivation of caspase-3 by inhibitor Ac-DEVD-CHO leads to significant fluorescence recovery

14

. The caspase-3-mediated proteolysis induces

30-fold decrease in fluorescence intensity at 532 nm (Figure 2B). It should be noted that the caspase-3 itself yields negligible background fluorescence (Figure 2A) and will not interfere with the fluorescence measurement. Moreover, this assay is highly specific to caspse-3. The treatment of Jun-CCC with active caspase-3 generates near-zero signal, in contrast to almost no change in the signal after treatment with three control proteases including SENP1, thrombin and trypsin (Figure 2C). Same results are obtained by quantitive fluorescence measurement (see Supporting Information, Figure S3), further confirming the specificity of the proposed assay. We further investigated the sensitivity of the proposed method by measuring the change in fluorescence intensity (∆F) in response to different concentration of caspase-3. As shown in Figure 2D, the value of ∆F increases gradually with increasing concentration of caspase-3. A linear correlation is obtained between ∆F and the caspase-3 concentration in the range from 1.87 × 10-4 to 1.17 × 10-2 µg/mL (inset of Figure 2D). The 12


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correlation equation is ∆F = 22.65 + 27452.86 C with a correlation coefficient of 0.999, where ∆F is the change of fluorescence intensity at 532 nm and C is the concentration of caspase-3 (µg/mL). The detection limit of the proposed method is determined to be 1.28 × 10-4 µg/mL based on the 3σ method. It should be noted that the sensitivity has improved by more than 156-fold, 56.6-fold, 39-fold, and 7.8-fold as compared with that of gold nanoparticle/quantum dot-based energy transfer assay (20 ng/mL)35, the graphene oxide/peptide-based

fluorescent

assay

(7.25

ng/mL)36,

the

unmodified

gold

nanoparticle-mediated colorimetric assay (0.005 µg/mL)18, and (Z-DEVD)2-R110-based fluorometric assay (1 ng/mL)29, respectively. The improved sensitivity might be attributed to following two factors: (i) the quantum yield of resultant bisarsenical-peptide complex is highly sensitive to the local conformation of tetra-cysteine motif 24. In the proposed assay, the precise arrangement of split tetracysteine motifs can be

readily disrupted by

caspase-3-mediated proteolysis; (ii) the probe Jun-CCC is made of amino acids without any modification, and this natural substrate-like characteristic may facilitate the efficient cleavage by caspase-3.

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Figure 2. Detection of caspase-3 activity in vitro using homodimerization-directed bipartite

tetracysteine display. (A) Fluorescence emission spectra of FlAsH–EDT2 upon binding to Jun-CCC (red), Jun-CCC cleaved by active caspase-3 (blue), and Jun-CCC cleaved by Ac-DEVD-CHO-inhibited caspase-3 (dark cyan). The caspase-3 protein itself does not bind to FlAsH-EDT2 (magenta), yielding only background fluorescence (λex = 510 nm). (B) Quantification of fluorescence intensity in A. (C) Fluorescence images of the probe in response to different proteases. A mixture without any protease was used as the control. (D) Variance of the fluorescence reduction at 532 nm (∆F) versus the concentration of caspase-3. The inset shows the linear relationship between the fluorescence reduction (∆F) and the caspase-3 concentration (C) in the range from 1.87 × 10-4 to 1.17 × 10-2 µg/mL. Error bars show the standard deviation of three independent experiments.

Detection of intracellular caspase-3 activity in RAW 264.7 cells The Jun-CCC probe can measure not only the recombinant caspase-3 in vitro (Figure 2) but 14


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also the endogenous caspase-3 in complex cell extract (Figure 3). We used RAW 264.7 cell, a macrophage-like cell line that derived from BALB/c mice, as the model. The cells were stimulated by staurosporine (STS, a nonspecific kinase inhibitor) to induce apoptosis

37

.

Meanwhile, the cells without any treatment were used as the control. As shown in Figure 3A, the STS-treated RAW 264.7 cells show strong signal of cleaved caspase-3, indicating the successful activation of caspase-3 within the cells. Consistent with the result of immunoblotting assay, the STS stimulation induces the decrease of fluorescence intensity in STS-treated cells (Figure 3B). Moreover, the STS-induced activation of caspase-3 is time-dependent, as indicated by the gradual increment of cleaved caspase-3 (Figure 3A) and the decrease of fluorescence intensity over time (Figure 3B). In contrast, neither remarkable increment of cleaved caspase-3 (Figure 3A) nor obvious change in fluorescence intensity (Figure 3B) is observed in control cells. Since the cell extract is a complex biological mixture with a large number of proteins and proteases, we need to make sure that the fluorescence reduction is derived from specific cleavage of the probe by caspase-3 in apoptotic cell lysates. We added 10 µM Ac-DEVD-CHO to the cell suspension before incubating with Jun-CCC. As shown in Figure 3D, the treatment of cells with STS induces significant decrease in fluorescence intensity, while the addition of Ac-DEVD-CHO causes the recovery of fluorescence intensity. Notably, the addition of Ac-DEVD-CHO does not alter the amount of caspase-3 protein (Figure 3C), and thus the fluorescence recovery is ascribed to the inhibition of caspase-3 activity. All together, these results demonstrate that the proposed method may specifically measure intracellular caspase-3 activity in complex biological samples.

