- Email: [email protected]
Determination of Caspase Specificities Using a Peptide Combinatorial Library
100
APOPTOSIS PROTEASES AND THEIR INHIBITORS
[9]
Procedure The assays are carried out in a total volume of 100/zl for detection in a 96-well plate reader, preferably operating in the kinetic mode. For analysis requiring larger volumes, increase proportionally. 1. Prepare working solutions of inhibitor titrant at 1-10/zM in caspase buffer, as required, from the 10 mM stock. The inhibitor is not fully soluble in aqueous solvents above 1 mM, and should be diluted in DMSO when working above 100/zM, before final dilution into caspase buffer. 2. Activate the enzyme to be measured in the usual way in assay buffer; 15 min at 37 ° is usually sufficient. 3. Add 20/zl of each working solution of inhibitor titrant to 80/.d of caspase solution (about 1.0/zM provisional concentration based on protein content) and incubate for 30 min at 37 °. 4. Assay samples from each reaction mixture, using an appropriate substrate in assay buffer to determine the residual activity. Plot residual activity as a function of inhibitor concentration. As with all enzyme assays, the rate of substrate hydrolysis must be in the linear range. If the reaction is too fast, and substrate is exhausted too rapidly, dilute the caspase/ZVAD-FMK reaction mixture to obtain a linear rate. 5. The plot should be linear with an intercept on the x axis equal to the concentration of active enzyme (Fig. 3). The curvature of the plot as it approaches this axis indicates that the reaction has not gone to completion. This can be overcome by longer incubation or by increasing the concentrations of enzyme and inhibitor. The concentration of the enzyme can only rarely be accurately determined by extrapolating a straight line drawn from partial inhibition.
[9] D e t e r m i n a t i o n o f C a s p a s e S p e c i f i c i t i e s U s i n g a Peptide Combinatorial Library
By NANCY A. THORNBERRY,KEVIN T. CHAPMAN, and D O N A L D W . N I C H O L S O N Introduction The caspase family of cysteine proteases is composed of at least 14 mammalian family members. Phylogenetically they can be divided into two distinct subgroups having markedly different functions in vivo. Members of the interleukin 1/3-converting enzyme (ICE) subfamily [ICE/caspase 1
METHODS IN ENZYMOLOGY, VOL. 322
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
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was originally identified as the protease responsible for the proteolytic maturation of proinflammatory interleukin 1/3 (prolL-1/3)] cleave and activate multiple cytokines (e.g., prolL-1/3, prolL-18) and thus participate in mounting an inflammatory response. Members of the CED-3 subfamily, on the other hand, mediate a cascade of proteolytic cleavage events that culminate in cell death and the apoptotic phenotype (CED-3 was originally identified as a protease in Caenorhabditis elegans that is necessary for developmental cell deaths). The central role that these proteases play in both of these important physiological processes has been resolved in part by inhibitor studies, knockout animals, substrate identification, and extensive in vitro studies with purified or recombinant caspase family members. 1'2 In this chapter we describe a strategy for defining the precise substrate specificity of individual caspases and discuss how this information can contribute to our understanding of the function that each enzyme serves in its particular biochemical pathway. Two of the most biologically distinct caspases, caspase 1 and caspase 3, have been purified from natural sources, cloned, and their structures determined by X-ray crystallography. 3-7 The results of these studies, together with a comparison of the sequences of all caspases, indicate that these enzymes are synthesized as zymogens that contain three distinct domains: an N-terminal polypeptide (3 to 25 kDa), a large subunit ( - 2 0 kDa), and a small subunit ( - 1 0 kDa). Activation involves proteolytic pro1 D. W. Nicholson and N. A. Thornberry, Trends Biochem. Sci. 22, 299 (1997). 2 N. A. Thornberry and Y. Lazebnik, Science 281, 1312 (1998). 3 j. Rotonda, D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry, and J. W. Becker, Nature Struct. Biol. 3, 619 (1996). 4 D. W. Nicholson, A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, Y. A. Lazebnik, N. A. Munday, S. M. Raju, M. E. Smulson, T.-T. Yamin, Y. L. Yu, and D. K. Miller, Nature (London) 376, 37 (1995). 5 N. A. Thornberry, H. G. Bull, J. R. Calaycay, K. T. Chapman, A. D. Howard, M. J. Kostura, D. K. Miller, S. M. Molineaux, J. R. Weidner, J. Aunins, K. O. Elliston, J. M. Ayala, F. J. Casano, J. Chin, G. J.-F. Ding, L. A. Egger, E. P. Gaffney, G. Limjuco, O. C. Palyha, S. M. Raju, A. M. Rolando, J. P. Salley, T.-T. Yamin, T. D. Lee, J. E. Shively, M. MacCross, R. A. Mumford, J. A. Schmidt, and M. J. Tocci, Nature (London) 356, 768 (1992). 6 N. P. Walker, R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett, L. D. Hammill, L. Herzog, M. Hugunin, W. Houy, J. A. Mankovich, L. McGuiness, E. Orlewicz, M. Paskind, C. A. Pratt, P. Reis, A. Summani, M. Terranova, J. P. Welch, L. Xiong, A. Moller, D. E. Tracey, R. Kamen, and W. W. Wong, Cell 78, 343 (1994). 7 K. P. Wilson, J. A. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A. Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J. Livingston, Nature (London) 370, 270 (1994).
