Substrate Specificities of Caspase Family Proteases*

(Received for publication, December 16, 1996, and in revised form, February 17, 1997)

Robert V. Talanian Dagger , Christopher Quinlan , Simone Trautz , Maria C. Hackett , John A. Mankovich , David Banach , Tariq Ghayur , Kenneth D. Brady and Winnie W. Wong

From BASF Bioresearch Corp., Worcester, Massachusetts 01605

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.


INTRODUCTION

A growing body of evidence supports important roles for the interleukin-1beta converting enzyme (ICE)1 (1, 2) and its homologs (recently renamed caspases (3)) in cytokine maturation and apoptosis. The caspase gene family, defined by protein sequence homology but also characterized by conservation of key catalytic and substrate-recognition amino acids, includes caspase-2 (4), caspase-3 (5-7), caspase-4 (8-10), caspase-5 (10), caspase-6 (11), caspase-7 (12-14), caspase-8 (15-17), caspase-9 (18, 19), and caspase-10 (17). Each is an intracellular cysteine protease that shares with the serine protease granzyme B specificity for Asp in the P1 position of substrates. The specific biological roles and interrelationships of these enzymes are for the most part unknown and are areas of active investigation in many laboratories.

A role for caspase-1 in inflammation is supported by several lines of evidence. Caspase-1-deficient mice, and cells derived from those animals, are deficient in IL-1beta maturation and are resistant to endotoxic shock (20, 21). Peptidic inhibitors of caspase-1 can be effective in blocking maturation and release of IL-1beta by cultured cells (1) and in whole animals (22, 23) and of inflammation in animal models (24, 25). The selectivity of the inhibitors employed in these studies among the caspases has not been demonstrated, and so the precise role of each caspase in inflammation is uncertain. Nevertheless the results uphold the promise of caspase-1 and/or its homologs as targets for anti-inflammatory drug discovery.

Caspases play important roles in apoptosis signaling and effector mechanisms. Sequence alignments reveal homology with Ced-3 (26), a nematode cysteine protease (27, 28) that is required for cell death. The viral proteins CrmA and p35 are antiapoptotic and act by inhibition of caspases (29, 30). A bacterial invasin induces apoptosis by binding to and activating caspase-1 specifically (31). Caspase-3 is necessary and sufficient for apoptosis in one acellular model (6); however, in mice the essential function of this enzyme is limited to apoptosis in the brain (32). A hallmark of apoptosis is the proteolytic inactivation of poly(ADP-ribose) polymerase (33), and several caspases can catalyze that cleavage (7, 11-13, 18, 34, 35). Lamin A cleavage during apoptosis is catalyzed by caspase-6 (34, 36). Cleavage of other proteins at Asp residues, including DNA-protein kinases C, protein kinase C-delta , and Gas2, also accompanies apoptosis (37-41), although in most cases the enzyme(s) responsible for those reactions have not been identified.

Caspases can transduce or amplify signals by mutual activation. The "death domain" motifs of caspase-8 (15-17, 19) and -10 (17, 19) and their association with Fas or tumor necrosis factor receptors via interaction with FADD, suggests that they are upstream activators that proteolytically mature other caspases. Fas-induced apoptosis is characterized by an early, transient "caspase-1-like" protease activity followed by a "caspase-3-like" activity (42), suggesting an ordered activation cascade. Reconstitution experiments suggest that both caspase-3 and -7 are activated by caspase-6 (43) and caspase-10 (17). Little is known regarding caspase regulation or inactivation. Caspase-1 contains a site that is rapidly autodegraded in vitro, and cleavage at this site may represent a physiological mechanism of down-regulation (44).

To facilitate studies of the roles of caspase family proteases in inflammation and apoptosis, and to help elucidate protease-mediated mechanisms of signal transduction and regulation, we have characterized in detail the substrate preferences of caspase-1 and five of its homologs. We present here our findings of the specificities of each enzyme, which suggest the design of novel specific peptidic substrates and inhibitors, and help to predict the biological roles of these enzymes.


MATERIALS AND METHODS

Peptides

Underivatized peptides (Quality Controlled Biochemicals, Hopkinton, MA) were prepared by standard solid-phase methods, purified to >= 95% by HPLC, and confirmed by low resolution mass spectrometry. Concentrations of stock solutions in Me2SO were determined in duplicate as described (45). Chromogenic and fluorogenic peptides were from California Peptide Research (Napa, CA). Concentrations of chromogenic peptide stock solutions were determined in duplicate as described (46). Concentrations of fluorogenic peptide stocks were determined in duplicate by UV absorbance using epsilon 325 = 15,990 in aqueous buffer at pH 7.5.

