Control of Apoptosis by Proteases

Nancy A. Thornberry" Antony Rosent Donald W. Nicholson$ *Department of Biochemistry Merck Research Laboratories Rahway, New jersey 07065 tDepartment of Medicine Johns Hopkins University School of Medicine Baltimore, Maryland 2 I 205 $Department of Biochemistry and Molecular Biology Merck Frosst Centre for Therapeutic Research Pointe Claire-Dorval, Quebec, Canada H9R 4P8

Control of Apoptosis by Proteases

1. Introduction The discovery that CED-3, the product of a gene necessary for programmed cell death in the nematode Cuenorhubditis eleguns, belongs to the interleukin-lp converting enzyme (ICE) family of cysteine proteases has led to intense interest in the role of these enzymes in apoptosis. It is now generally accepted that members of this family do, in fact, play key roles in at least some models of mammalian cell death, although the precise nature of their involvement remains obscure. Other proteases that have been implicated in apoptosis in vertebrates are the serine protease granzyme B and members of the calpain cysteine protease family. These findings have led to an abundance of research aimed at understanding the biochemical role of these enzymes, and their regulation, in the hope that the results of these studies will identify appropriate targets for therapeutic intervention in diseases resulting from inappropriate apoptosis. Advances m Pharmacology, Volume 41 Copyright 0 1997 by Academic Prrss. All rights of reproduction in any form reserved.

1054-3589197 $25.00

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II. ICE/CED-3 Family of Proteases A. Programmed Cell Death in C. elegans Elegant genetic studies in C. elegans (reviewed in the chapter by Desnoyers and Hengartner) have led to important insights into the biochemical mechanisms of cell death in vertebrates. In the nematode, 131 of the 1090 somatic cells formed during development of an adult undergo programmed cell death 111. Two genes have been identified, ced-3 and ced-4, that are required for this cell suicide in that mutations in either of these genes result in the survival of almost all cells that normally die [2]. The product of another nematode gene, ced-9, appears instead to protect cells from undergoing cell death [ 3 ] . The mechanism of cell death in the worm appears to be conserved, at least in part, in vertebrates. The first evidence for this came with the discovery that bcl-2, a gene that is a negative regulator of apoptosis in mammals, prevented cell death when expressed in C. eleguns 141. It was later discovered that the C. eleguns death repressor, ced-9, is in fact homologous to bcl-2 [ 5 ] .At about the same time, it was reported that ced-3, one of the two genes required for cell death in the nematode, is related to the gene encoding mammalian ICE 161. These findings strongly suggested that proteases, in particular cysteine proteases of the ICEICED-3 family, play a principal role in the biochemical events governing apoptosis in both nematodes and mammals. 6. Mammalian Homologs of CED-3

ICE was originally identified as the enzyme responsible for production of interleukin-1 /3 (IL-lP), a key mediator of inflammation. This cytokine

has long been considered an attractive target for therapeutic intervention in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and endotoxic shock. IL-lP is synthesized as a 31-kDa precursor (pro-IL-1/3) that requires proteolytic processing at Asp1 16-Alall7 to generate the 17.5-kDa mature, biologically active molecule. Upon its discovery in 1989, the enzyme responsible for this unusual cleavage (ICE)immediately presented itself as an attractive target for the interdiction of IL-1 activity [7,8]. Recently, mice deficient in this enzyme were shown to be defective in the production of IL-Ip, confirming its essential role in the production of this cytokine [ 9,101. Importantly, these mice are resistant to lipopolysaccharideinduced endotoxic shock, justifying interest in this protease as a potential target in inflammation. The enzyme has been purified and cloned and its structure determined by X-ray diffraction [ll-141. It employs a typical cysteine protease mechanism and has a near absolute requirement for aspartic acid in the PI position

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of both macromolecular and peptide substrates [12,15,16]. The enzyme is composed of subunits of 10 kDa (p10) and 2 0 kDa (p20), both of which are derived from a 45-kDa proenzyme [12]. These subunits are flanked by Asp-X sites, suggesting that activation is mediated by ICE itself or by a protease with similar specificity. In the mature enzyme the two subunits are intimately associated, with both contributing key residues t o the active site to form a heterodimer with a single catalytic domain [13,14]. When ICE was first cloned and sequenced in 1992, it was found to be unrelated to any known protein [11,12]. The subsequent finding that it is related to the product of ced-3 prompted an intense search for other mammalian homologs. To date, 10 family members of human origin have been identified (Fig. 1).A phylogenetic analysis indicates that these proteins fall into two major subfamilies, with ICE and CED-3 as representative examples of each. Members of the ICE subfamily include ICE, ICErel-II(TX and ICH-2), and ICE,,I-III (TY) [11,12,17-201. Family members more similar

“ri-

Subfamily

ICE,,,-III

(N)

ICE,,I-II

(TX. ICH-2)

- 1

ICE

CED-3 Subfami/y

-

ICE-LAP6

(Mch6)

ICH-1

(rnNedd2)

I

Mch4 FLICE

I

-

y

-2 1

10

-1 8

10

=I7

V

(MACH, Mch5)

Mch2

ceCED-3

FIGURE I The ICWCED-3 protease family. Known members of the ICE/CED-3 protease family include the C. eleguns enzyme, ceCED-3, and 10 members of human origin. The proteins can be tentatively grouped into two subfamilies based on their sequence homologies to ICE and CED-3. All family members are synthesized as proenzymes (shown)that are proteolytically processed at Asp-X sites (indicated by arrowheads) to generate the heterodirneric, mature enzymes.