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APOPTOSlSPROTEASESANDTHEIRINHIBITORS
[9]
cessing between domains, removal of the N-terminal polypeptide, and association of the large and small subunits to form a heterodimer. The active site, which is formed by amino acids from both subunits, contains a catalytic diad composed of cysteine and histidine, suggesting that these enzymes employ a typical cysteine protease mechanism. Studies of caspase specificity by traditional approaches indicated that two highly conserved properties of these enzymes dictate hydrolysis of substrates. First, they have a near-absolute requirement for aspartic acid in the P1 position, and second, they require at least four amino acids N terminal to the cleavage site. These observations, together with the finding that substrates with the general structure Ac-XXXD-aminomethylcoumarin (AMC) are efficiently hydrolyzed by caspases, led to the design of the approach described below for the systematic determination of caspase specificity (see Fig. 1). Positional Scanning Synthetic Combinatorial Library Positional scanning peptide libraries (PS-SCLs) are used for the identification of peptide sequences that have high affinity for a target protein. These libraries are generally composed of several sublibraries. In each sublibrary, one position is defined with an amino acid, while the remaining positions contain a mixture of amino acids present in equimolar concentrations. Analysis of the library results in an understanding of the specificity of the target protein for amino acids in each position. Positional scanning libraries have been used for the identification of receptor ligands, enzyme inhibitors, specific antigens, and protease substrates. 8-I°
Design of Caspase Positional Scanning Synthetic Combinatorial Library The PS-SCL used in this study, with the general structure Ac-[P4]-[P3][P2]-Asp-AMC (Fig. 2), permits the determination of caspase amino acid preferences in P2, P3, and P4 positions. The decision to keep aspartic acid invariant at P1 was based on the stringent P1-Asp specificity of all caspases. The fluorescent group AMC is incorporated in the P~ position; hydrolysis of the compounds in the library is monitored simply by monitoring the increase in fluorescence that results from release of the AMC moiety. This 8N. A. Thomberry,T. A. Rano, E. P. Peterson, D. M. Rasper,T. Timkey,M. Garcia-Calvo, V. M. Houtzager,P. A. Nordstrom,S. Roy,J. P. Vaillancourt,K. T. Chapman,and D. W. Nicholson,J. Biol. Chem. 272, 17907 (1997). 9T. A. Rano, T. Timkey,E. P. Peterson,J. Rotonda,D. W. Nicholson,J. W. Becker,K. T. Chapman, and N. A. Thornberry, Chem. Biol. 4, 149 (1997). lo C. Pinilla,J. R. Appel, P. Blanc, and R. A. Houghten,BioTechniques13, 901 (1992).
[9]
CASPASE SPECFICITY BY P S - S C L
103
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104
APOPTOSIS PROTEASES A N D T H E I R INHIBITORS
=z
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[9]
CASPASESPECFICITYBY PS-SCL
105
PS-SCL is composed of three separate sublibraries, each containing 20 mixtures (Fig. 2B). In each of the mixtures, one position (P2, P3, o r P4) contains 1 of 20 amino acids, whereas the other two contain a mixture of amino acids present at approximately equimolar concentrations. The entire library thus contains 60 total mixtures of 400 compounds (20 × 20), thus yielding 8000 distinct peptides.
Synthesis As a representative example, the P3 spatially addressed library is prepared as follows9: N-allyloxycarbonyl-L-aspartic acid-a-AMC is loaded onto a Rapp (Tuebingen, Germany) Polymere TentaGel S NH2 resin containing the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) handle via the Mitsunobu reaction [diisopropyl azodicarboxylate (DIAD)/triphenylphosphine (TPP)]. The allyloxycarbonyl (Alloc) group is removed with P d ( P h 3 ) 4 and 1,3-dimethylbarbituric acid (DMBA) in dichloromethane (DCM). The isokinetic mixture of protected amino acids is then prepared by dissolving the requisite amounts of each monomer in N,N-dimethylacetamide (DMA) along with 1-hydroxybenzotriazole hydrate (HOBT) followed by addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The isokinetic mixture is added to the resin, followed by agitation for 2 hr. The resin is washed with DMA and the procedure is repeated. The resin is washed with DMA, DCM, and N,N-dimethylformamide (DMF). Elimination of Fmoc (25% piperidine in DMF for 15 min) is followed by washing with DMA, tetrahydrofuran (THF), isopropyl alcohol (IPA), and DCM. The resin is transferred into 20 individual reaction vessels by the isopycnic slurry method and washed with DMA. Position P3 is "spatially addressed" by preactivating the 20 individual amino acids with EDC/HOBT as described above, followed by addition to the 20 reaction vessels. After 2 hr the resin is washed with DMA and the procedure re-
Fl~. 2. Strategy for synthesizing a positional-scanning combinatorial substrate library for caspases. Asp-aminomethylcoumarin (AMC), tethered to a solid support, is combined with an isokinetic mixture of proteinogenic amino acids ([20aa]) to establish all 20 amino acids in P2 coupled to the P1 Asp-AMC in a single reaction mixture (A). The mixture is then recombined with a fresh isokinetic mixture of the same amino acids to establish all 20 amino acids in P3, resulting in a single solid-phase reaction mixture containing all 400 permutations (20 × 20) of P3-P2-Asp-AMC tripeptides. This mixture is then separated into 20 individual allquots and coupled with a known single amino acid in P4 (the positionally defined amino acid). Thus, after release from the solid support, each well of the P4 sublibrary contains a known amino acid in P4 coupled to all 400 possible permutations of adjacent amino acids (in P3-P2) linked to the P1 Asp-AMC (a total of 8000 fluorogenic tetrapeptides). Similar strategies are used to generate the equivalent P3 and P2 sublibraries (B).