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Figure 3. Measurement of the activation and inhibition of caspase-3 in RAW 264.7 cells. (A)

Western blotting analysis of cleaved caspase-3 in RAW 264.7 cells after the cells were stimulated with staurosporine (STS) for 1 h, 2 h, 3 h, and 4 h, respectively. The cells without any treatment were used as the control. The tubulin was used as the loading control. (B) Variance of the fluorescence intensity with time. (C) Western blotting analysis of the inhibition of caspase-3 by Ac-DEVD-CHO. The cell lysate was pre-incubated with 10 µM Ac-DEVD-CHO for 30 minutes before incubating with probe Jun-CCC. (D) Fluorescence measurement of the inhibition of caspase-3 by Ac-DEVD-CHO. Error bars represent the standard deviation of three independent experiments.

Conclusion In summary, we have developed a simple and label-free method for sensitive detection of caspase-3 like proteases. This assay relies on the homodimerization-mediated spatial 16


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proximity of two Cys-Cys pairs and the subsequent abrogation of bipartite tetracysteine display by protease cleavage. Due to the natural substrate-like characteristic of the probe and the high sensitivity of biarsenical dye toward local structure of tetra-cysteine motif, excellent performance is achieved for caspase-3 assay with a detection limit of as low as 1.28 × 10-4 µg/mL. Notably, the proposed strategy has several distinctive advantages: (I) the sensing probe does not require any labeling; (II) the assay can be carried out in a “mix-and-read” manner without washing and separation steps, and thus is quite simple and convenient; (III) this strategy can be readily applied to other protease by adopting an appropriate recognition sequence in the probe (see Supporting Information, Figure S4); (IV) the probe can be stained by a set of biarsenical dyes with different excitation and emission spectra, making the detection system flexible; (V) this assay can be further extended to measure proteases activity in live cells by using genetically expressed probe. Therefore, the proposed method might find wide applications in the screening of protease-targeted drugs and the diagnosis of protease-associated diseases.

ASSOCIATED CONTENT Supporting Information MALDI-TOF MS analyses for the validation of the cleavage by protease, the fluorescent measurement of the cleavage by different proteases, and the domain organization of Jun-TCC are shown in supplementary material. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION 17



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Corresponding Author * E-mail: [email protected]

Phone: +86 0531-86186033.

Fax: +86 0531- 8261

5258. Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 21527811, 21675168 and 41373141), the Natural Science Foundation of Shenzhen City (Grant Nos. JCYJ20120615124830232 and JCYJ20140509174140691), the Guangdong Science and Technology Department (2013B030800001), and the Award for the Youth Innovation Promotion Association of the Chinese Academy of Sciences.