Enzyme Expression and Purification

Caspase-1, -2, -3, and -4, containing N-terminal His tags to facilitate purification, were prepared as described (9, 30, 47). Caspase-1 contained the stability-enhancing point mutation D381E (47). Caspase-6 (Mch2alpha ) (11) beginning at Ala-2, or caspase-7 beginning at Ala-23, with amino-terminal polyhistidine tags (MGHHHHHHGSG), were subcloned into a pBluescript II KS(+) (Stratagene, La Jolla, CA) derivative under control of the Lambda PL promoter. Amino-terminal coding sequences were modified to reflect Escherichia coli codon preferences. To increase soluble expression the proteases were coexpressed with the bacterial chaperones groES and groEL (48). The groESL operon, under control of the isopropyl-1-thio-beta -D-galactopyranoside-inducible Ptac promoter, was cloned into a pACYC177 (49) derivative encoding LacI and the temperature-sensitive repressor cI857 inserted into the beta -lactamase gene. Both plasmids were stably maintained in the same bacterial cell. Simultaneous expression of the chaperones and proteases were induced by addition of isopropyl-1-thio-beta -D-galactopyranoside to 2 mM and temperature shift from 30 °C to 40 °C at A600 = 0.6. The bacteria were harvested at t = 5 h, and the proteases were purified as described (47).

Enzyme Assays

Peptide cleavage assays were modeled on those of Howard et al. (50). Substrate concentration depletion during the reaction is described by Equation 1.
<FR><NU>d[<UP>S</UP>]</NU><DE>dt</DE></FR>=<FR><NU>V<SUB><UP>max</UP></SUB>[<UP>S</UP>]</NU><DE>[<UP>S</UP>]+K<SUB>m</SUB></DE></FR> (Eq. 1)
which solves to the general case.
<FR><NU>[<UP>S</UP>]<SUB>t</SUB></NU><DE>[<UP>S</UP>]<SUB>0</SUB></DE></FR> e<SUP>([<UP>S</UP>]<SUB>t</SUB>−[<UP>S</UP>]<SUB>0</SUB>)/K<SUB>m</SUB></SUP>=e<SUP><UP>−</UP>V<SUB><UP>max</UP></SUB>t/K<SUB>m</SUB></SUP> (Eq. 2)
Assuming [S] <<  Km, Equation 2 reduces to the following equation.
<FR><NU>[<UP>S</UP>]<SUB>t</SUB></NU><DE>[<UP>S</UP>]<SUB>0</SUB></DE></FR>=e<SUP><UP>−</UP>kt</SUP> (Eq. 3)
where k is the apparent first order substrate cleavage rate constant equal to Vmax/Km. Assays contained in 810 µl: 100 mM sodium acetate at pH 6.2 (for caspase-2) or 100 mM HEPES at pH 7.5 (all others), 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA, and 0.3-10 µg of purified enzymes. After a 30-min preincubation at 30 °C, substrates were added to 10 µM (except as noted). At 10-min intervals for 60 min, 110-µl aliquots were stopped with 11 µl of 3 M HCl. Samples were analyzed by HPLC using a 250 × 4.6 mm C18 reverse phase column (Vydak, Hesperia, CA) and a linear gradient of MeCN/H2O with 0.1% (v/v) trifluoroacetic acid, monitoring at 214 nm. The identical elution times of one of the two product peaks for each peptide in a series confirmed cleavage at the same site despite amino acid variations. The area under the substrate peaks (arbitrary units) was plotted versus time and fitted to Equation 3. Relative Vmax/Km values (unitless) were obtained by normalizing apparent Vmax/Km values to 1.00 for a chosen peptide. Km values were determined for selected substrates and enzymes by fitting initial rates of peptide cleavage at various substrate concentrations from 10 to 250 µM to the Michaelis-Menten equation.

Assays of chromogenic or fluorogenic substrate cleavage contained in 100 µl: 100 mM HEPES (pH 7.5), 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA, 0.1% (w/v) bovine serum albumin, and 0.3 (fluorogenic substrates) or 3.0-200 (chromogenic substrates) ng of purified enzymes. After a 30-min preincubation at 30 °C, substrates in Me2SO were added to various concentrations. For chromogenic substrates, enzyme-catalyzed release of p-nitroanilide was monitored at 405 nm in a microtiter plate reader (Molecular Devices, Menlo Park, CA). For fluorogenic substrates, Amc release was monitored at 460 nm using 385 nm excitation in a Labsystems (Needham Heights, MA) Fluoroskan Ascent fluorescence plate reader. Km and kcat values were determined from plots of activity versus substrate concentration. Absolute kcat values for chromogenic substrates were calculated using a standard curve determined with p-nitroanilide and are uncorrected for enzyme purity and percent activity.

Assays of inhibitor potency were performed with fluorogenic substrates using nominal enzyme concentrations of 0.1 nM or less. Apparent Ki values were calculated by dividing IC50 values by (1 + [S]/Km).


RESULTS

Assays of ICE Homolog Specificity

Building on previous work (1, 50, 51) we studied the substrate specificity of several caspases by measuring relative Vmax/Km values, a measure of enzymatic specificity (52), toward a series of defined peptide sequence variants. We chose this rather than a combinatorial approach to obtain quantitative data on both optimal and suboptimal substrate sequences. Peptides were generally of minimal length, contained C-terminal Trp residues to facilitate detection and concentration determination, and were acetylated and amidated to avoid introduction of nonnatural charges. Sequence variants were selected to test hypotheses of substrate recognition and to explore the structural range of natural amino acids. Application of Equation 3 requires that [S]0 <<  Km. Km values of each enzyme toward preferred peptide substrates were determined from the initial rates of peptide hydrolysis as a function of peptide concentration (Table I). All observed values were at least 10-fold greater than the peptide concentration utilized in the specificity studies (10 µM), confirming that the use of Equation 3 is appropriate.