106
APOPTOSIS P R O T E A S E S A N D T H E I R I N H I B I T O R S
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peated. After Fmoc removal and washing, P4 is installed by adding the isokinetic mixture of amino acids to each vessel. After Fmoc removal, the N terminus is acetylated with A c 2 0 / p y r i d i n e / D M F (1 : 2 : 3, by volume) for 1 hr. The acetylation is repeated, followed by washing with DMA, H20, THF, IPA, and DCM. The resin-bound mixtures are then twice cleaved for 30 min with using trifluoroacetic acid (TFA)/H20/phenol (PhOH)/ triisopropylsilane (TIS) (88 : 5 : 5 : 2, by volume). The cleavage solution is aged for 1 hr and 20 min before solvent removal in vacuo. The tetrapeptideAMC derivates are twice precipitated from cold Et20 before being lyophilized from CH3CN/H20 (2 : 1, v/v). The yields for the individual wells range from 30 to 49%. Each of the 60 samples is prepared as approximately 10 mM stocks in dimethyl sulfoxide (DMSO) in a 96-well plate format.
Preparation of Caspases The positional scanning approach to defining caspase substrate specificity is dependent on a preparation of enzyme that is (I) homogeneous with respect to other caspase family members (e.g., analysis of a crude cell extract containing multiple active caspases would be of limited value), (2) is free of degradative proteases that would nonspecifically degrade peptide-AMC substrates, and (3) is sufficiently concentrated to provide a robust fluorogenic signal. The source of the enzyme can be either recombinant 11 or purified. 4'5 Methods for preparing caspases suitable for positional substrate scanning have been extensively detailed elsewhere. 4'5'11
Specificity Determinations To determine protease specificity, enzyme is added to reaction mixtures containing 100/zM substrate mix, i00 mM HEPES, 10 mM dithiothreitol (DTT), pH 7.5, in a total volume of 100 izl. Under these conditions the final concentration of each individual compound is approximately 0.25/zM. The amount of enzyme required depends on the catalytic efficiency of the caspase of interest for cleavage of tetrapeptide fluorogenic substrates. In general, the final concentration of enzyme required is comparable to that used for an assay employing the best tetrapeptide substrate. Production of AMC is monitored continuously at ambient temperature in a Tecan (Hombrechtikon, Switzerland) Fluostar 96-well plate reader, using an exci-
11M. Garcia-Calvo, E. P. Peterson, D. M. Rasper, J. P. Vaillancourt, R. Zamboni, D. W. Nicholson, and N. A. Thornberry, Cell Death Differ. 6, 362 (1999).
[9]
CASPASE SPECFICITY BY P S - S C L Caspase
P4
P3
107 P2
Optimal Sequence WEHD
caspase-1
0CE)
8
caspase-4
(W/L)EHD
caspase-5
(W/L)EHD
(ICErel-II,TX, ICH-2)
(ICErel-III,TY)
F[6. 3. Optimal sequences of human group I caspases as determined by the PS-SCL. tation wavelength of 380 nm and an emission wavelength of 460 nm. Representative data f r o m the analysis of h u m a n caspases 8 are shown in Figs. 3-5. I n t e r p r e t i n g R e s u l t s a n d F u n c t i o n a l Significance A comparison of the specificities of the h u m a n caspase family reveals several key features. The results of this PS-SCL divide these enzymes Caspase
P4
P3
P2
Optimal Sequence
C. elegans CED-3
DETD
caspase-3
DEVD
(apopain,CPP32,Yama)
II
~^nNooea.i
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(Mch3, ICE-LAP3,CMH-1) s
s
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caspase-2
(ICH-1,mNedd2) ~A~.DOeeNIL~SrWVV
~A..DOeGH(t~FPBTWVV
~.~,DOeG.PLK~FelTWVV
FIG. 4. Optimal sequences of human group II caspases as determined by the PS-SCL.
108
APOPTOSIS PROTEASES AND THEIR INHIBITORS
Caspase
P4
P3
P2
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~*a.