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to CED-3 are ICH-1 (Nedd-2),CP1’32 (apopain, YAMA, and prICE), Mch2,

Mch3 (ICE-LAP3 and CMH-1), MACH (FLICE and MchS), ICE-LAP-6, and Mch4 [21-331. The crystal structure of ICE has defined those residues that are important for binding and catalysis 113,141. A comparison of the primary sequences of all homologs identified to date reveals the following similarities, First, the absolute specificity of ICE for aspartic acid in the PI position of substrates appears to be a distinguishing feature of this protease family in that the four residues forming the S, subsite are strictly conserved (Arg179, Gln283, Arg341, Ser347). Second, the catalytic diad (Cys285 and His237), and those amino acids implicated in stabilization of the tetrahedral reaction intermediates (Gly238, and Cys285), are also conserved, confirming that all family members employ a cysteine protease mechanism. The catalytic Cys285 is located in the middle of a conserved pentapeptide motif (GlnAla-Cys-Xxx-Gly) in what is the most highly homologous region of the proenzyme. Third, the aspartic acid at the C terminus of the 20-kDa subunit of ICE is conserved, suggesting that all family members have heterodimeric catalytic domains. Despite the apparent similarities in cysteine protease mechanism and specificity for Asp in P I , there are also obvious and important differences in the primary sequences of these enzymes. Most notably, the amino acids implicated in binding the P4 amino acid of substrates are not conserved. This residue had earlier been shown to be a critical determinant of specificity for ICE itself, which has a preference for hydrophobic amino acids in this position (121. In contrast, CPP32, one of the proteases most closely related to CED-3, requires aspartic acid in P4 (see below). The differences between the S4 subsites of ICE and CPP32 appear to be responsible almost entirely for their distinct macromolecular specificities and, consequently, functions. This has recently been confirmed with the solution of the X-ray crystal structure for CPP32 [34]. In this regard, members of the CED-3 subfamily (with the exception of ICE-LAP6) are distinguished by the presence of a span of 10 amino acids in the smaller subunit (in CPP32, Phe380-Phe389), which does not have a counterpart in those homologs related to ICE. The crystal structure of CPP32 indicates that these amino acids comprise a loop that plays a crucial role in defining the geometry of S4. Other significant differences between family members are evident in a comparison of their proenzyme structures (Fig. 1). At least five members of the CED-3 subfamily (CPP32 and Mch3) do not appear to have a linker peptide in the proenzyme between the two subunits of the heterodimer. The significance of the linker peptide is currently unknown. In addition, the length of the prodomain is highly variable (Fig. 1).The function of the prodomain has not yet been established for any of the family members; however, one possibility is suggested by the recent findings that the prodomains of MACH and Mch4 are homologous to the death effector domain

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(DED) of FADD, a protein that is one component of the Fas receptor signaling complex [30-321. It has also been shown that MACH binds to the DED of FADD, presumably via interactions between the two homologous domains [30,31]. Thus, in the case of MACH, the prodomain appears to be involved in physically engaging the enzyme to the signaling machinery of the apoptotic cell. C. Evidence for a Role in Mammalian Apoptosis A number of strategies have been employed in attempts to determine whether ICE and/or other mammalian family members play an essential role in cell death, as is suggested by their homology with CED-3. Studies with ICE-deficient mice, antisense constructs, and inhibitors all support the view that at least some of these enzymes, specifically those most closely related to CED-3, play an important role in the biochemical events that govern apoptosis in vertebrates. 1. ICE-Deficient Mice

The results of studies with ICE-deficient mice suggest that this family member is not a key mediator in most models of apoptosis. First, these animals develop normally, indicating that it does not play a role in the apoptosis that occurs during development [9,10]. Second, thymocytes from ICE-deficient mice undergo apoptosis normally in response to dexamethasone or ionizing radiation [9,10], and there is no defect in apoptosis in macrophages treated with ATP [ 101. All these observations argue against a central, nonredundant role for ICE in apoptosis. This conclusion is further supported by the findings that ICE protein is not even present in many cell types that are used in models of apoptosis [24,35]. However, thymocytes from ICE-deficient mice do appear to be resistant to apoptosis induced by anti-Fas antibody [9]. This observation, together with the results described below using inhibitors and ICE antisense, suggests that ICE itself may be involved in Fas-mediated apoptosis. However, the recent report that there is no detectable ICE protein in a Jurkat T cell model of Fas-induced apoptosis suggests that its role is limited to only some T cell types [35]. 2. Overexpression of ICEKED-3 Proteases

All the ICE family members induce apoptosis when overexpressed in healthy cells and, initially, this was viewed as evidence for their role in this process [17-19,21-23,26,27,29,36,37]. It is now clear that the results of such studies are difficult to interpret for the following reasons. Cytoplasmic overexpression of many proteases, including those that are not likely t o be involved in physiological programmed cell death, induces apoptosis [38,391. Moreover, it has been shown recently that ICE itself is a reasonable catalyst

