Chen, Ning

Huang, Yun

Liu, Rihe

Yang, Jennyâ  J.

Yang, Lily

PubMed

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Designing Caspase-3 Sensors for Imaging of Apoptosis in LivingCellsNing Chen[a], Yun Huang[a], Lily Yang[b], Rihe Liu[c], and Jenny J. Yang[a]Jenny J. Yang: chejjy@langate.gsu.edu[a]Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University,Atlanta, GA 30303 (USA), Fax: (+1)404-413-5551[b]Department of Surgery and Winship Cancer Institute, School of Medicine, Emory University,Atlanta, GA 30322 (USA)[c]School of Pharmacy, The University of North Carolina at Chapel Hill, NC 27599 (USA)Keywordsapoptosis; caspase-3; enhanced green fluorescent proteins; protease sensors; ratiometricmeasurementsApoptosis is an essential physiological process of tissue development and homeostasis andis associated with pathogenesis of human diseases, such as neurodegenerative diseases andcancers.[1,2] The caspase–cascade system plays a vital role in the induction, signaltransduction, and amplification of intracellular apoptotic signals.[3-5] One of the challengesin monitoring apoptosis pathways includes the serial involvement of multiple caspases,complicated activation or inhibition mechanisms, and the heterogeneous cell population at agiven time. Therefore, the mechanism of caspase activation during apoptosis is not fullyunderstood and investigations have been hampered by a lack of real-time sensors with highenzymatic specificity to quantitatively detect caspase activity in live cells or in vivo.[6,7]Currently, the majority of methods for detecting caspase activity include Western blot todetect the cleaved products, or synthetic substrates conjugated to a chromogenic dye tomonitor caspase activity in cell lysates during apoptosis.[6-8] Some newly developed peptideprobes with cell permeability allow one time snap-shots of caspase activity.[9] A DNA dye,DEVD-NucView 488 substrate with a DEVD cleavage linker, allows an indirect report ofthe activation of caspase-3 after diffusion to nuclei in living cells.[10] To achieve real-timeratiometric signal changes during caspase activation, progress has been made to developFRET-based (FRET, fluorescence resonance energy transfer) probes by connecting twofluorescent protein (FP) pairs with a caspase cleavage linker.[11,12]Here, we describe a sensitive and specific caspase-3 sensor, based on a single enhancedgreen fluorescent protein (EGFP) to avoid the cross-talk from the overlap of cyanfluorescent protein (CFP) excitation or yellow fluorescent protein (YFP) emission spectra asobserved in most FRET probes.[11] A ratiometric protease sensor was designed by grafting acaspase-3-specific cleavage linker at a sensitive loop location (Glu172) relative to thechromophore, taking advantage of the EGFP’s high resistance to proteases (Figure 1a).[13]© 2009 Wiley-VCH Verlag GmbH&Co. KGaA, WeinheimCorrespondence to: Jenny J. Yang, chejjy@langate.gsu.edu.Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901439.NIH Public AccessAuthor ManuscriptChemistry. Author manuscript; available in PMC 2012 October 24.Published in final edited form as:Chemistry. 2009 September 21; 15(37): 9311–9314. doi:10.1002/chem.200901439.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPreviously we have shown that calcium and trypsin sensors can be created by grafting acalcium-binding loop or trypsin cleavage site at Glu172 of EGFP, respectively.[14,15]Addition of a protease-specific cleavage linker at Glu172 loop region of EGFP results information of dual chromophore states with absorption at both 397 and 490 nm.[16] Such anequilibrium between the protonated and deprotonated chromophore forms was proposed tooriginate from a complicated hydrogen-bond network allowing for proton transfer betweenthe chromophore and its neighboring side chains. The predominant protonated form of thechromophore with its maximum absorbance at 397 nm is governed by the carboxylation ofGlu222 through electrostatic repulsion and hydrogen bonding between a bound watermolecule and Ser205. On the other hand, the maximum absorbance at 490 nm of thedeprotonated form of the chromophore is likely to be due to the proton relay betweenGlu222 and Ser205. The side chains of Asp117, Thr118, Glu172, and Asp190 may also beinvolved in the alteration of different conformations of the chromophore under theobservation of electron density of crystal structure. Our observation of the switch of dualchromophore forms upon the insertion of a cleavage linker at position 172 is probably aresult of the alteration of the ionization state of the chromophore.