Table I.

Km values of caspases for preferred peptide substrates

Km values were obtained as described under "Materials and Methods."


Enzyme Substrate Km

µM
Caspase-1 Ac-YEVDGW-Am 105
Caspase-4 Ac-LEVDGW-Am 430
Caspase-3 Ac-VDQMDGW-Am 200
Caspase-2 Ac-VDVADGW-Am 150
Caspase-6 Ac-VQVDGW-Am 130
Caspase-7 Ac-VDQVDGW-Am 125

Caspase-1 and -4 Specificity

Caspase-1 and -4 specificities were tested using variants of Ac-YVADGW-Am, a preferred peptide substrate (1) based on the caspase-1 cleavage site within IL-1beta (YVHD117A) (53, 54). In P4, caspase-1 and -4 prefer hydrophobic amino acids (Fig. 1), with the former preferring aromatics (consistent with IL-1beta cleavage), and the latter, aliphatics. In P3, both enzymes prefer Glu. The crystal structure of caspase-3 in complex with Ac-DEVD-CHO (55) shows that the P3 Glu of the peptidic ligand makes an ionic interaction with the side chain of the conserved residue Arg-207. Ligands containing P3 Glu probably make analogous favorable contacts with caspase-1 and -4. In P2, a broad range of amino acids are tolerated by both enzymes, consistent with the observation from the caspase-1 and -3 crystal structures that side chains of amino acids in this position are solvent-exposed. As expected (1, 51), caspase-1 and also caspase-4 prefer Gly in the P1' position of substrates. In addition, we find that all of the aromatic and the thiol- and hydroxyl-containing amino acids were also efficiently cleaved.


Fig. 1. Peptide substrate preferences of caspase-1 and caspase-4. Peptide substrate cleavage was measured for caspase-1 (black bars) and caspase-4 (white bars) using sequence variants of Ac-YVADGW-Am. Each panel displays the results for variants at a single position in the peptide, with substitutions shown on the x axis. Relative (Vmax/Km) values (y axis) for both enzymes are normalized to 1.00 for the results obtained with Ac-YVADGW-Am.
[View Larger Version of this Image (30K GIF file)]


As predicted from the peptide cleavage results, the chromogenic tetrapeptide p-nitroanilides Ac-YEVD-pNA and Ac-LEVD-pNA were preferentially cleaved by caspase-1 and -4, respectively, with kcat/Km values considerably higher than related peptides, including in the former case the widely used substrate Ac-YVAD-pNA (Table II).

Table II.

Kinetic constants for chromogenic peptide substrates

Enzymes were assayed and kinetic constants were calculated as described under "Materials and Methods." kcat values are uncorrected for enzyme purity and fractional activity and so permit comparisons only between different substrates for a single enzyme. NC, no cleavage up to 1 mM substrate; ND, not done.


Substrate KmM), kcat (M-1 s-1), kcat/Km (s-1)
Caspase-1 Caspase-4 Caspase-2 Caspase-3 Caspase-7 Caspase-6

Ac-YVAD-pNA 23, 1.5, 63,000 874, 0.24, 280 NC 29,000, 3.0, 100 NC NC
Ac-YEVD-pNA 7.3, 0.57, 79,000 31, 0.08, 2,600 ND 370, 14.1, 39,000 490, 6.9, 14,000 1200, 0.1, 90 
Ac-LEVD-pNA 8.5, 0.40, 54,000 44, 0.14, 3,200 ND ND ND 160, 6.8, 42,000 
Ac-DEVD-pNA 18, 0.50, 30,000 32, 0.05, 1,800 NC 11, 2.4, 218,000 12, 0.43, 37,000 180, 0.4, 2,000 
Ac-DQMD-pNA 45, 0.94, 21,000 350, 0.09, 270 ND 44, 11, 262,000 130, 3.2, 25,000 1300, 0.3, 230 
Ac-VDQQD-pNA ND ND 530, 8.0, 15,000 ND 3100, 4.8, 1,600 7000, 0.08, 11 
Ac-VDVAD-pNA ND ND 53, 4.5, 84,000 67, 5.1, 76,000 200, 2.6, 13,000 ND
Ac-VEID-pNA 46, 0.56, 12,000 205, 0.15, 750 NC 250, 16, 61,000 570, 3.74, 6,600 30, 5.0, 168,000 
Ac-VQVD-pNA 120, 0.33, 2,800 720, 0.12, 170 NC 510, 15, 29,000 2100, 6.2, 3,000 580, 7.1, 12,000

Caspase-2 Specificity

In a survey of peptide substrates spanning known or suspected cleavage sites within caspases and their substrates, we found that only peptides containing VDQQD (caspase-2 residues 312-316) and LDVVD (caspase-6 residues 175-179) were cleaved by caspase-2, but peptides Ac-DQQDGW-Am and Ac-DVVDGW-Am were not. We examined the length dependence of peptide cleavage by caspase-2 (Table III) and found that, in contrast to caspase-1 (1), caspase-2 requires a P5 residue in peptide substrates for efficient cleavage. Like caspase-1 (1), efficient cleavage of peptides by caspase-2 did not require prime-side substrate residues (Table III). We therefore examined the substrate specificity of caspase-2 using sequence variants of Ac-VDQQDGW-Am (Fig. 2). Hydrophobic residues were preferred in the P5 position. Like caspase-3 and -7 (see below), caspase-2 displayed a strong preference for Asp in P4. In P3 and P2, Val and Ala, respectively, were preferred over Gln, but like caspase-1 and -4, Glu was a favored P3 substrate residue and in P2 a structurally wide range of amino acids were tolerated.

