Tài liệu Báo cáo khoa học: KIPase activity is a novel caspase-like activity associated with cell proliferation doc

KIPase activity is a novel caspase-like activity associated with cell proliferation Cahora Medina-Palazon, Emmanuelle Bernard, Victoria Frost, Simon Morley and Alison J. Sinclair Biochemistry Department, School of Life Sciences, University of Sussex, Brighton, UK A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27 KIP1 , in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100– 200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1–10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypo- thesis whereby KIPase is a currently unidentified caspase- like enzyme which regulates the abundance of p27 KIP1 in a proliferation-dependent manner. Keywords: caspase; cell cycle; cyclin dependent kinase inhibitor; KIPase; p27 KIP1 . We previously identified a caspase-like activity present in transformed lymphoid cells [1–3]. The activity is present in a cell proliferation-dependent manner [1], it is able to cleave a subset of caspase substrates [1,2], but it is insensitive to the broad specificity caspase inhibitor, z-VAD-fmk [1,2]. The activity also cleaves a tetra peptide substrate found within the cyclin-dependent kinase inhibitor, p27 KIP1 [1], and will hereafter be referred to as KIPase activity. p27 KIP1 has also been reported to be cleaved by caspases during apoptosis [4–6]. However, this process is sensitive to the caspase inhibitor z-VAD-fmk whereas cleavage by KIPase activity is not [1,2]. Cleavage at the cognate tetra peptide region in p27 KIP1 ,DPSD 139 , would render the resulting protein segments incapable of cdk inhibition, which implies that both caspases and KIPase have the potential to be key regulators of p27 KIP1 and thus the mammalian cell cycle. Both the abundance and function of p27 KIP1 are regulated by a myriad of mechanisms involving changes to transcription, transla- tion, phosphorylation, ubiquitin-mediated degradation, subcellular localization [7–11] and also by cleavage [12]. It is difficult to decipher which of these mechanisms are most relevant to determine the function and abundance of p27 KIP1 in a specific cell type within a given milieu of signal transduction events. However, Roberts and colleagues have recently defined a role for the phosphory- lation of T187 by generating a genetic replacement of the endogenous murine locus using knock-in technology [13]. We previously demonstrated that KIPase appears to contribute to the cell cycle-dependent regulation of p27 KIP1 abundance in human lymphoid cells. Specifically, an inverse correlation between KIPase activity and the abundance of p27 KIP1 was noted as the human B-lymphoid cell line BJAB enters and exits the proliferative state [1]. BecauseKIPaseisabletocleaveasubsetofcaspase substrates (IETD-AMC; YVAD-AMC and LEHD-AMC and DEVD-AMC) [1,2], it is important to define whether KIPase is a novel enzyme or one of the known human caspases. Indeed, caspase 8 has recently been implicated in cell cycle control in lymphoid cells [14–22] and would be a prime candidate. In order to address this important issue we undertook an extensive comparison of KIPase with the currently characterized human caspases. Experimental procedures Cell culture BJAB B-lymphoblastoid cells [23], DG75 cells [24], IB4 [25], Jurkat A3 T-lymphoblastoid cells and their derivative, Jurkat 9.2 [26], were grown in RPMI medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 m M glutamine and 100 IUÆmL )1 penicillin/streptomycin (as Correspondence to A. Sinclair, School of Life Sciences, University of Sussex, Brighton, BN1 9QG, UK. Fax: + 44 1273 678 433, Tel.: + 44 1273 678 194, E-mail: a.j.sinclair@sussex.ac.uk Abbreviations: Ac-DPSD-AMC, N-acetyl-Asp-Pro-Ser-Asp-AMC; AMC, 7-amino-4-methyl coumarin; cdk, cyclin dependent kinase; DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-AMC; DVPD-AMC, N-acetyl-Asp-Val-Pro-Asp-AMC; DPSD, N-acetyl-Asp-Pro-Ser-Asp; ESQD-AMC, N-acetyl-Glu-Ser-Gln-Asp-AMC; IETD-CHO, N-acetyl-Ile-Glu-Thr-Asp-aldehyde; IETD-AMC, N-acetyl-Ile-Glu- Thr-Asp-AMC; LEHD-AMC, N-acetyl-Leu-Glu-His-Asp-AMC; YVAD-AMC, N-acetyl-Tyr-Val-Ala-Asp-AMC; z-VAD-fmk, ben- zoyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone. (Received 4 March 2004, revised 29 April 2004, accepted 4 May 2004) Eur. J. Biochem. 