[14,15]Three EGFP-based caspase-3 sensor variants were genetically generated (Figure 1b). Toobtain optimal signal change and kinetic properties, two helical sequences were used toextend the caspase-3 cleavage linker and to improve enzymatic accessibility in solutionbased on our previous knowledge gained in developing calcium and trypsin sensors.[14,15]The insertion of sequence with helical properties results in a significantly greater signalchange, possibly due to its greater solvent accessibility for calcium binding and trypsincleavage. The comparison between dual cleavage sequence (DEVD) and a single sequencein caspase-3 sensors was also conducted. No caspase-3 cleavage was observed with additionof only a single DEVD or DEVD without using a helical sequence. Thus, two cleavagesequences (DEVD) grafted onto EGFP were used to obtain a sensor, designated EGFP-C3A,for achieving rapid cleavage. Moreover, to achieve enzymatic specificity, a recognitionsequence (VDEVDG) with preferable residues from P5 to P1′ was inserted into EGFP tocreate another sensor denoted as EGFP-C3B.[17] Finally, a negative control, EGFP-C3N,was created by grafting a loop lacking a caspase-3 cleavage site.Previous investigations showed that protease sensors designed by EGFP through the graftingof a cleavable linker for specific proteases can be cleaved at the designed location withoutthe release of other small fragments under physiological conditions; these cleavage siteswere identified by using protein sequence analysis, MALDI-MS and SDS-PAGE. Moreover,the cleaved fragments exhibited strong fluorescence complementation in living cells.[18]Similarly, both purified EGFP-C3A and EGFP-C3B were specifically cleaved by caspase-3into two major fragments in caspase-3 reaction buffer (20 mM HEPES, 100 mM NaCl, 1 mMEDTA, 10 mM DTT, 10% sucrose, pH 7.5), as revealed by SDS-PAGE (Figure 1c). Massspectrometry analysis reveals that the molecular mass of the cleavage fragments isconsistent with cleavage at the desired caspase-3 cleavage location (Figure 1d). In contrast,wild type EGFP (EGFP-wt) and EGFP-C3N did not exhibit similar cleavage upon overnightincubation with caspase-3. Moreover, our caspase-3 sensors can not be cleaved bycaspase-6, caspase-7, caspase-8, and caspase-9 (Figure 1c). Such strong enzymaticspecificity is very important for its further application in monitoring caspase-3 action in livesystems and understanding the mechanisms that control the apoptosis cascade.Cancer cell lines, HeLa or MIA PaCa-2 cells transfected with EGFP-C3A, EGFP-wt orEGFP-C3N exhibit strong fluorescence at 510 nm when excited at either 398 or 488 nmfollowing 18 to 48 h of expression (Figure 2a,b). Upon caspase-3 activation by 1 μMstaurosporine (STS), the fluorescence emission signal of EGFP-C3A at 510 nm exhibited asignificant increase with excitation of 398 nm and a concurrent decrease with excitation ofChen et al. Page 2Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript488 nm (Figure 2a–c). Such ratiometric fluorescence change (F398nm/F488nm) has a dynamicrange up to 3.2 in HeLa cells and 7.5 in MIA PaCa-2 cells (Figure 2d), which is consistentwith observations that lower caspase-3 activation was detected in cell lysates of HeLa cellsthan in MIA PaCa-2 cells using a fluorogenic caspase-3 substrate kit, Ac-DEVD-AFC(Supporting Information Figure S1a) or Western blot through caspase-3 antibody (Figure2e). Moreover, this result is also in agreement with qualitative detection of caspase-3activity by immunofluorescence methods[19] and the detection with DEVD-NucView 488caspase-3 substrate (Supporting Information Figure S1b). In contrast, at 510 nm, nosignificant fluorescence signal change of EGFP-wt or EGFP-C3N, which lacks cleavagesites, was observed with excitation of 398 and 488 nm (Figure 2d). Similar results can alsobe obtained by using fluorescence spectroscopy to monitor emission spectra changes