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REFERENCES (1) Sanman, L. E.; Bogyo, M. Annu. Rev. Biochem. 2014, 83, 249-273. (2) Turk, B.; Turk, D.; Turk, V. EMBO J. 2012, 31, 1630-1643. (3) Li, J.; Yuan, J. Oncogene 2008, 27, 6194-6206. (4) Crawford, E. D.; Wells, J. A. Annu. Rev. Biochem. 2011, 80, 1055-1087. (5) Creagh, E. M. Trends Immunol. 2014, 35, 631-640. (6) Friedlander, R. M. N. Engl. J. Med. 2003, 348, 1365-1375. (7) Favaloro, B.; Allocati, N.; Graziano, V.; Di Ilio, C.; De Laurenzi, V. Aging (Albany NY) 2012, 4, 330-349. (8) McIlwain, D. R.; Berger, T.; Mak, T. W. Cold Spring Harb. Perspect. Biol. 2013, 5, a008656. (9) Wang, X. J.; Cao, Q.; Zhang, Y.; Su, X. D. Annu. Rev. Pharmacol. Toxicol. 2015, 55, 553-572. (10) Huang, R.; Wang, X.; Wang, D.; Liu, F.; Mei, B.; Tang, A.; Jiang, J.; Liang, G. Anal. Chem. 2013, 85, 6203-6207. (11) Takano, S.; Shiomoto, S.; Inoue, K. Y.; Ino, K.; Shiku, H.; Matsue, T. Anal. Chem. 2014, 86, 4723-4728. (12) Vuojola, J.; Syrjanpaa, M.; Lamminmaki, U.; Soukka, T. Anal. Chem. 2013, 85, 1367-1373. (13) Li, S. Y.; Liu, L. H.; Cheng, H.; Li, B.; Qiu, W. X.; Zhang, X. Z. Chem. Commun. (Camb) 2015, 51, 14520-14523. (14) Dong, Y. P.; Chen, G.; Zhou, Y.; Zhu, J. J. Anal. Chem. 2016, 88, 1922-1929. (15) Poreba, M.; Szalek, A.; Kasperkiewicz, P.; Rut, W.; Salvesen, G. S.; Drag, M. Chem. Rev. 2015, 115, 12546-12629. (16) Prasuhn, D. E.; Feltz, A.; Blanco-Canosa, J. B.; Susumu, K.; Stewart, M. H.; Mei, B. C.; Yakovlev, A. V.; Loukov, C.; Mallet, J. M.; Oheim, M.; Dawson, P. E.; Medintz, I. L. ACS Nano 2010, 4, 5487-5497. (17) Li, J.; Li, X.; Shi, X.; He, X.; Wei, W.; Ma, N.; Chen, H. ACS Appl. Mater. Interfaces 2013, 5, 9798-9802. 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(18) Pan, Y.; Guo, M.; Nie, Z.; Huang, Y.; Peng, Y.; Liu, A.; Qing, M.; Yao, S. Chem. Commun. (Camb) 2012, 48, 997-999. (19) Zhang, J.; Wang, X.; Cui, W.; Wang, W.; Zhang, H.; Liu, L.; Zhang, Z.; Li, Z.; Ying, G.; Zhang, N.; Li, B. Nat. Commun. 2013, 4, 2157. (20) Hoffmann, C.; Gaietta, G.; Zurn, A.; Adams, S. R.; Terrillon, S.; Ellisman, M. H.; Tsien, R. Y.; Lohse, M. J. Nat. Protoc. 2010, 5, 1666-1677. (21) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272. (22) Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002, 124, 6063-6076. (23) Martin, B. R.; Giepmans, B. N.; Adams, S. R.; Tsien, R. Y. Nat. Biotechnol. 2005, 23, 1308-1314. (24) Luedtke, N. W.; Dexter, R. J.; Fried, D. B.; Schepartz, A. Nat. Chem. Biol. 2007, 3, 779-784. (25) Scheck, R. A.; Schepartz, A. Acc. Chem. Res. 2011, 44, 654-665. (26) Krishnan, B.; Gierasch, L. M. Chem. Biol. 2008, 15, 1104-1115. (27) Cornell, T. A.; Fu, J.; Newland, S. H.; Orner, B. P. J. Am. Chem. Soc. 2013, 135, 16618-16624. (28) Gurtu, V.; Kain, S. R.; Zhang, G. Anal. Biochem. 1997, 251, 98-102. (29) Liu, J.; Bhalgat, M.; Zhang, C.; Diwu, Z.; Hoyland, B.; Klaubert, D. H. Bioorg. Med. Chem. Lett. 1999, 9, 3231-3236. (30) Lozanov, V.; Ivanov, I. P.; Benkova, B.; Mitev, V. Amino Acids 2009, 36, 581-586. (31) Yu, K.; Kennedy, C. A.; O'Neill, M. M.; Barton, R. W.; Tatake, R. J. Apoptosis 2001, 6, 151-160. (32) Spagnuolo, C. C.; Vermeij, R. J.; Jares-Erijman, E. A. J. Am. Chem. Soc. 2006, 128, 12040-12041. (33) Hagemann, U. B.; Mason, J. M.; Muller, K. M.; Arndt, K. M. J. Mol. Biol. 2008, 381, 73-88. (34) Crooks, R. O.; Rao, T.; Mason, J. M. J. Biol. Chem. 2011, 286, 29470-29479. (35) Kim, Y. P.; Oh, Y. H.; Oh, E.; Ko, S.; Han, M. K.; Kim, H. S. Anal. Chem. 2008, 80, 4634-4641. 20


Page 20 of 22

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(36) Wang, H.; Zhang, Q.; Chu, X.; Chen, T.; Ge, J.; Yu, R. Angew. Chem. Int. Ed. Engl. 2011, 50, 7065-7069. (37) Nicholls, S. B.; Chu, J.; Abbruzzese, G.; Tremblay, K. D.; Hardy, J. A. J. Biol. Chem. 2011, 286, 24977-24986.

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Label-Free and Homogenous Detection of Caspase-3-Like Proteases

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