271, 2716–2723 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04200.x described in [27]). The cell lines, T98G [28], 293-HEK (ECACC) and U2OS [29], were maintained at between a density of 2 and 10 · 10 6 cellsÆmL )1 to maintain exponential growth. An antagonistic antibody to FAS (TCS Biologicals, Bucks, UK) was added at a final concentration of 1 lgÆmL )1 to cells at a density of 1 · 10 7 per mL. Following a 30 min incubation at 4 °C, cells were diluted to a final concentration of 5 · 10 6 per mL and incubated for between 1 and 24 h. Etoposide (Sigma) was added to exponentially growing cells at a final concentration of 10 lgÆmL )1 and the cells were incubated for between 1 and 24 h. To synchronize cells, exponentially growing Jurkat 9.2 cells were seeded at a density of 1 · 10 6 per mL. The proliferative status of the cells was determined by assessing the extent of DNA synthesis. [ 3 H]Thymidine (0.5 lCi; Amersham Biosciences) was added to 200 lL of cells and the cells were incubated for 4 h at 37 °C (in triplicate). The cells were then harvested onto filters and the incorporation of labelled thymidine into insoluble material was determined [1]. To prepare total protein extracts, cells were washed with NaCl/P i ,lysedin SDS sample buffer [4% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, 100 lgÆmL )1 bromophenol blue, 0.12 M Tris/HCl, pH 6.8] and heated for 5 mins at 95 °C. Following clarification, proteins were fractionated on a 12% Tris-Bis gel (Invitrogen) and transferred to nitrocellu- lose membrane by Western blot. The filters were probed for p27 KIP1 and cdk2 expression with rabbit polyclonal antisera specific for p27 KIP1 (ABCAM, Cambridge, UK) and cdk2 (SantaCruz). After incubation with protein-A coupled to HRP (Amersham Biosciences), the signals were developed with ECL reagents (Amersham Biosciences). Extract preparation and fluorogenic caspase assays Briefly, cells were washed with NaCl/P i and lysed (1 · 10 7 cellsÆmL )1 ) in cold lysis buffer [130 m M NaCl, 1% (v/v) Triton X-100, 10 m M NaPPi, 10 m M Tris/HCl, 1m M EDTA, 1 m M phenylmethanesulfonyl fluoride, 0.25 lgÆmL )1 pepstatin A, 10 m M NaH 2 PO 4 /Na 2 HPO 4 , pH 7.5]. An alternate buffer containing 50 m M NaCl was used for some experiments, as indicated in the figure legends. Debris was removed from the resultant extracts by centrifugation (18 000 g). Caspase assays were carried out in duplicate in 96 well plates by mixing 50 lLof extract with 200 lL reaction buffer (10% glycerol, 2 m M dithiothreitol, 1 m M EDTA, 1 m M phenylmethanesulfo- nyl fluoride, 25 lgÆmL )1 pepstatin A, 200 m M Hepes, pH 7.5 and 25 lgÆmL )1 tetra peptide-AMC substrate). Caspase inhibitors such as Ac-IETD-CHO were added to a final concentration of 2 lgÆmL )1 and those such as z-VAD-fmk were added to a final concentration of 3 l M , where indicated. Reactions were allowed to proceed for 90 min at 37 °C. Measurement of AMC liberated from the tetra peptide-AMC substrates was carried out using a SpectraMax Gemini spectrofluorometer (Molecular Devices) with an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Background measurements generated from the reaction completed in the absence of substrate were assessed and subtracted from experimental values. Typical values of the background reading were 305 relative fluorescent units and values for a DPSD-cleavage assay were 1165 relative fluorescent units. Caspases and inhibitors Caspase substrates and their inhibitors were purchased from Biomol. Ac-DEVD-AMC is a substrate for caspases 3 and 7; Ac-YVAD-AMC is a substrate for caspase 1; Ac-IETD-AMC is a substrate for caspase 8 and 10; Ac-LEHD-AMC is a substrate for caspases 2, 4, 5 and 9. Ac-DVPD-AMC, Ac-DPSD-AMC and Ac-ESQD-AMC are tetra peptide substrates representing mdm-2, p27 KIP1 and a nonspecific site, respectively, and were synthesized on a custom basis by QCB Inc. (Hopkinton, MA, USA). Ac-DEVD-CHO