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[9] Optimal Sequence
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~*RNDO~Q.:LX~PpSTWVV
caspase-9
LEHD
caspase-8
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(ICE-LAP6,Mch6)
III (MACH, FLICE,Mch5) 0
caspase-10
(Mch4)
A
.JLK~F~
TWVV
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FIG. 5. Optimal sequences of human group III caspases as determined by the PS-SCL.
into three major groups, and indicate that S 4 is the single most important determinant of specificity among caspases. Group I caspases (1, 4, and 5), all favor hydrophobic amino acids in $4, with an optimal sequence of WEHD. Group II enzymes (2, 3, 7, C. elegans CED-3) have a strict requirement for Asp in $4, preferring the sequence DEXD. Caspases in group III tolerate many amino acids in $4, but have a marked preference for those with branched, aliphatic side chains, and an optimal sequence of (I, V, L)EXD. Within each group the specific amino acid preferences are exceedingly similar, in some cases identical, implying that at least some of these enzymes (1) have redundant functions or (2) are cell type- or tissuespecific isoforms. It is clear from several lines of evidence that tetrapeptide specificity extends to macromolecules. It is most compelling that, when endogenous substrates for particular caspases are known, the tetrapeptide sequence at the cleavage sites is similar or identical to the optimal sequence determined by the PS-SCL. In addition, for caspases 1 and 3, the kcat/Km for cleavage of tetrapeptide substrates (>10 6 M -1 sec -1) is greater than or equal to the corresponding rate of cleavage of their authentic macromolecular substrates. Consequently, the tetrapeptide specificities provide important clues regarding the biological functions and relationships between these enzymes. These results, together with those from numerous biochemical and genetic studies, suggest that the caspases may be functionally grouped as follows:
[9]
CASPASESPECFICITYBY PS-SCL
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(1) Group I caspases function primarily as mediators of inflammation, where they are involved in the proteolytic activation of proinflammatory cytokines; (2) group II caspases, and probably caspase 6, are mediators in the effector phase of apoptosis, where they are responsible for cleavage of key structural and homeostatic proteins; (3) group III caspases, with the possible exception of caspase 6, are involved in signaling pathways, where they function as upstream activators of the effector caspases. Virtues and Limitations of Positional Scanning Synthetic Combinatorial Library Several methods have been described for determinations of protease specificity. Traditional approaches typically involve synthesis of large numbers of peptides and/or peptide-based inhibitors, which are individually evaluated for hydrolysis. More recently, strategies for the systematic study of protease specificity have emerged. The most notable examples are substrate phage display, and the positional scanning approach described here. The virtues of positional scanning as a method to determine protease specificity are severalfold. First and most important, it produces an accurate description of specificity, as demonstrated by the findings that the optimal sequences obtained for the caspases are similar or identical to the cleavage site sequences found in known endogenous substrates for these enzymes (described above). Second, it is extremely rapid; using 96-weU plate technology, a complete analysis can be obtained in the length of time required to run an enzyme assay. Third, it requires only catalytic quantities of enzymes. The most significant limitation of this approach is the relatively small number of positions that can be investigated simultaneously. This number is determined by a number of factors, including the selectivity of the enzyme, the solubility of the compounds, and the sensitivity of the detection method employed. For example, in the PS-SCL described here, where 3 positions are explored, each mixture contains 400 compounds, each present at a final concentration of 0.25 ~M. Thus, in the event that only one compound in the mixture is efficiently hydrolyzed by the enzyme of interest, the method of detection must be sensitive enough to detect <0.25 ~M product (AMC in this case). The utility of the method can also be limited by the design of the library. For example, this PS-SCL, in which a fluorescent leaving group is incorporated in PI, restricts its utility to proteases that can tolerate this substitution, and only permits analysis of specificity N terminal to the cleavage site. This method will also fail to provide insights into substituent effects that may be significant in substrate and inhibitor binding. Finally, chemical synthesis of such libraries are labor intensive, a practical obstacle to widespread use of this technology.
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APOPTOSIS PRO'I'EASES AND THEIR INHIBITORS
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Despite these caveats, the results described here with the caspases dearly demonstrate the power of this approach for obtaining an intimate understanding of specificity that can be exploited to identify substrates and inhibitors, and to provide clues to the biological functions of these enzymes. Systematic methods such as these are expected to facilitate efforts to elucidate functions for the many orphan proteins that are identified in genome sequencing projects.
[ 1 O] C r i t e r i a f o r I d e n t i f y i n g A u t h e n t i c C a s p a s e Substrates during Apoptosis By
SOPHIE ROY and DONALD W. NICHOLSON
During apoptotic cell death, members of a discrete and highly limited subset of cellular polypeptides are cleaved by caspases. 1'2 The collective contributions of these proteolytic events manifest the apoptotic phenotype. Despite the enormous biochemical and morphological changes that accompany this form of cell death, most cellular polypeptides escape at least the preengulfment stages of apoptosis unscathed; a survey of protein-banding patterns by sodium dodecyl sulfate (SDS)-polyacrylamide or two-dimensional (2-D) gels, for example, reveals that only a small percentage of the cellular proteome is cleaved during apoptosis. This chapter outlines a series of experimental approaches that can be used to identify and authenticate legitimate caspase substrates and to delineate which caspase family member or subtype is likely to account for proteolysis in vivo. An initial identification of cellular protein constituents that are cleaved by caspases during apoptosis can be undertaken by several approaches. Validation, by the criteria described below, can then aid in substantiating the protein as a legitimate caspase substrate and define the biochemical characteristics of its proteolysis during cell death. Caspases recognize within their polypeptide substrates a core tetrapeptide motif that, in every case, contains an essential aspartic acid in the P1 position (see Fig. 1). The caspase superfamily contains three specificity subgroups, which differentiate their substrates largely on the basis of the residue in P4 (Fig. 1A). 3 Tetrapeptides 1 N. A. T h o r n b e r r y and Y. Lazebnik, Science 281, 1312 (1998). 2 D. W. Nicholson, Cell Death Differ. 6, 1028 (1999). 3 N. A. Thornberry, T. A. Rano, E. P. Peterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom, S. Roy, J. P. VaiUancourt, K. T. C h a p m a n , and D. W. Nicholson, J. BioL Chem. 272, 17907 (1997).