Table III.

Length dependence of peptide cleavage by caspase-2

Peptide cleavage assays were performed, and relative (Vmax/Km) values were calculated as described under "Materials and Methods." Peptides are aligned with respect to the P1 residue, which is underlined for the first peptide.


Peptide Relative (Vmax/Km)

Ac-RGVDQQ GKNHW-Am 1.00
 Ac-GVDQQD GKNW-Am 0.65
  Ac-VDQQD GKNW-Am 0.74
   Ac-DQQD GKNW-Am 0.07
    Ac-QQD GKNW-Am 0.02
  Ac-VDQQD GKW-Am 0.75
  Ac-VDQQD GW-Am 0.73
  Ac-VDQQD W-Am 0.69


Fig. 2. Peptide substrate preferences of caspase-2, caspase-3, and caspase-7. Peptide substrate cleavage was measured for caspase-2 (black bars), caspase-3 (gray bars), and caspase-7 (white bars) using sequence variants of Ac-VDQQDGW-Am. Relative (Vmax/Km) values are normalized to 1.00 for the results obtained with Ac-VDQQDGW-Am, with the exception of P2, where the caspase-3 results are normalized to Ac-VDQADGW-Am and the caspase-7 results to Ac-VDQMDGW-Am.
[View Larger Version of this Image (31K GIF file)]


The results suggested that optimal caspase-2 peptidic substrates and inhibitors would contain the sequence VDVAD, and consistent with that, Ac-VDVADGW-Am was cleaved at a correspondingly greater efficiency than either of the singly substituted peptides (not shown). We tested Ac-VDVAD-pNA as a caspase-2 substrate and found it was preferred by caspase-2, cleaved with a kcat/Km value similar to those of other enzymes and their preferred substrates (Table II). Ac-DEVD-pNA, widely used as a caspase-3 substrate, was poorly cleaved by caspase-2. Using the fluorogenic substrate Ac-VDVAD-Amc (Km = 80 µM), the pentapeptide aldehyde Ac-VDVAD-CHO inhibited caspase-2 with a Ki value of 3.5 nM. In contrast, the Ki of Ac-DEVD-CHO was 1.75 µM, again supporting the observed in vitro requirement of caspase-2 for a P5 peptide residue.

Caspase-3 and -7 Specificity

Caspase-3 and -7 were probed in P4-P2 using the peptide variants designed for caspase-2. Length dependence experiments showed that for these enzymes a P5 residue was unnecessary for efficient cleavage, and the presence of the P5 Val residue in particular did not significantly affect cleavage rates.2 The two enzymes displayed many similarities in their primary specificities. The P4 Asp preference of caspase-3 was almost absolute (Fig. 2), with peptides containing charge-conserving (Glu) or isosteric (Asn) substitutions cleaved poorly. Caspase-7 also displayed a strong preference for Asp in P4. Like the other caspases, both enzymes tolerated a broad range of amino acids in P3 but preferred Glu, again consistent with the structure of caspase-3 bound to Ac-DEVD-CHO (55). In P2, caspase-3 and -7 were also similar to other caspases in their preferences for hydrophobic residues, but for these enzymes the preference was stronger. The favorable interaction of caspase-3 for Met in P2 probably contributes to its potent inhibition by p35, which inactivates caspase-3 by binding at a site containing DQMD (30, 56).

Consistent with the peptide cleavage assay results, the preferred chromogenic substrate for caspase-7 was Ac-DEVD-pNA (Table II). Caspase-3 also efficiently cleaved this peptide, and, as expected, cleaved the p35-based substrate Ac-DQMD-pNA with greater efficiency. The corresponding fluorogenic peptide Ac-DEVD-Amc displayed Km values of 10 and 11 µM for caspase-3 and -7, respectively. With this substrate, Ac-DEVD-CHO inhibited both enzymes potently, with apparent Ki values of 0.2 and 0.3 nM, respectively. The caspase-2-designed inhibitor Ac-VDVAD-CHO also potently inhibited caspase-3 and -7, with apparent Ki values of 1.0 and 7.5 nM, respectively.

