A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B

There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 andCaenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal. There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 andCaenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal. Apoptosis is a form of cell death that is essential for morphogenesis, tissue homeostasis, and host defense (for review see Ref. 1Ellis R.E. Yuan J. Horvitz H.R. Annu. Rev. Cell Biol. 1991; 7: 663-698Crossref PubMed Scopus (2019) Google Scholar). There is accumulating evidence that defects in apoptosis may lead to several pathologies including some neurodegenerative disorders, ischemic injury, and cancer (2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6244) Google Scholar). The discovery that CED-3, the product of a gene necessary for programmed cell death in the nematodeCaenorhabditis elegans, is related to the cysteine protease interleukin-1β converting enzyme (ICE, caspase-1) 1The abbreviations used are: ICE, interleukin-1β converting enzyme; PS-SCL, positional scanning synthetic combinatorial library; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; AMC, aminomethylcoumarin. established proteases to be key mediators in this process (3Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2301) Google Scholar). Although the precise biochemical pathways involved in mammalian cell death remain ill-defined, it is now clear that proteases play an essential role both in the initial signaling events and in the downstream processes that result in the apoptotic phenotype. Those that are known to be involved include members of the ICE/CED-3 or caspase (4Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2160) Google Scholar) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease, granzyme B. The identification of potential endogenous substrates has provided important clues to their molecular role(s); however, the identities of the enzymes involved and their relationships to each other remain obscure. To better understand the roles of proteases in apoptosis, to identify appropriate fluorogenic substrates, and to facilitate inhibitor design, peptide substrate specificities for the caspases and granzyme B were determined using a positional scanning synthetic combinatorial library (PS-SCL) (5Pinilla C. Appel J.R. Blanc P. Houghten R.A. Biotechniques. 1992; 13: 901-905PubMed Google Scholar). The results from this study establish new functional relationships between these important biological mediators. The method used for production of caspases 1, 2, 3, 4, 5, 7, 8, and 9 involves folding of active enzymes from their constituent large and small subunits, which are expressed separately in Escherichia coli. The details of the methods used for preparation of caspases 1 and 3 are described elsewhere (6Rano T.A. Timkey T. Peterson E.P. Rotonda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 7Rotonda 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. Becker J.W. Nat. Struct. Biol. 1996; 3: 619-625Crossref PubMed Scopus (403) Google Scholar). The other homologs were engineered in a similar manner by polymerase chain reaction-directed template modification to generate the following constructs: caspase-2 as a MetAsn150-Asp316 large subunit with a MetSerGly332-Thr435 small subunit; caspase-4 as a MetSerGly95-Asp270 large subunit with a MetSerVal291-Asn377 small subunit; caspase-5 as a MetSerGly136-Asp311 large subunit with a MetSer331-Asn418 small subunit; caspase-6 as a MetSerAla34-Asp179 large subunit with a MetSerGln181-Asn293 small subunit; caspase-7 as a MetSerLys25-Asp198 large subunit with a MetSer199-Gln303 small subunit; caspase-8 as a MetSer217-Asp374 large subunit with a MetSer375-Asp479 small subunit; and caspase-9 as a MetSerGly140-Asp305 large subunit with a MetSerIle332-Ser416 small subunit. To obtain active enzyme, the individual subunits from purified inclusion bodies were solubilized in 6 m guanidine HCl and then rapidly diluted to a final concentration of 100 μg/ml at room temperature under conditions determined to be optimal for each enzyme. Caspase-2 was folded in 100 mm Hepes, 10 mm DTT, pH 7.5. Caspase-4 and caspase-5 were folded in 100 mm Hepes, 10% sucrose, 1% Triton X-100, 10 mm DTT, pH 7.5. Caspase-7 and caspase-8 were folded in 100 mm Hepes, 10% sucrose, 10 mm DTT, pH 7.5. Caspase-9 was folded in 100 mmHepes, 20% sucrose, 1% Triton X-100, 10 mm DTT, pH 