METHODS IN ENZYMOLOGY,VOL. 322
Copyright © 2000by AcademicPress All rights of reproduction in any form reserved. 0076-6879/00$30.00
APOPTOSIS PROTEASES AND THEIR INHIBITORS
[9]
Procedure The assays are carried out in a total volume of 100/zl for detection in a 96-well plate reader, preferably operating in the kinetic mode. For analysis requiring larger volumes, increase proportionally. 1. Prepare working solutions of inhibitor titrant at 1-10/zM in caspase buffer, as required, from the 10 mM stock. The inhibitor is not fully soluble in aqueous solvents above 1 mM, and should be diluted in DMSO when working above 100/zM, before final dilution into caspase buffer. 2. Activate the enzyme to be measured in the usual way in assay buffer; 15 min at 37 ° is usually sufficient. 3. Add 20/zl of each working solution of inhibitor titrant to 80/.d of caspase solution (about 1.0/zM provisional concentration based on protein content) and incubate for 30 min at 37 °. 4. Assay samples from each reaction mixture, using an appropriate substrate in assay buffer to determine the residual activity. Plot residual activity as a function of inhibitor concentration. As with all enzyme assays, the rate of substrate hydrolysis must be in the linear range. If the reaction is too fast, and substrate is exhausted too rapidly, dilute the caspase/ZVAD-FMK reaction mixture to obtain a linear rate. 5. The plot should be linear with an intercept on the x axis equal to the concentration of active enzyme (Fig. 3). The curvature of the plot as it approaches this axis indicates that the reaction has not gone to completion. This can be overcome by longer incubation or by increasing the concentrations of enzyme and inhibitor. The concentration of the enzyme can only rarely be accurately determined by extrapolating a straight line drawn from partial inhibition.
[9] D e t e r m i n a t i o n o f C a s p a s e S p e c i f i c i t i e s U s i n g a Peptide Combinatorial Library
By NANCY A. THORNBERRY,KEVIN T. CHAPMAN, and D O N A L D W . N I C H O L S O N Introduction The caspase family of cysteine proteases is composed of at least 14 mammalian family members. Phylogenetically they can be divided into two distinct subgroups having markedly different functions in vivo. Members of the interleukin 1/3-converting enzyme (ICE) subfamily [ICE/caspase 1
METHODS IN ENZYMOLOGY, VOL. 322
Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
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was originally identified as the protease responsible for the proteolytic maturation of proinflammatory interleukin 1/3 (prolL-1/3)] cleave and activate multiple cytokines (e.g., prolL-1/3, prolL-18) and thus participate in mounting an inflammatory response. Members of the CED-3 subfamily, on the other hand, mediate a cascade of proteolytic cleavage events that culminate in cell death and the apoptotic phenotype (CED-3 was originally identified as a protease in Caenorhabditis elegans that is necessary for developmental cell deaths). The central role that these proteases play in both of these important physiological processes has been resolved in part by inhibitor studies, knockout animals, substrate identification, and extensive in vitro studies with purified or recombinant caspase family members. 1'2 In this chapter we describe a strategy for defining the precise substrate specificity of individual caspases and discuss how this information can contribute to our understanding of the function that each enzyme serves in its particular biochemical pathway. Two of the most biologically distinct caspases, caspase 1 and caspase 3, have been purified from natural sources, cloned, and their structures determined by X-ray crystallography. 3-7 The results of these studies, together with a comparison of the sequences of all caspases, indicate that these enzymes are synthesized as zymogens that contain three distinct domains: an N-terminal polypeptide (3 to 25 kDa), a large subunit ( - 2 0 kDa), and a small subunit ( - 1 0 kDa). Activation involves proteolytic pro1 D. W. Nicholson and N. A. Thornberry, Trends Biochem. Sci. 22, 299 (1997). 2 N. A. Thornberry and Y. Lazebnik, Science 281, 1312 (1998). 3 j. Rotonda, D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry, and J. W. Becker, Nature Struct. Biol. 3, 619 (1996). 4 D. W. Nicholson, A. Ali, N. A. Thornberry, J. P. Vaillancourt, C. K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, Y. A. Lazebnik, N. A. Munday, S. M. Raju, M. E. Smulson, T.-T. Yamin, Y. L. Yu, and D. K. Miller, Nature (London) 376, 37 (1995). 5 N. A. Thornberry, H. G. Bull, J. R. Calaycay, K. T. Chapman, A. D. Howard, M. J. Kostura, D. K. Miller, S. M. Molineaux, J. R. Weidner, J. Aunins, K. O. Elliston, J. M. Ayala, F. J. Casano, J. Chin, G. J.-F. Ding, L. A. Egger, E. P. Gaffney, G. Limjuco, O. C. Palyha, S. M. Raju, A. M. Rolando, J. P. Salley, T.-T. Yamin, T. D. Lee, J. E. Shively, M. MacCross, R. A. Mumford, J. A. Schmidt, and M. J. Tocci, Nature (London) 356, 768 (1992). 6 N. P. Walker, R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett, L. D. Hammill, L. Herzog, M. Hugunin, W. Houy, J. A. Mankovich, L. McGuiness, E. Orlewicz, M. Paskind, C. A. Pratt, P. Reis, A. Summani, M. Terranova, J. P. Welch, L. Xiong, A. Moller, D. E. Tracey, R. Kamen, and W. W. Wong, Cell 78, 343 (1994). 7 K. P. Wilson, J. A. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A. Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J. Livingston, Nature (London) 370, 270 (1994).