was obtained from Biomol. Ac-DPSD- CHO and Ac-ESQD-CHO were synthesized on a custom basis by QCB (INC). z-VAD-fmk was purchased from Calbiochem. Human recombinant caspases 1–10 were purchased from Biomol and were used at a final concen- tration of 0.3 UÆlL )1 . In the experiments presented in this study z-VAD-fmk routinely inhibited human recombinant caspase by 3–25% of its normal value. Fig. 1. KIPase is active in a range of human cell lineages. Extracts were prepared from the indicated cell lines during logarithmic growth. (A) KIPase activity was determined in duplicate using an in vitro fluorogenic tetra peptide cleavage assay, using DPSD-AMC as substrate. Activity was normalized for protein concentration and is shown in the histogram as relative fluorescent units (RFU) per lg protein, together with the standard deviation. (B) The ability of the broad specificity caspase inhibitor, z-VAD-fmk, to inhibit KIPase activity in the indicated cell lines was assessed using the same assay. The data are expressed relative to the activity measured in the absence of z-VAD-fmk. In test assays z-VAD-fmk inhibited human recombinant caspase 3 to only 25% of its normal activity. Ó FEBS 2004 KIPase – a novel caspase-like enzyme (Eur. J. Biochem. 271) 2717 Gel filtration chromatography Superdex 200 (prep grade) was obtained from Sigma. A 21 mL column was prepared and equilibrated with 50 m M Hepes, pH 7.0, 0.1% (w/v) Chaps, 2 m M EDTA, 10% (v/v) glycerol, 2 m M dithiothreitol and 50 m M NaCl. An extract was prepared from BJAB cells using a lysis buffer contain- ing 50 m M NaCl, as described above. Four point nine milligrams (1.0 mL) were applied to the column at 0.25 mLÆmin )1 and 40 · 0.5 mL fractions were subse- quently collected. KIPase activity was measured using the in vitro fluorogenic assay with Ac-DPSD-AMC as substrate, as described above. Results We previously identified KIPase activity in the transformed B-lymphoid cell lines BJAB and IB4 [1,2]. Here we question Fig. 3. Caspase and KIPase activity in BJAB cells in response to FAS ligation. Proliferating BJAB cells were incubated with an antagonistic antibody to FAS and cells harvested at the indicated times (shown in hours poststimulation). Extracts were prepared from the cells and their ability to cleave a variety of tetra peptide substrates determined in duplicate. Activity was normalized for protein concentration and is shown in the histogram as relative fluorescent units (RFU) per lg protein, together with the standard deviation. Fig. 2. Native molecular mass of KIPase. An extract was prepared from Jurkat J6 cells and 1 mL (4.9 mg) was applied to a 21 mL Supadex 200 column. Fractions were collected and subject to an in vitro cleavage assay, in duplicate, with Ac-DPSD-AMC as substrate. The relative fluorescent units (RFU) and the standard deviation are plotted on the y-axis and the migration of protein standards of known molecular mass are shown above the relevant fractions. 2718 C. Medina-Palazon et al.(Eur. J. Biochem. 271) Ó FEBS 2004 whether KIPase expression is restricted to this lineage or whether its expression is more widespread. An in vitro cleavage assay based on the tetra peptide from p27 KIP1 , DPSD-AMC, revealed that KIPase activity can be identified in proliferating cells from six further human cell lines which include cells of fibroblast, epithelial and T-lymphoid origin (Fig. 1A). A further characteristic of KIPase that we identified in the BJAB and IB4 cells is its insensitivity to the caspase inhibitor, z-VAD-fmk (Fig. 1A). In Fig. 1B we demonstrate that z-VAD-fmk is unable to inhibit KIPase in a broad spectrum of cell types. Caspases are a family of low molecular mass proteins, processed from zymogens to approximately 10 kDa and 20 kDa subunits; these associate homodimeric complexes which migrate with a native molecular mass of approxi- mately 150 kDa [30–32]. To identify the native molecular mass of KIPase we undertook gel filtration analysis (Fig. 2). This revealed that KIPase activity is found in fractions containing proteins of native molecular masses of approxi- mately 150–200 kDa, similar