7.5. Active, recombinant CED-3 and caspase-6 were prepared by expressing a construct encoding the entire proenzyme, minus the N-terminal peptide, in E. coli, under conditions where a portion of the protein produced is cytosolic and undergoes self-maturation (C. elegans CED-3 was engineered for expression as a MetAla222-Val503 prodomainless construct and caspase-6 as a MetSerPhe25-Asn293 prodomainless construct). To prepare homogeneous, human granzyme B, granules were isolated from cultured human natural killer leukemia YT cells and extracted with NaCl as described previously (8Hanna W.L. Zhang X. Turbov J. Winkler U. Hudig D. Froelich C.J. Protein Expression Purif. 1993; 4: 398-404Crossref PubMed Scopus (67) Google Scholar, 9Quan L.T. Caputo A. Bleackley R.C. Pickup D.J. Salvesen G.S. J. Biol. Chem. 1995; 270: 10377-10379Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) except that calcium was omitted. The resulting extract was diluted then loaded onto a Mono-S cation exchange column (0.5 × 5 cm; Pharmacia Biotech Inc.) that had been pre-equilibrated in 50 mm MES, pH 6.1, 25 mmNaCl. After washing with 12 volumes of equilibration buffer, proteins were eluted with a linear gradient up to 1 m NaCl (in 50 mm MES, pH 6.1) over 40 column volumes. Granzyme B eluted at approximately 0.6 m NaCl and was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. The preparation of the PS-SCL is described elsewhere (6Rano T.A. Timkey T. Peterson E.P. Rotonda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (242) Google Scholar). Each of the 60 samples (20 amino acids × 3 sublibraries) of the PS-SCL was prepared as a stock of approximately 10 mm in Me2SO. To determine protease specificity, enzyme was added to reaction mixtures containing 100 μm substrate mix, 100 mm Hepes, 10 mm DTT, pH 7.5, in a total volume of 100 μl. Under these conditions the final concentration of each individual compound is approximately 0.25 μm. Production of AMC was monitored continuously at room temperature in a Tecan Fluostar 96-well plate reader using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. A PS-SCL with the general structure Ac-X-X-X-Asp-AMC was synthesized to determine the specificities of members of the caspase family and granzyme B (Scheme FS1). The design of this library was based on several catalytic properties of the caspases, including their near absolute specificity for cleavage after aspartic acid and their ability to utilize tetrapeptides terminating in Asp-AMC as efficient fluorogenic substrates. Reports that granzyme B has a similar preference for aspartic acid in P1 (10Shi L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (422) Google Scholar, 11Odake S. Kam C.-M. Narasimhan L. Poe M. Blake J.T. Krahenbuhl O. Tschopp J. Powers J.C. Biochemistry. 1991; 30: 2217-2227Crossref PubMed Scopus (247) Google Scholar) suggested that this library would also be useful for defining the specificity of this enzyme. This PS-SCL is composed of three separate sublibraries of 8,000 compounds each. In each sublibrary, one position is defined with one of 20 amino acids (Scheme FS1, O) (excluding cysteine), whereas the remaining two positions (Scheme FS1, X) contain a mixture of amino acids present in approximately equimolar concentrations. Using this strategy, analysis of the three sublibraries (20 samples each) affords a complete understanding of the amino acid preferences across S2, S3, and S4 subsites. To validate this approach as providing a reliable measure of specificity, we have used it to determine the amino acid preferences of caspase-1/ICE (6Rano T.A. Timkey T. Peterson E.P. Rotonda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (242) Google Scholar). The results of these studies indicated that the preferred recognition motif for ICE is WEHD and led to the synthesis of a fluorogenic substrate, Ac-WEHD-AMC, that is cleaved 50-fold more efficiently than that previously believed to be optimal for this enzyme, with a second order rate constant (k cat/K m) of 3.3 × 106m−1 s−1. The corresponding tetrapeptide aldehyde, Ac-WEHD-CHO, was found to have aK i for inhibition of caspase-1 of 56 pm, making it the most potent reversible, small molecule inhibitor described for any caspases. These results clearly demonstrated that this novel method furnishes an accurate identification of primary specificity that can be exploited to produce efficient substrates and potent inhibitors. We have now extended