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APOPTOSlSPROTEASESANDTHEIRINHIBITORS
[9]
cessing between domains, removal of the N-terminal polypeptide, and association of the large and small subunits to form a heterodimer. The active site, which is formed by amino acids from both subunits, contains a catalytic diad composed of cysteine and histidine, suggesting that these enzymes employ a typical cysteine protease mechanism. Studies of caspase specificity by traditional approaches indicated that two highly conserved properties of these enzymes dictate hydrolysis of substrates. First, they have a near-absolute requirement for aspartic acid in the P1 position, and second, they require at least four amino acids N terminal to the cleavage site. These observations, together with the finding that substrates with the general structure Ac-XXXD-aminomethylcoumarin (AMC) are efficiently hydrolyzed by caspases, led to the design of the approach described below for the systematic determination of caspase specificity (see Fig. 1). Positional Scanning Synthetic Combinatorial Library Positional scanning peptide libraries (PS-SCLs) are used for the identification of peptide sequences that have high affinity for a target protein. These libraries are generally composed of several sublibraries. In each sublibrary, one position is defined with an amino acid, while the remaining positions contain a mixture of amino acids present in equimolar concentrations. Analysis of the library results in an understanding of the specificity of the target protein for amino acids in each position. Positional scanning libraries have been used for the identification of receptor ligands, enzyme inhibitors, specific antigens, and protease substrates. 8-I°
Design of Caspase Positional Scanning Synthetic Combinatorial Library The PS-SCL used in this study, with the general structure Ac-[P4]-[P3][P2]-Asp-AMC (Fig. 2), permits the determination of caspase amino acid preferences in P2, P3, and P4 positions. The decision to keep aspartic acid invariant at P1 was based on the stringent P1-Asp specificity of all caspases. The fluorescent group AMC is incorporated in the P~ position; hydrolysis of the compounds in the library is monitored simply by monitoring the increase in fluorescence that results from release of the AMC moiety. This 8N. A. Thomberry,T. A. Rano, E. P. Peterson, D. M. Rasper,T. Timkey,M. Garcia-Calvo, V. M. Houtzager,P. A. Nordstrom,S. Roy,J. P. Vaillancourt,K. T. Chapman,and D. W. Nicholson,J. Biol. Chem. 272, 17907 (1997). 9T. A. Rano, T. Timkey,E. P. Peterson,J. Rotonda,D. W. Nicholson,J. W. Becker,K. T. Chapman, and N. A. Thornberry, Chem. Biol. 4, 149 (1997). lo C. Pinilla,J. R. Appel, P. Blanc, and R. A. Houghten,BioTechniques13, 901 (1992).
[9]
CASPASE SPECFICITY BY P S - S C L
103
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104
APOPTOSIS PROTEASES A N D T H E I R INHIBITORS
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[9]
CASPASESPECFICITYBY PS-SCL
105
PS-SCL is composed of three separate sublibraries, each containing 20 mixtures (Fig. 2B). In each of the mixtures, one position (P2, P3, o r P4) contains 1 of 20 amino acids, whereas the other two contain a mixture of amino acids present at approximately equimolar concentrations. The entire library thus contains 60 total mixtures of 400 compounds (20 × 20), thus yielding 8000 distinct peptides.
Synthesis As a representative example, the P3 spatially addressed library is prepared as follows9: N-allyloxycarbonyl-L-aspartic acid-a-AMC is loaded onto a Rapp (Tuebingen, Germany) Polymere TentaGel S NH2 resin containing the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) handle via the Mitsunobu reaction [diisopropyl azodicarboxylate (DIAD)/triphenylphosphine (TPP)]. The allyloxycarbonyl (Alloc) group is removed with P d ( P h 3 ) 4 and 1,3-dimethylbarbituric acid (DMBA) in dichloromethane (DCM). The isokinetic mixture of protected amino acids is then prepared by dissolving the requisite amounts of each monomer in N,N-dimethylacetamide (DMA) along with 1-hydroxybenzotriazole hydrate (HOBT) followed by addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The isokinetic mixture is added to the resin, followed by agitation for 2 hr. The resin is washed with DMA and the procedure is repeated. The resin is washed with DMA, DCM, and N,N-dimethylformamide (DMF). Elimination of Fmoc (25% piperidine in DMF for 15 min) is followed by washing with DMA, tetrahydrofuran (THF), isopropyl alcohol (IPA), and DCM. The resin is transferred into 20 individual reaction vessels by the isopycnic slurry method and washed with DMA. Position P3 is "spatially addressed" by preactivating the 20 individual amino acids with EDC/HOBT as described above, followed by addition to the 20 reaction vessels. After 2 hr the resin is washed with DMA and the procedure re-
Fl~. 2. Strategy for synthesizing a positional-scanning combinatorial substrate library for caspases. Asp-aminomethylcoumarin (AMC), tethered to a solid support, is combined with an isokinetic mixture of proteinogenic amino acids ([20aa]) to establish all 20 amino acids in P2 coupled to the P1 Asp-AMC in a single reaction mixture (A). The mixture is then recombined with a fresh isokinetic mixture of the same amino acids to establish all 20 amino acids in P3, resulting in a single solid-phase reaction mixture containing all 400 permutations (20 × 20) of P3-P2-Asp-AMC tripeptides. This mixture is then separated into 20 individual allquots and coupled with a known single amino acid in P4 (the positionally defined amino acid). Thus, after release from the solid support, each well of the P4 sublibrary contains a known amino acid in P4 coupled to all 400 possible permutations of adjacent amino acids (in P3-P2) linked to the P1 Asp-AMC (a total of 8000 fluorogenic tetrapeptides). Similar strategies are used to generate the equivalent P3 and P2 sublibraries (B).