to active caspases. Interest- ingly, multimers of caspase 9 are assembled into larger multicomponent complexes, specifically a 1.4 MDa com- plex, termed the apoptosome which contains procaspase 9, Apaf-1, cytochrome c and ATP/dATP [33–37]. The assem- bled caspase 9 has far higher activity than the remaining monomers and dimers [36]. From this analysis we can conclude that while KIPase is not a component of a high molecular mass complex such as the apoptosome, it is of similar native molecular mass to active caspase 3 [38]. Because KIPase cleaves a subset of caspase substrates, we queried whether KIPase is associated with apoptosis. In all cases apoptosis was measured by comparing the percentage of poly (ADP-ribose) polymerase (PARP) cleavage within the cells. As previously described in Frost et al.[3]this varied between 50% and 100% cleavage (data not shown). The initial experiments were undertaken in the human B- lymphoid cell line BJAB. Proliferating cells were stimulated to undergo apoptosis by ligating FAS on the surface of the cells using an antagonistic antibody. Over the following 24 h period the activity of KIPase and of the apoptotic caspases were determined. As can be seen in Fig. 3, cleavage of the apoptotic caspase substrates DEVD-AMC, DVPD- AMC, IETD-AMC and LEHD-AMC were greatly stimu- lated over basal activity, peaking between 2 and 4 h poststimulation. In contrast, cleavage of the p27 KIP1 - DPSD-AMC substrate was not increased over the basal level. The effect of the topoisomerase II inhibitor, etoposide, was then analysed in order to compare two quite distinct routes to apoptosis. This resulted in a slower induction of apoptosis with the peak of the activities of the apoptotic caspases activity evident 24 h post exposure (data not shown). These data support a model whereby the apoptotic caspases are induced in BJAB cells following the initiation of apoptosis, but KIPase activity remains constant. In order to ascertain whether KIPase behaves in a proliferation dependent manner in cells other than BJAB, we synchronized a T-lymphoma cell line, Jurkat, and collected proliferating and arrested populations of cells. [ 3 H]Thymidine incorporation assays confirmed the status of the populations; arrested cells incorporated four-fold less [ 3 H]thymidine than the proliferating cells (P ¼ 0.0003) (Fig. 4A). Furthermore the abundance of the substrate for KIPase, p27 KIP1 , is inversely related to both proliferation status (Fig. 4B) and to KIPase activity, having 1.7-fold less (P ¼ 0.011) (Fig. 4C). Thus KIPase activity is regulated in a proliferation dependent manner in non-B-lymphoid cell lineage. We next questioned how the activites of KIPase and the caspases alter during apoptosis in the Jurkat cells. Both ligation of FAS and exposure to etoposide resulted in the activation of apoptotic caspases with very similar kinetics to those observed with BJAB cells (data not shown). A summary of these data are shown in Fig. 5 from which it can be clearly observed that cleavage of DEVD-AMC, IETD-AMC, LEHD-AMC and DVPD-AMC are increased when apoptosis is induced. In contrast, cleavage of the p27 KIP1 DPSD-AMC substrate remained constant under these assay conditions. Thus it appears that KIPase maintains a basal level of p27 KIP1 cleavage activity in B- and T-lymphocytes under- going apoptosis. From this we can deduce that KIPase is Fig. 4. Synchronized T-cells express proliferation-dependent KIPase activity. Jurkat cells were synchronized as described in Materials and methods. (A) During the final four hours a [ 3 H]thymidine incorpor- ation assay was undertaken in triplicate. The total amount of thymi- dine label incorporated per 10 000 cells is shown together with the standard deviation. (B) Total protein extracts were prepared, fractionated on a 12% Tris-Bis gel, transferred to nitrocellulose and incubated with antibodies to the indicated proteins. HRP-linked to protein-A was used to visualize the proteins by ECL. (C) Extracts were prepared from Jurkat cells and subjected to an in vitro cleavage assay, in duplicate, using Ac-DPSD-AMC as substrate. z-VAD-fmk was included in the assay where indicated. The RFU per lgproteinare plotted on the y-axis together with the standard deviation. Ó FEBS 2004 KIPase – a novel caspase-like enzyme (Eur. J. Biochem. 