Caspase-6 Specificity

Sequence variants of a peptide based on the caspase-6 cleavage site in lamin A (34, 36) were used in a limited study of the substrate preference of caspase-6 (Fig. 3). Supporting its proposed role in cleavage of lamin A at VEID230N during apoptosis, the results show a favorable interaction of caspase-6 with peptides containing Val in P4. The similar preference for Thr in P4 suggests that, for this enzyme, it is not necessarily hydrophobics that are preferred in P4, but rather the beta -branched amino acids. Unexpectedly, the observed preference in peptide substrates for Gln and Val in P3 and P2, respectively (Fig. 3), was not reflected in the corresponding chromogenic peptides, where Ac-VEID-pNA displayed a kcat/Km value more than 10 times that of Ac-VQVD-pNA (Table II).


Fig. 3. Peptide substrate preferences of caspase-6. Substrate cleavage by caspase-6 was measured using variants of Ac-LEVDGW-Am. Relative (Vmax/Km) values are normalized to 1.00 for the results obtained with Ac-VEVDGW-Am.
[View Larger Version of this Image (16K GIF file)]



DISCUSSION

We are interested in the development of caspase-1 inhibitors as anti-inflammatory drugs. We are studying the biological roles of caspases to predict the consequences of cross-inhibition by our compounds and to reveal new promising drug discovery targets. The present study resulted in new research tools with which to probe the structure and function of caspases, explanations for observed activities toward specific natural substrates, and information that can be used to predict activation pathways and which enzyme(s) are likely or not to cleave newly discovered substrates.

Consistent with previous studies, we find the most significant differences in caspase specificities at the P4 positions. P3 specificities are similar between these enzymes, and in P2 a wide range of amino acids is tolerated. These observations are consistent with the structures of caspase-1 and -3 (55, 57, 58), which show that the P3 and P2 side chains of peptidic inhibitors are relatively solvent-exposed, and that the P4 side chains occupy defined pockets that vary significantly between those enzymes. The P4 preferences can be categorized as hydrophobic (caspase-1, -4, and -6) or Asp (caspase-2, -3, and -7). This categorization is at odds with sequence alignment (3), which predicts that caspase-6 is more closely related to caspase-3 and -7 than to caspase-1, and that caspase-2 is more distantly related. The caspase-3 structure (55) shows that the P4 Asp side chain of a cocrystallized peptidic inhibitor makes polar contacts with the backbone amide nitrogen of Phe-250 (referred to as Phe-381B in Ref. 55 by alignment with caspase-1) and the Ndelta 2 of Asn-208 (referred to as Asn-342). By alignment, the analogous residue in caspase-6 is Glu-221, which might make an unfavorable ionic interaction with Asp in P4 of a substrate.

Caspase-1 and -4 display very similar specificities in each substrate position including P1', and preliminary results suggest that caspase-5 is also similar.2 The P4 specificity of caspase-1 suits it to activation of pro-IL-1beta , and the defect in pro-IL-1beta maturation in caspase-1-deficient mice (20, 21) suggests that little or no pro-IL-1beta is matured by other proteases. This does not rule out a requirement for a second protease in IL-1beta maturation, for example by regulating maturation of caspase-1. Murine Ich-3 stimulates pro-IL-1beta maturation by ICE (59), and caspase-4 and -5, for which clear roles have not been established, may function analogously. The P4 hydrophobic preference of caspase-4 is consistent with activation of caspase-1, where cleavages occur at AVQD119N, WFKD297S, and FEDD316A. Although caspase-1 is capable of autoactivation in vitro (60), it exists largely as its inactive precursor in vivo (61) and therefore may require activation by another enzyme in cells. Since caspase-1 knockout mice display normal apoptosis, the transient caspase-1-like activity (defined as cleavage activity toward a P4 Tyr-containing peptide substrate), that precedes caspase-3-like activity and is required for Fas-induced apoptosis in mouse W4 cells (42) might include caspase-4 or -5.

The primary specificity of caspase-6 suits it well to cleavage of one known natural substrate, lamin A (34, 36). The caspase-6 preference for the beta -branched amino acids in P4 distinguishes it from caspase-1 and -4 and suggests cleavage of other substrates with sites containing beta -branched P4 residues, including maturation sites within caspase-6 (19) (TETD23A and TEVD193A) and caspase-3 (IETD175S). The latter is consistent with proposals based on cotransfection (43) and in vitro maturation experiments (19, 43). Intolerance for P4 Tyr (Fig. 3) would prevent caspase-6 maturation of IL-1beta .

Caspase-2, -3, and -7 each display similar specificities, which suggests that their roles in cells, if not completely redundant, are at least overlapping. They share a strong requirement for Asp in P4, which qualifies them for cleavage of several known P4 Asp-containing apoptosis substrates. The relationships between these enzymes are largely unknown. In one model system, caspase-3 is required for apoptosis (6), but other enzymes may participate in other tissues or after different apoptotic stimuli. Despite the in vitro cleavage of a peptide spanning a known maturation site of caspase-6, the intact protein is not a caspase-2 substrate,3 demonstrating that the structural context of a protein sequence also contributes to recognition as a caspase substrate. Notably, the caspase-3-like proteolytic activity often measured in apoptotic cells is usually defined by cleavage of Ac-DEVD-pNA or similar substrates. Caspase-2, if active, would not be observed using that substrate, but could be observed along with caspase-3 and -7 by using Ac-VDVAD-pNA.