106
APOPTOSIS P R O T E A S E S A N D T H E I R I N H I B I T O R S
[9]
peated. After Fmoc removal and washing, P4 is installed by adding the isokinetic mixture of amino acids to each vessel. After Fmoc removal, the N terminus is acetylated with A c 2 0 / p y r i d i n e / D M F (1 : 2 : 3, by volume) for 1 hr. The acetylation is repeated, followed by washing with DMA, H20, THF, IPA, and DCM. The resin-bound mixtures are then twice cleaved for 30 min with using trifluoroacetic acid (TFA)/H20/phenol (PhOH)/ triisopropylsilane (TIS) (88 : 5 : 5 : 2, by volume). The cleavage solution is aged for 1 hr and 20 min before solvent removal in vacuo. The tetrapeptideAMC derivates are twice precipitated from cold Et20 before being lyophilized from CH3CN/H20 (2 : 1, v/v). The yields for the individual wells range from 30 to 49%. Each of the 60 samples is prepared as approximately 10 mM stocks in dimethyl sulfoxide (DMSO) in a 96-well plate format.
Preparation of Caspases The positional scanning approach to defining caspase substrate specificity is dependent on a preparation of enzyme that is (I) homogeneous with respect to other caspase family members (e.g., analysis of a crude cell extract containing multiple active caspases would be of limited value), (2) is free of degradative proteases that would nonspecifically degrade peptide-AMC substrates, and (3) is sufficiently concentrated to provide a robust fluorogenic signal. The source of the enzyme can be either recombinant 11 or purified. 4'5 Methods for preparing caspases suitable for positional substrate scanning have been extensively detailed elsewhere. 4'5'11
Specificity Determinations To determine protease specificity, enzyme is added to reaction mixtures containing 100/zM substrate mix, i00 mM HEPES, 10 mM dithiothreitol (DTT), pH 7.5, in a total volume of 100 izl. Under these conditions the final concentration of each individual compound is approximately 0.25/zM. The amount of enzyme required depends on the catalytic efficiency of the caspase of interest for cleavage of tetrapeptide fluorogenic substrates. In general, the final concentration of enzyme required is comparable to that used for an assay employing the best tetrapeptide substrate. Production of AMC is monitored continuously at ambient temperature in a Tecan (Hombrechtikon, Switzerland) Fluostar 96-well plate reader, using an exci-
11M. Garcia-Calvo, E. P. Peterson, D. M. Rasper, J. P. Vaillancourt, R. Zamboni, D. W. Nicholson, and N. A. Thornberry, Cell Death Differ. 6, 362 (1999).
[9]
CASPASE SPECFICITY BY P S - S C L Caspase
P4
P3
107 P2
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caspase-1
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8
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(ICErel-III,TY)
F[6. 3. Optimal sequences of human group I caspases as determined by the PS-SCL. tation wavelength of 380 nm and an emission wavelength of 460 nm. Representative data f r o m the analysis of h u m a n caspases 8 are shown in Figs. 3-5. I n t e r p r e t i n g R e s u l t s a n d F u n c t i o n a l Significance A comparison of the specificities of the h u m a n caspase family reveals several key features. The results of this PS-SCL divide these enzymes Caspase
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C. elegans CED-3
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FIG. 4. Optimal sequences of human group II caspases as determined by the PS-SCL.
108
APOPTOSIS PROTEASES AND THEIR INHIBITORS
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FIG. 5. Optimal sequences of human group III caspases as determined by the PS-SCL.