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of cleavage of poly(ADP-ribose) polymerase (PARP),a protein that is cleaved during apoptosis and that is not likely to be a substrate of this family member in vivo [40]. Thus, it is likely that the apoptosis observed upon overexpression of these enzymes is due to proteolytic events that occur only as a result of their high, nonphysiologic concentrations. 3. Inhibition of Apoptosis by Proteare Inhibitors

A number of laboratories have attempted to use both small molecule and macromolecular protease inhibitors to implicate a particular protease or protease family in apoptosis. In general, the results of these investigations strongly implicate ICEKED-3 proteases, particularly those most closely related to CED-3, as key mediators in this process. Many of the early studies employed reagents generally considered to be nonspecific cysteine or serine protease inhibitors, for example, N-tosyl-I.-Phe chloromethyl ketone, Na-p-tosyI-L-Lys chloromethyl ketone, iodoacetate, and N-ethylmaleimide. Because these reagents are extremely nonspecific and highly unstable, the results from such studies are difficult to interpret and often irreproducible. Recently, peptide-based inhibitors designed exclusively for ICE/CED-3 proteases have been used as probes of biological function of these enzymes in cells and cell lysates. The classes of inhibitor employed in these studies include aldehydes, which are reversible inhibitors of cysteine proteases, and a-substituted ketones (monohalomethylketones and acyloxymethylketones), which covalently modify the active site cysteine. The most commonly used inhibitors are summarized in Table I. Peptide recognition sequences in these inhibitors are based on either the cleavage site in pro-IL-lp, PS-YVAD (The actual cleavage site in human pro-IL-lP is YVHD. Alanine has replaced histidine in the P2 position of substrates and inhibitors to facilitate their synthesis), or the cleavage site in PARP, P5-DEVD, a protein that appears to be cleaved by one or more ICE/CED-3 proteases during apoptosis (see below). As summarized in the table, these inhibitors have a broad range of potencies and specificities for the two best characterized members of this family, ICE and CPP32. For example, Ac-YVAD-CHO is a potent, selective inhibitor of ICE itself (K, = 0.76 nM) and a weak inhibitor of CPP32

TABLE I Selectivity of Inhibitors for ICE and CPP32

ICE CPP32 Selectivity

0.76 10,000 13,000-fold

17.0 0.35 49-fold

2.8 X l o s 4.1 x 103 68-fold

c0.004

>loo >25,000-f0ld

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161

( K , = 10,000 n M ) [12,24]. In contrast, Ac-DEVD-CHO is a relatively potent inhibitor of both ICE and CPP32, with a 49-fold preference for CPP32 ( K ,Cpp32 = 0.35 nM; K, ICE = 17 n M ) (N. Thornberry, unpublished results). The difference in the selectivity of the two inhibitors reveals a fundamental difference between the two enzymes. For both, P4 is a critical determinant of specificity. However, the macromolecular substrate specificity of CPP32 (described below) suggests that this enzyme has a strict requirement for Asp in this position, explaining the poor performance of YVAD-based inhibitors. In contrast, ICE is more promiscuous; it prefers hydrophobic amino acids in P4 but can tolerate others (including Asp) [41]. As noted previously, this difference in specificity is consistent with the observation that residues implicated by the ICE crystal structure in the S4 subsite are not conserved between these enzymes. Not surprisingly, the monofluoroketone, Z-VAD-CH2-F, which lacks a P4 amino acid, is a respectable irreversible inhibitor of both enzymes, with a 68-fold preference for ICE (k,,,,JK, ICE = 2.8 X 10' M-ls-'; k,,,,,/K, cpp32 = 4.1 X lo3M-'s-' 1 (N. Thornberry, unpublished results). Irreversible inhibitors containing only the PI aspartic acid (Boc-Asp-CH2-C11421 and Z-Asp-CH2-OCO-2,6,-dichlorobenzene [43] have been prepared and are anticipated to be nonselective irreversible inhibitors of all ICE/CED-3 family members (and possibly granzyme B). Inhibition of cell death by these compounds has been demonstrated in both whole cell and in vitro models of apoptosis. In intact cells, inhibition of apoptosis triggered by diverse stimuli in a variety of cell types has been achieved with several of the peptide-based inhibitors [24,35,44-5 11. Without exception, relatively high levels of inhibitor (>1p M ) were required for efficacy in these models. This is probably due, at least in part, to poor cell penetration and instability. In permeabilized cells, potent inhibition (ICj0 < 5 n M ) of Fas-mediated cell death by the ICE inhibitor Ac-YVADCHO was observed [52]. This result is consistent with the finding that ICEdeficient mice have a defect in at least some models of Fas-mediated apoptosis. T o overcome the problem of poor cell penetration in whole cells, an increasing number of investigators are testing these inhibitors in in vitro models of apoptosis (for review, see the chapter by Takahashi and Earnshaw). The results of several studies demonstrate that ICEKED-3 protease inhibitors prevent the morphological changes in the nucleus that are characteristic of apoptosis [24,35,53-551. Studies comparing the effects of YVADand DEVD-containing inhibitors have been particularly enlightening [24,35]. In these experiments, Ac-DEVD-CHO was shown to be 1000-fold more effective than Ac-YVAD-CHO in preventing nuclear apoptosis. These results suggested that (i) CPP32, or a closely related homolog, is required for apoptosis in these models, and (ii) ICE itself is not involved. Two macromolecular inhibitors of ICE/CED-3 proteases have been described and shown to be negative effectors of apoptosis. Cytokine response