ofliving mammalian cells expressing the caspase-3 sensor gene in the coverslip following STSinduction (Supporting Information Figure S2). The cells transfected with EGFP-C3Afollowing incubation with the caspase-3 inhibitor, Ac-DEVD-CHO, exhibited no ratiometricsignal change.Our developed sensors exhibit a capability of monitoring the early stage of an apoptoticevent. MIA PaCa-2 cells exhibit a half time of 16.8±3.5 min for caspase-3 activation, whichis shorter than HeLa cells (30.2±5.4 min) as detected by EGFP-C3A (Figure 2d). Inaddition, the half time of caspase-3 activation is 16.3±5.9 min as detected by EGFP-C3B,which is shorter than 48.5±11.2 min with commercially available DEVD-NucView 488caspase-3 substrate detection under identical inducer concentration in HeLa cells (Figure 2f). This is due to the direct monitoring of caspase-3 action in the cytosol by our sensor,rather than detection of caspase-3 after its migration from cytosol to nuclei to accomplishDNA dye cleavage and cooperative DNA binding in an on/off mode. In addition, caspase-3activation was observed following the migration from the cytoplasm to the nucleus afterSTS induction based on the fluorescence signal change (Supporting Information: movie S1),which is in good agreement with previous reports of caspase-3 activation pattern in livingcells.[5,20]Table 1 summarizes kinetic parameters, turnover number (kcat), Michaelis constants (KM),and substrate specificity constants (kcat/KM) of our caspase-3 sensors and commerciallyavailable caspase-3 substrates determined in caspase-3 reaction buffer. EGFP-C3B exhibiteda smaller KM and a higher kcat/KM than commercial caspase-3 substrates, Bz-DEVD-pNAand Z-DEVD-AMC from previous reports.[21] Overall, EGFP-C3B with optimal cleavageresidues from P5 to P1′ exhibited a larger dynamic range as well as better kinetic propertiesas indicated by the kcat/KM value. As shown in Table 1, the kcat/KM value of EGFP-C3B(59600 ± 9300M−1 s−1) is fivefold larger than that of EGFP-C3A (12500 ± 150M−1 s−1).In summary, we have developed ratiometric caspase-3 sensors by grafting cleavage linkersat a location sensitive to chromophore optical signal ratiometric change of a single EGFP.The designed caspase-3 sensor can be used to detect caspase-3 activation or inhibition inliving cells with a large dynamic range and high enzymatic specificity due to high potentialaccessibility for caspase-3 at a lower basal caspase-3 activity or caspase-3 activation inliving cells at the early stage of apoptosis, which can eliminate interferences frombackground, expression level, and problems of orientation change or translocation. Inaddition, this strategy can also be specifically targeted to desired cellular compartments withthe aid of specific signal peptides for real-time monitoring of caspase-3 activation orinhibition, which will be valuable for investigations of physiological mechanisms orpathological processes of diseases related to caspase-3 activity at early stage of apoptosis inliving cells or in vivo. Because the newly developed sensors are ratiometric and not FRET-based, they are applicable over a slightly wider range to avoid cross-talk from the overlap ofCFP excitation or YFP emission spectra, which occurs in most FRET probes. Furthermore,Chen et al. Page 3Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptthis novel strategy for the design of caspase-3 sensors can also be extended to thedevelopment of other specific caspase sensors such as caspase-8, caspase-6 and caspase-9sensors, facilitating drug development and protease inhibitor screening.Experimental SectionThe detailed information related to design, expression, purification, kinetic studies ofcaspase sensors, real-time detection of caspase-3 activation using designed caspase-3 sensorin living cells and confirmation for caspase-3 in cells is given in the Supporting Information.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe would like to thank Dan Adams and Michael Kirberger for their critical review of this manuscript, Jin Zou,Shenghui Xue, Yiming Ye, and Shen Tang for helpful discussions; and other members of Dr. Yang’s researchgroup for their helpful discussions and suggestions. This work is supported in part by the following sponsors:GM-70555 to J.J.Y.; GSU Molecular Basis of Disease Predoctoral Fellowships to N.C.References1. Bredesen DE. Curr Mol Med. 2008; 8:173–186. [PubMed: 18473818]2. Jiang X, Wang X. Annu Rev Biochem. 