into three major groups, and indicate that S 4 is the single most important determinant of specificity among caspases. Group I caspases (1, 4, and 5), all favor hydrophobic amino acids in $4, with an optimal sequence of WEHD. Group II enzymes (2, 3, 7, C. elegans CED-3) have a strict requirement for Asp in $4, preferring the sequence DEXD. Caspases in group III tolerate many amino acids in $4, but have a marked preference for those with branched, aliphatic side chains, and an optimal sequence of (I, V, L)EXD. Within each group the specific amino acid preferences are exceedingly similar, in some cases identical, implying that at least some of these enzymes (1) have redundant functions or (2) are cell type- or tissuespecific isoforms. It is clear from several lines of evidence that tetrapeptide specificity extends to macromolecules. It is most compelling that, when endogenous substrates for particular caspases are known, the tetrapeptide sequence at the cleavage sites is similar or identical to the optimal sequence determined by the PS-SCL. In addition, for caspases 1 and 3, the kcat/Km for cleavage of tetrapeptide substrates (>10 6 M -1 sec -1) is greater than or equal to the corresponding rate of cleavage of their authentic macromolecular substrates. Consequently, the tetrapeptide specificities provide important clues regarding the biological functions and relationships between these enzymes. These results, together with those from numerous biochemical and genetic studies, suggest that the caspases may be functionally grouped as follows:
[9]
CASPASESPECFICITYBY PS-SCL
109
(1) Group I caspases function primarily as mediators of inflammation, where they are involved in the proteolytic activation of proinflammatory cytokines; (2) group II caspases, and probably caspase 6, are mediators in the effector phase of apoptosis, where they are responsible for cleavage of key structural and homeostatic proteins; (3) group III caspases, with the possible exception of caspase 6, are involved in signaling pathways, where they function as upstream activators of the effector caspases. Virtues and Limitations of Positional Scanning Synthetic Combinatorial Library Several methods have been described for determinations of protease specificity. Traditional approaches typically involve synthesis of large numbers of peptides and/or peptide-based inhibitors, which are individually evaluated for hydrolysis. More recently, strategies for the systematic study of protease specificity have emerged. The most notable examples are substrate phage display, and the positional scanning approach described here. The virtues of positional scanning as a method to determine protease specificity are severalfold. First and most important, it produces an accurate description of specificity, as demonstrated by the findings that the optimal sequences obtained for the caspases are similar or identical to the cleavage site sequences found in known endogenous substrates for these enzymes (described above). Second, it is extremely rapid; using 96-weU plate technology, a complete analysis can be obtained in the length of time required to run an enzyme assay. Third, it requires only catalytic quantities of enzymes. The most significant limitation of this approach is the relatively small number of positions that can be investigated simultaneously. This number is determined by a number of factors, including the selectivity of the enzyme, the solubility of the compounds, and the sensitivity of the detection method employed. For example, in the PS-SCL described here, where 3 positions are explored, each mixture contains 400 compounds, each present at a final concentration of 0.25 ~M. Thus, in the event that only one compound in the mixture is efficiently hydrolyzed by the enzyme of interest, the method of detection must be sensitive enough to detect <0.25 ~M product (AMC in this case). The utility of the method can also be limited by the design of the library. For example, this PS-SCL, in which a fluorescent leaving group is incorporated in PI, restricts its utility to proteases that can tolerate this substitution, and only permits analysis of specificity N terminal to the cleavage site. This method will also fail to provide insights into substituent effects that may be significant in substrate and inhibitor binding. Finally, chemical synthesis of such libraries are labor intensive, a practical obstacle to widespread use of this technology.
110
APOPTOSIS PRO'I'EASES AND THEIR INHIBITORS
[10]
Despite these caveats, the results described here with the caspases dearly demonstrate the power of this approach for obtaining an intimate understanding of specificity that can be exploited to identify substrates and inhibitors, and to provide clues to the biological functions of these enzymes. Systematic methods such as these are expected to facilitate efforts to elucidate functions for the many orphan proteins that are identified in genome sequencing projects.
[ 1 O] C r i t e r i a f o r I d e n t i f y i n g A u t h e n t i c C a s p a s e Substrates during Apoptosis By
SOPHIE ROY and DONALD W. NICHOLSON
During apoptotic cell death, members of a discrete and highly limited subset of cellular polypeptides are cleaved by caspases. 1'2 The collective contributions of these proteolytic events manifest the apoptotic phenotype. Despite the enormous biochemical and morphological changes that accompany this form of cell death, most cellular polypeptides escape at least the preengulfment stages of apoptosis unscathed; a survey of protein-banding patterns by sodium dodecyl sulfate (SDS)-polyacrylamide or two-dimensional (2-D) gels, for example, reveals that only a small percentage of the cellular proteome is cleaved during apoptosis. This chapter outlines a series of experimental approaches that can be used to identify and authenticate legitimate caspase substrates and to delineate which caspase family member or subtype is likely to account for proteolysis in vivo. An initial identification of cellular protein constituents that are cleaved by caspases during apoptosis can be undertaken by several approaches. Validation, by the criteria described below, can then aid in substantiating the protein as a legitimate caspase substrate and define the biochemical characteristics of its proteolysis during cell death. Caspases recognize within their polypeptide substrates a core tetrapeptide motif that, in every case, contains an essential aspartic acid in the P1 position (see Fig. 1). The caspase superfamily contains three specificity subgroups, which differentiate their substrates largely on the basis of the residue in P4 (Fig. 1A). 3 Tetrapeptides 1 N. A. T h o r n b e r r y and Y. Lazebnik, Science 281, 1312 (1998). 2 D. W. Nicholson, Cell Death Differ. 6, 1028 (1999). 3 N. A. Thornberry, T. A. Rano, E. P. Peterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom, S. Roy, J. P. VaiUancourt, K. T. C h a p m a n , and D. W. Nicholson, J. BioL Chem. 272, 17907 (1997).
METHODS IN ENZYMOLOGY,VOL. 322
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