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modifier A (CrmA) is a 38-kDa protein from cowpox virus that is a member of the serpin superfaniily [56]. As shown in Table I, CrmA is a potent ICE inhibitor ( K , = 4 pM; k,, = 1.7 X lO'M-ls-' ) and a weak inhibitor of CPP32 ( K , > 100 nM; k,, < 1 X l o 4 M-'s-' ) [24, 571. CrmA is also a relatively potent inhibitor of granzyme B (k,,,= 2.9 X los M-'s-' ~ 1 Microinjection of the cowpox serpin, CrmA, prevents apoptosis in chick dorsal root ganglion neurons deprived of nerve growth factor [59]. Similarly, overexpression of this viral gene product inhibits both Fas- and tumor necrosis factor-mediated apoptosis [52,60,61]. Because the concentrations of CrmA achieved in these experiments are likely to be high (>1 wM), any of the ICE/CED-3 homologs and/or granzyme B, and perhaps other proteases, are potential targets of CrmA in these models. Thus, although these data are consistent with a role for ICE/CED-3 proteases in apoptosis, they are not conclusive. Moreover, until it is established that the concentrations of CrmA used in these studies are similar to those attained during viral infection, it is premature to conclude that CrmA functions as a regulator of apoptosis in vivo. Another macromolecular inhibitor of ICEICED-3 family proteases is a 35-kDa protein from baculovirus (p35) [62]. This protein is an irreversible inhibitor of several family members, including ICE, ICH-1, ICEre,-II,CPP32, and CED-3 [63,64]. The potency and selectivity of inactivation of these enzymes by p35 have yet to be reported. Unlike CrmA, this protein does not appear to be a granzyme B inhibitor. Expression of p35 has been shown to prevent cell death not only in insect cells [62,65,66], but also in C. eleguns [67], and mammalian systems of apoptosis [68-701. Although the target of p35 inhibition in these models has yet to be definitively established, these results are consistent with a key, evolutionarily conserved role for at least some of these cysteine proteases in cell death. 4. Expression of Antisense Constructs

The small molecule and macromolecular inhibitors described are useful for defining a role for proteases of the ICEICED-3 family in models of apoptosis, but they clearly have limited utility as probes of biological function for a particular family member. An alternative approach involves expression of antisense constructs in models of apoptosis. To date, experiments have been reported using antisense for both ICE and Nedd2. An antisense ICE construct was found to reduce Fas/APO-1-mediated apoptosis 1521. Similarly, expression of Nedd2 antisense mRNA in a factor-dependent hematopoietic cell line (FDC-P1) significantly inhibited apoptosis induced by removal of cytokines [71]. These results are consistent with a role for ICE and Nedd2 in apoptosis, with the caveat that the full-length constructs employed in these studies may have also eliminated closely related homologs of these enzymes.

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D. Substrates for ICEICED-3 Proteases Taken together, the studies described in the previous section have established a prominent role for members of the ICEKED-3 family in apoptosis. Definitive identification of the particular homologs that are essential for cell death, and a precise understanding of their biochemical role(s), has been, and will continue to be, facilitated enormously through the identification of proteins that appear to be cleaved by these enzymes during apoptosis. Unfortunately, few systematic methods exist to facilitate such studies. The initial identification of PARP, an enzyme that is involved in genome surveillance and DNA repair [72], as a cleaved substrate in apoptotic cells [73,74] was fortuitous. Similar fortuitous observations have identified retinoblastoma protein [75],adenomatous polyposis coli protein [76],and sterol regulatory element-binding proteins [77] as substrates for ICEKED-3 proteases. Although it is likely that other chance discoveries will define several other proteins as potential substrates for these enzymes during apoptosis, systematic methods will inevitably lead to more rapid progress on this front. 1. ‘Candidate Substrate” Approach

Using this approach, several substrates for the apoptotic proteases have been identified in attempts to explain the observed downstream biochemical and morphologic features that are characteristic of apoptosis. For example, the striking surface blebbing characteristic of the apoptotic cell suggested an alteration in the membrane skeleton and led to the identification of fodrin (nonerythroid spectrin) cleavage as an early proteolytic event in apoptosis [78]. Although fodrin is a putative substrate for calpain, the actual activity responsible for cleaving fodrin during apoptosis has not yet been conclusively demonstrated. Nuclear lamina disassembly is observed during apoptosis, and specific cleavage of the nuclear lamins appears to correlate with this phenomenon [79,80]. Similarly, the cleavage of Gas2 at Asp279 by an as yet unidentified proteolytic activity might explain some of the striking rearrangements of the actin cytoskeleton observed during apoptosis [811. The recent demonstration that actin itself may be a substrate for the ICE family has led to the suggestion that actin cleavage may have several consequences during apoptosis, both on organization of the cytoskeleton and by release of DNAse complexed with actin [82]. Studies exploring the pathways involved in transducing ionizing radiation-induced signals to the nucleus have revealed that protein kinase C6 (PKCS) is activated by proteolytic cleavage during apoptosis [83]. The candidate substrate approach might be fruitfully extended to other phenomena observed in apoptosis, including phosphatidylserine redistribution [84,85], cytosolic shrinkage [86], intracellular acidification [87,88], and nuclear scaffold disassembly [89].