271) 2719 not an apoptotic caspase. Furthermore, it appears that the apoptotic caspases are incapable of recognizing DPSD- AMC. To directly test this hypothesis, and to examine whether any of the 10 known human caspases processed DPSD-AMC, a series of in vitro cleavage assays were undertaken with recombinant human caspases 1–10, com- paring their ability to cleave DPSD-AMC and their preferred substrate. Figure 6 shows that human caspases 1–8 and 10 cleave their preferred substrate efficiently but have poor activity towards the p27 KIP1 tetra peptide substrate, suggesting that DPSD-AMC is not recognized as a substrate by these caspases. Recombinant caspase 9 displayed a much lower level of activity against its substrate so it is more difficult to draw a conclusion about its lack of cleavage of DPSD-AMC. However, this is not unexpected as the activity of caspase 9 is greatly increased by its association with the apoptosome [33–39], which was not present in these assays. Based on these studies, the DPSD-AMC substrate appears to be specific for KIPase activity, which suggests that the DPSD tetra peptide could be used to design a specific inhibitor of KIPase. As reversible inhibitors for several caspases have been generated by synthesizing aldehyde-modified substrates [30], we generated an equiv- alent form of the DPSD tetra peptide, termed DPSD- CHO. The ability of DPSD-CHO to inhibit KIPase activity was evaluated. The dose–response curve of the aldehyde-modified version of DPSD was evaluated against KIPase isolated from BJAB and Jurkat cells. As a control, the ability of an aldehyde-modified form of an unrelated tetra peptide, ESQD, to inhibit KIPase was evaluated in these assays. As can be seen in Fig. 7, the aldehyde-modified form of DPSD inhibited KIPase activ- ity, whereas the aldehyde-modified form of ESQD displayed no inhibition of KIPase activity for either cell type. These data showed that DPSD-CHO is an efficient inhibitor of KIPase activity; however, the specificity of the Fig. 5. Comparison of the relative activity of caspases and KIPase during lymphoid cell apoptosis. BJAB cells (A) and Jurkat cells (C) were exposed to anti-FAS serum as described in Fig. 3 and the activity of caspases and KIPase determined using the in vitro fluorogenic assays. The maximum induction of activity, observed 4 h post induction is shown for each substrate. The horizontal line indicates an induction value of 1. BJAB cells (B) and Jurkat cells (D) were exposed to etoposide as des- cribed in Materials and methods and the activity of caspases and KIPase determined over a 24 h time course using in vitro fluoro- genic assays. The maximum induction of activity, observed 24 h post induction, is shown for each substrate. The horizontal line indicates an induction value of 1. Fig. 6. Comparison of the ability of recombinant human caspases to cleave DPSD-AMC. The human recombinant caspases were assayed for their ability to recognize tetra peptide substrates using in vitro flu- orogenic assays performed in duplicate. The preferred substrate (bar A) for each was as follows: caspase 1, WEHD-AMC; caspase 2, VDVAD- AMC; caspase 3, DEVD-AMC; caspase 4, WEHD-AMC; caspase 5, WEHD-AMC, caspase 6, VEID-AMC; caspase 7, DEVD-AMC; ca- spase 8, IETD-AMC; caspase 9, LEHD-AMC; caspase 10, LEHD- AMC. The p27 KIP1 substrate, DPSD-AMC (bar B), and a nonspecific peptide ESQD-AMC (bar C) were also assayed. The resulting activity (relative fluorescent units; RFUs) is shown together with the standard deviation. 2720 C. Medina-Palazon et al.(Eur. J. Biochem. 