The design of efficiently recognized novel peptidic substrates and inhibitors based on in vitro specificity studies was straightforward and successful, and several such compounds are described here. However, optimized peptides were recognized similarly by enzymes with similar specificities, limiting their usefulness as specific in vivo probes of these enzymes. The requirement of caspase-2 for a P5 residue allows for inhibitors and substrates (such as Ac-DEVD-CHO and Ac-DEVD-pNA) that are not well recognized by caspase-2. Unfortunately the presence of the P5 residue, while not required, was not detrimental to recognition by other enzymes such as caspase-3 and -7. It may be possible to design new peptidic probes that display greater specificity by using residues that are suboptimal for target enzymes and not tolerated by others.

The caspase proteases have overlapping substrate specificities that suggest at least partially overlapping functions. Despite evidence for a central role for caspase-3 in apoptosis, mice deficient in this enzyme display a defect in apoptosis that is limited to the brain (32), suggesting that in most tissues other enzymes such as caspase-1 and -7 function redundantly with it. Partial functional overlap may allow cells to modulate the degree or nature of the response to different proinflammatory or proapoptotic stimuli by differential regulation of the caspases. Alternatively, functional overlap might simply provide backup mechanisms for critical processes such as viral or tumor clearance. Some caspase functions suggested by in vitro studies may not occur because of tissue or subcellular compartmentalization or regulation of activation or expression during cell cycle or development. It should not be surprising that the systems that execute key steps in the processes of cytokine maturation and apoptosis are capable of both fine regulation and redundant function.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: BASF Bioresearch Corp., 100 Research Dr., Worcester, MA 01605. Tel.: 508-849-2581; Fax: 508-754-7784; E-mail: talanian{at}biovax.dnet.basf-ag.de.
1   The abbreviations used are: ICE, interleukin-1beta converting enzyme; IL-1beta , interleukin-1beta ; HPLC, high performance liquid chromatography; pNA, p-nitroanilide; Amc, aminomethylcoumarin; Ac, acetyl; Am, amide; CHO, aldehyde.
2   R. V. Talanian and C. Quinlan, unpublished results.
3   T. Ghayur, unpublished results.