this approach to determine the substrate specificities for C. elegans CED-3, nine of the ten known human caspase family members and granzyme B (Fig.1). The results obtained with the caspases divide these enzymes into three major groups. Surprisingly, the peptide preferences within each group are remarkably similar. Members of Group I (caspases 1, 4, and 5) all prefer the tetrapeptide sequence WEHD. In contrast, the optimal peptide recognition motif for Group II caspases (2, 3, and 7 and CED-3) is DEXD. The similarity between caspase-3 and caspase-7 is particularly striking; their specificity profiles are virtually indistinguishable. The caspases in Group III (6, 8, and 9) prefer the sequence (L/V)EXD. A comparison of the specificities of these enzymes reveals the following. First, aside from their stringent requirement for Asp in P1, P4 is the most critical determinant of specificity. Members of Group I can accommodate large aromatic/hydrophobic amino acids in this position, whereas those in Group II require Asp for efficient catalysis. The Group III caspases tolerate many different amino acids in P4 but prefer those with larger aliphatic side chains. Second, all of these enzymes prefer, to varying degrees, Glu in P3. Finally, in general, liberal substitutions are tolerated in P2. One notable exception is caspase-9, which has a stringent specificity for His in P2. The structural basis for the distinct specificities of these enzymes can be inferred from the crystal structures of caspases 1 and 3 in complex with tetrapeptide-based inhibitors (7Rotonda 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. 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Interestingly, there are several striking similarities between the specificities of granzyme B and the caspases beyond what appears to be a common requirement for Asp in the P1, including the importance of P4 and a preference for Glu in P3. These results suggest that the active sites in this serine protease and the cysteine proteases of the caspase family may be structurally similar, even though there is no significant overall sequence homology between these proteins. Several lines of evidence illustrate that the specificity observed with tetrapeptide substrates extends to macromolecules. Most compelling is the observation that the optimal tetrapeptide recognition motifs for some of these enzymes are identical or closely related to the sequences found in known macromolecular substrates (Fig. 2). In addition, with caspases 1 and 3, we have found that tetrapeptide substrates are cleaved just as efficiently or better than protein substrates (data not shown). We have also found that incorporation of the optimal tetrapeptide sequence into proteins that are not normally good substrates results in a substantial improvement in the rate of catalysis (data not shown). Clearly, other factors also influence the hydrolysis rate of macromolecules, such as tertiary structure. However, it appears that with these proteases primary sequence recognition is a necessary requirement for catalysis and that the results obtained with the PS-SCL approach accurately reflect macromolecular specificity. Accordingly, an intimate understanding of their specificities provides important insights into their biological functions. First, the observation that closely related caspases have similar and in some cases identical specificities suggests either that these enzymes represent tissue-specific isoforms or that they have related or redundant functions within the same cell type. 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Seshadri T. Cell. 1995; 80: 401-411Abstract Full Text PDF PubMed Scopus (1330) Google Scholar). In conclusion, the results presented here, which precisely define the specificities of the caspases and granzyme B, suggest previously unknown functional relationships between these important biological mediators. This information can now be exploited to produce selective, small molecule inhibitors that will not only help to further define the biological roles of these enzymes but may have clinical utility. Moreover, these specificity “fingerprints” can now be used to identify caspases in different cell types and in other species, leading to additional insights into the biochemical mechanisms that govern apoptosis. We thank Robert W. Myers and Antony Rosen for numerous helpful discussions and for critical evaluations of this manuscript.