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2. Lupus Autoantibodies Recognize Molecules Specifically Cleaved Early during Apoptosis

Systemic lupus erythematosus (SLE) is a prototype multisystem autoimmune disease that is characterized by a specific and exuberant humoral immune response to self-molecules [90]. A potential mechanism for breaking tolerance to self-antigens that is gaining increasing acceptance is that of exposure of previously cryptic epitopes in self-molecules [91], possibly by proteolysis or high-affinity binding to other molecules (e.g., antibodies and receptors) [92]. SLE is characterized clinically by intermittent exacerbations (flares) during which the immune system appears to be driven by the release of self-antigens, likely from apoptotic cells [93,94]. These autoantigens are clustered at the surface of apoptotic cells [94], leading to the proposal that this “clustered targeting” of unique autoantigens by the immune system might reflect the susceptibility of these particular molecules to proteolysis. The fact that PARP and lamin B, both established autoantigens in SLE 195,961, are known substrates for ICE-like enzymes during apoptosis [24,25,53,80] supports this proposal. The hypothesis is further sustained by the finding that several other autoantigens are united by their specific proteolytic cleavage early during apoptosis 197,981. Other autoantigens that have been identified to date include the 70-kDa subunits of the U1 small ribonucleoprotein (Ul-70kDa) and the catalytic subunit of DNA-dependent protein kinase (DNA-PK,,). Because the cleavage of proteins early during apoptosis is a highly selective process, involving only a very small minority of cellular proteins [98], the early cleavages recognized by these autoantibodies present a unique approach to defining the role of selective proteolysis in the apoptotic mechanism. Identification of the remaining lupus autoantigens is currently under investigation. 3. Cleavage of Potential Substrates by ICEICED-3 family Members

The potential endogenous substrates identified to date, and their respective cleavage sites (where known), are summarized in Table 11. In every case, these macromolecules contain aspartic acid in the PI position, suggesting that a member of the ICE/CED-3 family may be involved. Determining which ICE family member is responsible for physiologic cleavage of a particular substrate must quantitatively address the efficiency of catalysis, rather than merely demonstrate that large amounts of protease are able to cleave those substrates in vitro. Second-order rate constants ( k J K , ) for hydrolysis of PARP, U1-7OkDa, and DNA-PK,, by 0 3 2 have been determined 1991. All three proteins are excellent substrates for this enzyme, with k,,JK, values > 2.3 x lo6M-’s-’. In every case, the products of hydrolysis were derived from cleavage at a D-X-X-D site and appeared to be identical to those seen in apoptotic cells.

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TABLE II Proteins Cleaved during Apoptosis ~

Protein

Cleavage sequence

Poly(ADP-ribose) polymerase (PARP) 70-kDa U1 small ribonucleoprotein (Ul-70kDa) DNA-dependent protein kinase,, (DNA-PK,,) Protein kinase C 6 (PKCG) Lamin A Gas2 fl-Actin a-Fodrin Topoisomerase 1 Adenomatous polyposis coli (APC) Retinoblastoma protein (RB)

DEVD-G DGPD-G DEVD-N DMQD-N VEID-N SRVD-G ELPD-G NA" NA NA NA

NA, not available.

These results suggest that Asp in P4 is a critical determinant of specificity for CPP32. The magnitude of these k,,,/K, values leaves little doubt that these proteins are physiologic substrates for this enzyme. Several other observations support this conclusion. First, purification of the enzyme responsible for specific PARP cleavage from apoptotic cell cytosols resulted in the isolation of CPP32 [24]. Second, the ICSO values for inhibition of cleavage of PARP, U1-7OkDa, and DNA-PK,, by the tetrapeptide aldehyde inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO in extracts from apoptotic cells ( ICsOAc.DEVD.CHO = 0.5 nM; ICSO Ac.YVAD.CHO 2 10 p M ) are virtually identical to those obtained with the purified enzyme (Table I) [99]. Finally, as described previously, Ac-DEVD-CHO is much more effective than Ac-YVAD-CHO in preventing in vitvo apoptosis. Taken together, these results constitute overwhelming evidence that CPP32 (and/or a closely related homolog) is responsible for the cleavage of these proteins observed during apoptosis. Kinetic constants for cleavage of the other putative substrates have not been reported. PKCS is an obvious candidate for cleavage by CPP32 because cleavage occurs at a D-X-X-D site. Conversely, the structural proteins that are not cleaved at this motif (lamins, actin, and Gas2) are probably not physiologic substrates for this enzyme. This is consistent with the findings that these proteins appear to be cleaved later than PARP, DNA-PK,,, U170kDa, and PKCS during apoptosis. Identification of the enzyme(s) responsible for these proteolytic events must await further studies.