REFERENCES

  1. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774 [CrossRef][Medline] [Order article via Infotrieve]
  2. Cerretti, D. P., Kozlosky, C. J., Mosley, B., Nelson, N., Van, N. K., Greenstreet, T. A., March, C. J., Kronheim, S. R., Druck, T., Cannizzaro, L. A., Huebner, K., and Black, R. A. (1992) Science 256, 97-100 [Medline] [Order article via Infotrieve]
  3. Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A., Wong, W. W., and Yuan, J. (1996) Cell 87, 171 [Medline] [Order article via Infotrieve]
  4. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994) Cell 78, 739-750 [Medline] [Order article via Infotrieve]
  5. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1994) J. Biol. Chem. 269, 30761-30764 [Abstract/Free Full Text]
  6. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37-43 [CrossRef][Medline] [Order article via Infotrieve]
  7. Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beider, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801-809 [Medline] [Order article via Infotrieve]
  8. Faucheu, C., Diu, A., Chan, A. W. E., Blanchet, A.-M., Miossec, C., Hervé, F., Collard-Dutilleul, V., Gu, Y., Aldape, R. A., Lippke, J. A., Rocher, C., Su, M. S.-S., Livingston, D. J., Hercend, T., and Lalanne, J.-L. (1995) EMBO J. 14, 1914-1922 [Abstract]
  9. Kamens, J., Paskind, M., Hugunin, M., Talanian, R. V., Allen, H., Banach, D., Bump, N., Hackett, M., Johnston, C. G., Li, P., Mankovich, J. A., Terranova, M., and Ghayur, T. (1995) J. Biol. Chem. 270, 15250-15256 [Abstract/Free Full Text]
  10. Munday, N. A., Vaillancourt, J. P., Ali, A., Casano, F. J., Miller, D. K., Molineaux, S. M., Yamin, T.-T., Yu, V. L., and Nicholson, D. W. (1995) J. Biol. Chem. 270, 15870-15876 [Abstract/Free Full Text]
  11. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1995) Cancer Res. 55, 2737-2742 [Abstract]
  12. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., Krebs, J., Fritz, L., Tomaselli, K. J., Wang, L., Yu, Z., Croce, C. M., Salveson, G., Earnshaw, W. C., Litwack, G., and Alnemri, E. S. (1995) Cancer Res. 55, 6045-6052 [Abstract]
  13. Lippke, J. A., Gu, Y., Sarnecki, C., Caron, P. R., and Su, M. S.-S. (1996) J. Biol. Chem. 271, 1825-1828 [Abstract/Free Full Text]
  14. Duan, H., Chinnaiyan, A. M., Hudson, P. L., Wing, J. P., He, W.-W., and Dixit, V. M. (1996) J. Biol. Chem. 271, 1621-1625 [Abstract/Free Full Text]
  15. Muzio, M., Chinnaiyan, A. M., Kischel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827 [Medline] [Order article via Infotrieve]
  16. Boldin, M. P., Varfolomeev, E. E., Pancer, Z., Mett, I. L., Camonis, J. H., and Wallach, D. (1995) J. Biol. Chem. 270, 7795-7798 [Abstract/Free Full Text]
  17. Fernandes-Alnemri, T., Armstrong, R. C., Krebs, J., Srinivasula, S. M., Wang, L., Bullrich, F., Fritz, L. C., Trapani, J. A., Tomaselli, K. J., Litwack, G., and Alnemri, E. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7464-7469 [Abstract/Free Full Text]
  18. Duan, H., Orth, K., Chinnaiyan, A. M., Poirier, G. G., Froelich, C. J., He, W.-W., and Dixit, V. M. (1996) J. Biol. Chem. 271, 16720-16724 [Abstract/Free Full Text]
  19. Srinivasula, S. M., Fernandes-Alnemri, T., Zangrilli, J., Robertson, N., Armstrong, R. C., Wang, L., Trapani, J. A., Tomaselli, K. J., Litwack, G., and Alnemri, E. S. (1996) J. Biol. Chem. 271, 27099-27106 [Abstract/Free Full Text]
  20. Li, P., Allen, H., Banerjee, S., Franklin, S., Herzog, L., Johnston, C., McDowell, J., Paskind, M., Rodman, L., Salfeld, J., Towne, E., Tracey, D., Wardwell, S., Wei, F.-Y., Wong, W., Kamen, R., and Seshadri, T. (1995) Cell 80, 401-411 [Medline] [Order article via Infotrieve]
  21. Kuida, K., Lippke, J. A., Ku, G., Harding, M. W., Livingston, D. J., Su, M. S.-S., and Flavell, R. A. (1995) Science 267, 2000-2003 [Medline] [Order article via Infotrieve]
  22. Fletcher, D. S., Agarwal, L., Chapman, K. T., Chin, J., Egger, L. A., Limjuco, G., Luell, S., MacIntyre, D. E., Peterson, E. P., Thornberry, N. A., and Kostura, M. J. (1995) J. Interferon Cytokine Res. 15, 243-248 [Medline] [Order article via Infotrieve]
  23. Miller, B. E., Krasney, P. A., Gauvin, D. M., Holbrook, K. B., Koonz, D. J., Abruzzese, R. V., Miller, R. E., Pagani, K. A., Dolle, R. E., Ator, M. A., and Gilman, S. C. (1995) J. Immunol. 154, 1331-1338 [Abstract/Free Full Text]
  24. Elford, P. R., Heng, R., Révészn, L., and MacKenzie, A. R. (1995) Br. J. Pharmacol. 115, 601-606 [Abstract]
  25. Ku, G., Faust, T., Lauffer, L. L., Livingston, D. J., and Harding, M. W. (1996) Cytokine 8, 377-386 [CrossRef][Medline] [Order article via Infotrieve]
  26. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell 75, 641-652 [Medline] [Order article via Infotrieve]
  27. Hugunin, M., Quintal, L. J., Mankovich, J. A., and Ghayur, T. (1996) J. Biol. Chem. 271, 3517-3522 [Abstract/Free Full Text]
  28. Xue, D., Shaham, S., and Horvitz, H. R. (1996) Genes Dev. 10, 1073-1083 [Abstract]
  29. Gagliardini, V., Fernandez, P. A., Lee, R. K., Drexler, H. C., Rotello, R. J., Fishman, M. C., and Yuan, J. (1994) Science 263, 826-828 [Medline] [Order article via Infotrieve]
  30. Bump, N. J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T., Li, P., Licari, P., Mankovich, J., Shi, L., Greenberg, A. H., Miller, L. K., and Wong, W. W. (1995) Science 269, 1885-1888 [Medline] [Order article via Infotrieve]