2004; 73:87–106. [PubMed: 15189137]3. Degterev A, Boyce M, Yuan J. Oncogene. 2003; 22:8543–8567. [PubMed: 14634618]4. Salvesen GS, Riedl SJ. Adv Exp Med Biol. 2008; 615:13–23. [PubMed: 18437889]5. Sundararajan R, Chen G, Mukherjee C, White E. Oncogene. 2005; 24:4908–4920. [PubMed:15824730]6. Gurtu V, Kain SR, Zhang G. Anal Biochem. 1997; 251:98–102. [PubMed: 9300088]7. Kohler C, Orrenius S, Zhivotovsky B. J Immunol Methods. 2002; 265:97–110. [PubMed:12072181]8. Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM,Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT, Nicholson DW. J Biol Chem. 1997;272:17907–17911. [PubMed: 9218414]9. Wang ZQ, Liao J, Diwu Z. Bioorg Med Chem Lett. 2005; 15:2335–2338. 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J Biotechnol. 2009;142:205–213. [PubMed: 19500621]19. Yang L, Cao Z, Yan H, Wood WC. Cancer Res. 2003; 63:6815–6824. [PubMed: 14583479]Chen et al. Page 4Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript20. Ramuz O, Isnardon D, Devilard E, Charafe-Jauffret E, Hassoun J, Birg F, Xerri L. Int J ExpPathol. 2003; 84:75–81. [PubMed: 12801281]21. Fang B, Boross PI, Tozser J, Weber IT. J Mol Biol. 2006; 360:654–666. [PubMed: 16781734]Chen et al. Page 5Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Design of EGFP-based caspase-3 sensors. a) The model and b) cleavage linkers ofratiometric EGFP-based caspase-3 sensors via grafting approach are demonstrated. c) Thespecificity of caspase-3 sensors was detected in SDS-PAGE. d) The cleavage sites wereidentified by MALDI-MS. Lanes M, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 are: proteinmarker, EGFP-C3A, EGFP-C3A with caspase-3 digestion, EGFP-C3A with caspase-8digestion, EGFP-C3B, EGFP-C3B with caspase-3 digestion, EGFP-C3B with caspase-8digestion, EGFP-wt, EGFP-wt with caspase-3 digestion, EGFP-C3A with caspase-6digestion, EGFP-C3A with caspase-9 digestion, EGFP-C3A with caspase-7 digestion,EGFP-C3B with caspase-6 digestion, EGFP-C3B with caspase-9 digestion and EGFP-C3Bwith caspase-7 digestion, respectively. The molecular masses of intact EGFP-C3A, cleavedmajor large fragment and major small fragment are 34440.02, 25607.73 and 8226.51 Da,respectively.Chen et al. Page 6Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.Live-cell imaging and Western blot analysis of caspase-3 activity. HeLa cells transientlytransfected with EGFP-C3A were examined by fluorescence microscope with excitations ofa) 398 and b) 488 nm. A significant fluorescence increase was observed between 0-32 minpost-induction of 1 μM STS at 398 nm, with a concurrent fluorescence decrease from 0-32min post-induction of 1 μM STS at 488 nm. c) The relative fluorescence changes atexcitations of 398 (●) and 488 nm (■) were shown as a ratiometric mode upon addition of 1μM STS. d) Fluorescence ratio change among EGFP-wt (○), EGFP-C3A with Ac-DEVD-CHO inhibition (◇), EGFP-C3N without cleavage sites (Δ), EGFP-C3A in HeLa cells (●)and in MIA PaCa-2 cells (■) following STS induction reveals a significantly shorter halftime for caspase-3 activation in MIA PaCa-2 cells. e) Caspase-3 activation in HeLa andMIA PaCa-2 cells exhibited a faster caspase-3 activation and a larger proportion of activatedcaspase-3 in MIA PaCa-2 cells following 1 μM STS induction by means of a Western blotanalysis. f) EGFP-C3B with an optimal cleavage linker was observed to have a shorter halftime (16.3±5.9 min) than DEVD-NucView 488 caspase-3 substrate (48.5±11.2 min) inHeLa cells following 1 μM STS stimulation.Chen et al. Page 7Chemistry. Author manuscript; available in PMC 2012 October 24.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptChen et al. Page 8Table 1Catalytic kinetic parameters of caspase-3 sensors.Sensor kcat [s−1] KM [μM] kcat/KM [M−1 s−1]EGFP-C3A 0.13±0.01 10.03±1.19 12500±150EGFP-C3B 0.39±0.03 6.55±1.53 59600±9300Bz-DEVD-pNA 0.73±0.02 26.91±1.20 2730±120Chemistry. Author manuscript; available in PMC 2012 October 24.

Designing Caspase-3 Sensors for Imaging of Apoptosis in Living Cells

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