E. Proposed Biochemical Role(s) Proteases in Apoptosis

of ICE/CED-3

From the substrates thus far shown to be specifically cleaved during apoptosis it is suggested that protease action may alter the function of two

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groups of proteins: (i) those involved in organization and maintenance of cell structure (e.g., lamins, actin, Gas2, and fodrin) and (ii) catalytic proteins functioning in homeostatic pathways (e.g., PARP, DNA-PK,,, U1-7OkDa, and PKCG). The cleavage of these and other as yet undefined proteins by members of the ICEKED-3 family may be responsible for many of the stereotyped morphologic features that define the apoptotic state and may determine the fate of the apoptotic cell within tissues. The specific proteolytic cleavages and DNA breaks that proceed rapidly during the execution phase of apoptosis imply that macromolecule degradation far outpaces repair [loo]. It is likely that the efficient cleavage of nuclear proteins involved in homeostatic pathways eliminates essential repair functions. The focused crippling of homeostasis by CPP32 (and/or potentially other closely related homolog) may be a fundamental feature ensuring the rapid irreversibility of the apoptotic process. For example, because the machinery to sense and repair DNA damage would counteract the DNA degradation characteristic of apoptosis, the destruction of this machinery would be physiologically essential if the decision to die is to be irreversible and rapidly executed. Mutations in several of the proteins involved in DNA repair (e.g., DNA-PK,, and ATM) have been associated with severe phenotypes and/or susceptibility to malignancy, suggesting that abolition of their function severely impairs DNA repair processes [lOl-1031. Although PARP has also been implicated in DNA repair pathways 1721, its actual function in this process has yet to be defined [104], and the phenotype observed in PARP knockout mice was minimal [105]. This suggests that PARP function in DNA repair is redundant and, hence, that its proteolysis in apoptosis would not exert any functional effect by loss of function alone. The possibility that one of the fragments of PARP generated during apoptosis has a dominant-negative role in abrogating DNA repair has been suggested 11041 but remains to be directly addressed. Overexpression of the C-terminal domain of U1-7OkDa [which contains two arginineherine-rich (SR) regions] has a dominant-negative effect on splicing and transport of mRNA to the cytoplasm [106]. Interestingly, the 22-kDa fragment generated by CPP32 contains one of these SR domains and would likely have a similar effect. Because repair pathways are facilitated by new mRNA synthesis, inhibition of mRNA splicing during apoptosis might impair expression of the homeostatic transcriptional response. The cleavage of nuclear lamins during the later phases of apoptosis might promote nuclear condensation and fragmentation. Because these intermediate filament proteins have a role in nuclear envelope integrity and the organization of interphase chromatin 11071, the altered nuclear structure might also limit those functions (e.g., transcription and mRNA splicing) that are topographically organized. Clearly, identification of additional substrates for the ICEKED-3 proteases will rapidly lead to a greater understanding of the biological roles of these enzymes in apoptosis.

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Recently, compelling evidence has emerged to suggest that ICE/CED-3 proteases play a role not only in the effector phase of apoptosis but also in the upstream signaling events, As described previously, MACH has been shown to be physically associated with the Fas signaling complex via its interaction with the DED of the adaptor protein FADD [30,31]. This finding strongly suggests that MACH participates in the signaling activity of the receptor complex during Fas-mediated cell death and represents the most upstream component in a cascade that serves to amplify the death signal. It is anticipated that Mch4, which also contains a DED in its prodomain, will have an analogous function [32].

111. Other Proteases Implicated in Apoptosis A. Granzyme B

Cytotoxic lymphocytes (CLs) invoke the apoptotic suicide of other cells by two distinct mechanisms that can be distinguished by their dependence on calcium. The calcium-independent (nonsecretory) pathway involves the Fas receptor and its ligand [108-111; see also the chapter by Eischen and Leibson] and appears to involve a cascade of ICEKED-3 cysteine proteases as described previously [30,31,44,52,54,112]. The calcium-dependent (secretory) pathway, on the other hand, utilizes a discrete subset of cytotoxic granule proteins that invade the target cell and trigger apoptosis [113-1161. The proposed model for granule-mediated CL cytotoxicity proceeds via the synergistic contribution of perforin (cytolysin) and a family of serine proteases, the granzymes. Both perforin and the granzymes are contained within cytoplasmic granules. Binding of a CL to an appropriate target cell leads t o a calcium-dependent degranulation process, which results in the discharge of these granule constituents into the intercellular space. The secreted perforin undergoes calcium-dependent polymerization into transmembrane channels that form 10 to 20-nm pores in the target cell membrane. Although there is no direct evidence, these pores are believed to facilitate the entry of the granzymes into the target cell cytoplasm, where they trigger an apoptotic response. The principal granzyme involved in provoking apoptosis appears to be granzyme B (fragmentin 2), which is by far the most abundant of the granule proteases and also the most efficient at initiating a suicide response [ 117,1181. The important roles played by both granzyme B and perforin in granulemediated CL cytotoxicity are supported by several observations. First, cells from mice deficient in granzyme B or perforin are defective in their ability to induce apoptosis in target cells 139,1191. Second, antisense elimination of either granzyme B or perforin from a CL cell line resulted in a dramatic reduction in granule-mediated cytotoxicity [120]. Third, myelocytic leuke-