  31. Chen, Y., Smith, M. R., Thirumalai, K., and Zychlinsky, A. (1996) EMBO J. 15, 3853-3860 [Abstract]
  32. Kuida, K., Zheng, T. S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P., and Flavell, R. A. (1996) Nature 384, 368-372 [CrossRef][Medline] [Order article via Infotrieve]
  33. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347 [CrossRef][Medline] [Order article via Infotrieve]
  34. Orth, K., Chinnaiyan, A. M., Garg, M., Froelich, C. J., and Dixit, V. M. (1996) J. Biol. Chem. 271, 16443-16446 [Abstract/Free Full Text]
  35. Gu, Y., Sarnecki, C., Aldape, R. A., Livingston, D. J., and Su, M. S.-S. (1995) J. Biol. Chem. 270, 18715-18718 [Abstract/Free Full Text]
  36. Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri, T., Litwack, G., Moir, R. D., Goldmen, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8395-8400 [Abstract/Free Full Text]
  37. Song, Q., Lees-Miller, S. P., Kumar, S., Zhang, N., Chan, D. W., Smith, G. C. M., Jackson, S. P., Alnemri, E. S., Litwack, G., Khanna, K. K., and Lavin, M. F. (1996) EMBO J. 15, 3238-3246 [Abstract]
  38. Song, Q., Burrows, S. R., Smith, G., Lees-Miller, S. P., Kumar, S., Chan, D. W., Trapani, J. A., Alnemri, E., Litwack, G., Lu, H., Moss, D. J., Jackson, S., and Lavin, M. F. (1996) J. Exp. Med. 184, 619-626 [Abstract]
  39. Wang, X., Pai, J., Wiedenfeld, E. A., Medina, J. C., Slaughter, C. A., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 18044-18050 [Abstract/Free Full Text]
  40. Brancolini, C., Benedetti, M., and Schneider, C. (1995) EMBO J. 14, 5179-5190 [Abstract]
  41. Emoto, Y., Manome, Y., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W. W., Kamen, R., Weichselbaum, R., and Kufe, D. (1995) EMBO J. 14, 6148-6156 [Abstract]
  42. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996) Nature 380, 723-726 [CrossRef][Medline] [Order article via Infotrieve]
  43. Orth, K., O'Rourke, K., Salvesen, G. S., and Dixit, V. M. (1996) J. Biol. Chem. 271, 20977-20980 [Abstract/Free Full Text]
  44. Talanian, R. V., Dang, L. C., Ferenz, C. R., Hackett, M. C., Mankovich, J. A., Welch, J. P., Wong, W. W., and Brady, K. D. (1996) J. Biol. Chem. 271, 21853-21858 [Abstract/Free Full Text]
  45. Edelhoch, H. (1967) Biochemistry 6, 1948-1954 [Medline] [Order article via Infotrieve]
  46. Lottenberg, R., and Jackson, C. M. (1983) Biochim. Biophys. Acta 742, 558-564 [Medline] [Order article via Infotrieve]
  47. Dang, L. C., Talanian, R. V., Banach, D., Hackett, M. H., Gilmore, J. L., Hays, S. J., Mankovich, J. A., and Brady, K. D. (1996) Biochemistry 35, 14910-14916 [CrossRef][Medline] [Order article via Infotrieve]
  48. Amrein, K. E., Takas, B., Stieger, M., Molnos, J., Flint, N. A., and Burn, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1048-1052 [Abstract]
  49. Chang, A. C. Y., and Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156 [Medline] [Order article via Infotrieve]
  50. Howard, A. D., Kostura, M. J., Thornberry, N., Ding, G. J., Limjuco, G., Weidner, J., Salley, J. P., Hogquist, K. A., Chaplin, D. D., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1991) J. Immunol. 147, 2964-2969 [Abstract/Free Full Text]
  51. Sleath, P. R., Hendrickson, R. C., Kronheim, S. R., March, C. J., and Black, R. A. (1990) J. Biol. Chem. 265, 14526-14528 [Abstract/Free Full Text]
  52. Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd Ed., W. H. Freeman & Co., New York
  53. Black, R. A., Kronheim, S. A., and Sleath, P. R. (1989) FEBS Lett. 247, 386-390 [CrossRef][Medline] [Order article via Infotrieve]
  54. Kostura, M. J., Tocci, M. J., Limjuco, G., Chin, J., Cameron, P., Hillman, A. G., Chartrain, N. A., and Schmidt, J. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5227-5231 [Abstract]
  55. Rotonda, J., Nicholson, D. W., Fazil, K. M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E. P., Rasper, D. M., Ruel, R., Vaillancourt, J. P., Thornberry, N. A., and Becker, J. W. (1996) Nature Struct. Biol. 3, 619-625 [Medline] [Order article via Infotrieve]
  56. Xue, D., and Horvitz, R. (1995) Nature 377, 248-251 [CrossRef][Medline] [Order article via Infotrieve]
  57. Wilson, K. P., Black, J. A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275 [CrossRef][Medline] [Order article via Infotrieve]
  58. Walker, N. P., Talanian, R. V., Brady, K. D., Dang, L. C., Bump, N. J., Ferenz, C. R., Franklin, S., Ghayur, T., Hackett, M. C., Hammill, L. D., Herzog, L., Hugunin, M., Houy, W., Mankovich, J. A., McGuiness, L., Orlewicz, E., Paskind, M., Pratt, C. A., Reis, P., Summani, A., Terranova, M., Welch, J. P., Xiong, L., Möller, A., Tracey, D. E., Kamen, R., and Wong, W. W. (1994) Cell 78, 343-352 [Medline] [Order article via Infotrieve]
  59. Wang, S., Miura, M., Jung, Y., Zhu, H., Gagliardini, V., Shi, L., Greenberg, A. H., and Yuan, J. (1996) J. Biol. Chem. 271, 20580-20587 [Abstract/Free Full Text]
  60. Malinowski, J. J., Grasberger, B. L., Trakshell, G., Huston, E. E., Helaszek, C. T., Smalwood, A. M., Ator, M. A., Banks, T. M., Brake, P. G., Ciccarelli, R. B., Jones, B. N., Koehn, J. A., Kratz, D., Lundberg, N., Stams, T., Rubin, B., Alexander, R. S., and Stevis, P. E. (1995) Protein Sci. 4, 2149-2155 [Abstract/Free Full Text]
  61. Ayala, J. M., Yamin, T.-T., Egger, L. A., Chin, J., Kostura, M. J., and Miller, D. K. (1994) J. Immunol. 153, 2592-2599 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.