271) Ó FEBS 2004 inhibitor was uncharacterized. Initial experiments to evaluate the specificity of DPSD-CHO were undertaken in vitro using human recombinant caspases. DPSD-CHO showed negligible ability to inhibit the recombinant caspases that can be readily assayed (Fig. 8A). Further- more, DPSD-CHO had little ability to inhibit the apoptotic caspases present in extracts from BJAB cells undergoing apoptosis in response to ligation of FAS (Fig. 8B). Taken together the above experiments support our model whereby KIPase activity, the proliferation-dependent caspase-like activity, which is found in a wide distribution of cell lineages, is distinct from the activity of human caspases 1–10 and it represents a currently unidentified caspase-like enzyme that plays a role in the regulation of cell proliferation. Discussion KIPase activity has previously been identified in trans- formed human lymphoid cells [1–3]. Here we demonstrate that KIPase activity is not restricted to this lineage; specifically it is also detected in glioblastoma cells, fibro- blasts, embryonic kidney cells and osteosarcoma cells. Furthermore, KIPase activity is regulated in a proliferation dependent manner in the human T-lymphoma cell line, Jurkat, in an inverse manner to the abundance of its substrate p27 KIP1 . This resembles the correlation between p27 KIP1 abundance and KIPase activity described for the human B-lymphoma cell line BJAB [1]. In this case, inhibition of KIPase activity using a cell-permeable inhibitor resulted in an increase in p27 KIP1 abundance and a decrease in cell cycle progression [1]. KIPase has some of the characteristics of caspases, such as its ability to cleave DXXD tetra peptides [1], its inhibition by aldehyde-modified versions of the substrate [1], its inhibition by iodoacetimide and its resistance to inhibition by phenylmethanesulfonyl fluoride or E64 (V. Frost & A. J. Sinclair, unpublished data). However, KIPase is also resistant to inhibition by the broad specificity caspase inhibitor, z-VAD-fmk. This raises the question of whether KIPase is a member of the caspase family or not. Here we have demonstrated that KIPase is distinct from human caspases 1–10. The evidence supporting this is three fold: (a) KIPase is not inhibited by z-VAD-fmk whereas Fig. 7. DPSD-CHO inhibits KIPase activity. Extracts were prepared from proliferating BJAB (A,B) and Jurkat (C) cells and the ability of the aldehyde-modified version of DPSD and ESQD to inhibit KIPase activity determined using in vitro fluorogenic tetra peptide cleavage assays, performed in duplicate. Assays were undertaken at the inhibitor concentration indicated and the percentage inhibition relative to no inhibitor presented together with the standard deviation. Fig. 8. DPSD-CHO does not inhibit the in vitro activity of known human caspases. (A) The indicated human recombinant caspases were each assayed with their optimal substrate (Fig. 6) and the ability of DSPD-CHO or z-VAD-fmk to inhibit each is shown. The results are presented as percentage inhibition relative to no inhibitor present. (B) Extracts were prepared from BJAB cells 4 h after stimulation with anti-FAS serum. The activity of apoptotic caspases was measured using an in vitro fluorogenic assay with DEVD-AMC as substrate. The ability of DEVD-CHO, DPSD- CHO and ESQD-CHO to inhibit caspase activity was determined and is shown as percentage inhibition together with the standard deviation. The ability of DSPD-CHO to inhibit KIPase activity was assayed as in Fig. 7 (data not shown). Ó FEBS 2004 KIPase – a novel caspase-like enzyme (Eur. J. Biochem. 271) 2721 caspases 1–10 are; (b) recombinant versions of human caspases 1–10 do not recognize the KIPase substrate DPSD- AMC; (c) the aldehyde form of the DPSD tetra peptide does not inhibit human caspases but it does inhibit KIPase in vitro. Thus, it appears that KIPase must be encoded by a currently uncharacterized gene. Interestingly, a distant relative of the caspases, termed paracaspase, was identified in Homo sapiens using a PSI - BLAST search [39]. Paracaspase could be a candidate gene for KIPase although no caspase activity from its product has been reported to date [39]. An alternative hypothesis is that KIPase represents a modified version of one of the known caspases, although its insensitivity to z-VAD-fmk suggests that this is less likely. Purification and sequencing of KIPase will be required to answer these questions. 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