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mia cells became cytotoxic when transfected with both perforin and granzyme but not with either alone [121]. Finally, granzyme B and perforin induced rapid apoptosis when added exogenously to target cells, but only when both were added together [117,122]. Collectively, these studies support a critical role for both perforin and granzyme B in apoptosis. Granzyme B is a 27-kDa serine protease that is synthesized as a proenzyme in cytotoxic lymphocytes. The mature enzyme is derived from the proenzyme by removal of an N-terminal leader sequence and a putative activation dipeptide [123-125]. Although the specificity of this enzyme is ill-defined, some preliminary mapping has been accomplished using peptide thioester substrates [117,126]. These results suggest that granzyme B has a preference for Asp in the PI position. It is important to note, however, that this is clearly not a stringent requirement because other residues, such as Asn, Met, or Ser, are also reasonably well tolerated in PI. For example, the k J K , values for cleavage of Boc-Ala-Ala-Asp-SBzl (2.3 X l o 5 M - Y ' ) and Boc-Ala-Ala-Asn-SBzl(4.9 X lo4M-ls-' ) differ by only4.7-fold. (In contrast, with ICE, substitution of Asp with Asn in substrates results in >1000-fold Despite this promiscuity, two potential mechanisms for decrease in kLat/Km.) granzyme B-mediated apoptosis are suggested by its modest preference for Asp in PI. One possibility is that granzyme B is responsible for proteolysis of one or more of the proteins that are known to be cleaved during apoptosis. Alternatively, because ICE/CED-3 proteases are all synthesized as dormant proenzymes that are activated by hydrolysis at Asp-X bonds, granzyme B may catalyze the activation of existing proapoptotic ICEICED-3 cysteine proteases. Regarding the latter hypothesis, granzyme B has been shown to process and activate the proapoptotic CED-3-related protease CPP32 in vitro by catalyzing the proteolytic separation of the large (p17) and small (p12) subunits [127,128]. Recently, granzyme B has also been shown to catalyze the in vitro activation of several other members of the ICE/CED-3 protease family [28,32,33,129-1311. In both intact cells and a reconstituted in vitro apoptosis system, this granzyme B-mediated activation resulted in a pattern of subsequent cleavage events that were indistinguishable from those attributable to the activity of ICE/CED-3 proteases, including the specific breakdown of PARP, a-fodrin, U1-70kDa, snRNP, and lamins. The inhibition profile of these downstream cleavage events is consistent with the involvement of CPP32 or a closely related homolog, suggesting that the principal role of granzyme B is to activate ICEKED-3 proteases.

B. Calpains The calpains are a family of highly related calcium-activated neutral endopeptidases that are implicated in membrane and cytoskeletal modification [132-1341. Although a role for calpains in apoptosis has not been

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firmly established, there is a considerable body of circumstantial evidence to indicate that these proteases might somehow be involved. First, the dysregulation of calcium homeostasis is a prominent feature of many cells undergoing apoptosis, and Ca2+fluxes have been shown to result in calpain activation. Second, many human diseases in which inappropriate apoptosis is prominent have also been linked with calpain overactivation, including ischemic brain and cardiovascular damage, Alzheimer’s disease, and other neurodegenerative disorders [132-1361. Third, putative substrates for calpain include polypeptides that are known to be cleaved during apoptosis, such as a-fodrin [137-1391. Finally, inhibitors of calpain can prevent apoptosis in thymocytes [140]. Each heterodimeric calpain isoform is composed of a unique large subunit (=SO kDa) that contains a papain-like cysteine protease domain and a common small subunit ( ~ 3 kDa) 0 that is shared by all the known calpain family members. The C terminus of both subunits contains a calmodulin-like domain that includes several EF-hand Ca2+binding sites, which presumably mediate calcium-dependent activation of the protease [141]. The N terminus of each subunit, on the other hand, contains a short peptide that is proteolytically removed by an apparently autolytic mechanism following activation of the enzyme in response to the presence of appropriate concentrations of calcium. During or following activation, calpains translocate from the cytosol to the membrane fraction, where their proteolytic targets are presumed to reside. Several proteins have been proposed to be endogenous substrates for the calpains based on cleavages that occur in vitro. These putative substrates include cytoskeletal proteins (e.g., fodrin and MAP2), transcription factors (e.g., Fos and Jun), signaling enzymes (e.g., protein kinase C and calcineurin), and many others [132]. Although there is no compelling evidence to suggest that these cleavages are physiologically relevant, these and other studies have helped define the general substrate requirements for calpain [142,143], which appears to prefer amino acids with large aliphatic or aromatic side chains in the P3, P2, and PI positions and a basic or large aliphatic residue in PI’. These proteases also have a bias for Leu or Val in Pz. Calpain has been proposed to be the enzyme responsible for the proteolytic maturation on interleukin-la, suggesting an intriguing link between calpains and ICE-like proteases [144,145]. At least two of the proposed substrates for the calpains, a-fodrin and PKC, are cleaved during apoptosis, and this is viewed as evidence for the role of these enzymes in cell death. The cleavage of a-fodrin by purified calpain I (p-calpain) [ 137,1461 resulted in fragments that are indistinguishable in size from those that were observed in vivo in apoptotic cells or in ischemic tissue [78,1381. Recently, however, a-fodrin cleavage in apoptotic extracts has been shown to be prevented by the tetrapeptide aldehyde, AcDEVD-CHO, suggesting that either calpain activation is downstream of and dependent on an ICEICED-3-like proteases or that an ICEICED-3-like

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protease is, in fact, responsible for a-fodrin cleavage during apoptosis [128]. The latter is certainly possible, especially given that within nine residues of the in vitro calpain cleavage site there is a consensus sequence for CPP32 recognition ( DETD1'8S-S"86)that would also result in a-fodrin fragments that correspond in size to those observed in viva PKC is another putative calpain substrate. Several isozymes have been shown to be cleaved by both calpains I and I1 in vitro, resulting in PKC activation (1471. Calpain-mediated PKC cleavage occurs in the V3 region that separates the PKC regulatory domain from its catalytic domain. In apoptotic U937 cells, only the S isoform of PKC is proteolytically activated; this cleavage, which also occurs in the V3 region (at DMQD330-N331), is attributable to an ICE/CED-3-like proteolytic activity [ 831. Taken together, the evidence so far fails to establish a definitive role for the calpains in apoptosis.

IV. Proteases as Therapeutic Targets in Apoptosis The results described in this review confirm an important role for proteases in mammalian cell death. The available evidence indicates that ICE/ CED-3 proteases are essential components of a common cell death mechanism that is triggered in response to diverse stimuli. The identification of some of the potential victims of proteolysis by these enzymes has provided important insights into the actual pathways that may be altered to produce the apoptotic phenotype. Granzyme B is clearly a key mediator of the apoptosis that occurs during granule-mediated CL cytotoxicity. Although there is increasing evidence that this serine protease mediates cell death through activation of the ICEICED-3 cysteine proteases, this has yet to be definitively established. Of the known ICE/CED-3 family members, CPP32 and/or closely related homologs are clearly key players in apoptosis, whereas ICE itself is relatively unimportant in this process. This is not surprising given the fact that CPP32 is one of the homologs most highly related to CED-3. The conclusion that CED-3 subfamily members are important in mammalian apoptosis is supported by recent reports that activation of CPP32, Mch3, and Mch2 occurs early in apoptosis [28,35,131]. Currently, the relationship between the functions of these proapoptotic homologs during cell death remains unclear. Because they are all derived from cleavage of their respective proenzymes at Asp-X sites, it is possible that they are components of a proteolytic cascade that is triggered in response to a death stimulus. This is supported by the recent finding that MACH is associated with the Fas signaling complex [30,31]. Alternatively, mammalian cells may have evolved to produce several redundant pathways to control this important physiologic process. It is also conceivable that some of these proteins are tissue-specific isoforms. Further

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experiments are required to distinguish between these and perhaps other possibilities. Despite the finding that inhibitors of ICE/CED-3 proteases prevent apoptosis in several models, serious issues regarding the suitability of these enzymes as targets for therapeutic intervention remain to be addressed. First, in many diseases resulting from excess cell death, particularly in chronic diseases, it will probably be desirable to target a specific cell type (e.g., hippocampal pyramidal neurons in Alzheimer’s disease). Early indications are that ICEKED-3 family members play necessary roles in one or more biochemical pathways of apoptosis that are common to many or all cell types. Even if different homologs are involved in distinct tissues, selective inhibition of these enzymes (particularly those with similar functions) may be relatively difficult to achieve, as described below. Thus, unless there are major advances in our ability to deliver drugs to specific tissues, preferable therapeutic targets may be those mediators that are unique to a particular cell type. Second, because members of the ICE/CED-3 family appear to have key functions in distinct physiologic processes, it will probably be important to selectively inhibit only the proapoptotic homologs (e.g., CPP32). There is some evidence that this will not be easy to accomplish, at least for some family members. For example, CPP32 is a highly specific enzyme with a strict requirement for the sequence D-X-X-D in substrates and inhibitors. However, because this sequence is also reasonably well tolerated by ICE, a more promiscuous enzyme with a key role in inflammation, potent peptidebased inhibitors of CPP32 also inhibit ICE. It is hoped that a comparison of the crystal stuctures of the two enzymes will reveal opportunities for development of selective inhibitors. Finally, the current inhibitors have several limitations in addition to lack of selectivity that must be overcome to produce compounds that are effective in vivo. The poor performance of the existing inhibitors against intact cells is one outstanding problem. Clearly, the development of nonpeptide inhibitors that are stable in vivo, penetrate cells, and reach their target enzyme(s) is not a trivial task. Nonetheless, several reversible and irreversible peptide-based inhibitors have been developed for ICE family members [41,148] using motifs that have proven successful for inhibition of other cysteine proteases, and at least some of these are attractive starting points for future development. Reversible inhibitors include aldehydes, nitriles, and ketones. The most interesting of these are the ketones because of their anticipated stability in vivo. Irreversible inhibitors contain the general structure peptide-CO-CH2-X, where X is a halide ion (chloromethylketones and fluoromethylketones), -Nz (diazomethylketones), - 0 C O R [(acyloxy)methylketones], or -OR [a-(pyrazo1oxy)methylketonesand (phosphiny1oxy)methylketones]. Of these inactivators, the most promising are the latter three because of their intrinsic low reactivity with biological nucleophiles [ 1491.

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