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:
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.
Finally, it is clear that although the in vitro tetra peptide
cleavage assays are a valuable means to follow enzyme
activities, not all substrates are recognized by caspases in
this manner. It has been previously shown that caspases can
cleave p27
KIP1
at the DPSD site [5,6], yet none of the human
recombinant caspases cleave the DPSD tetra peptide
substrate used in this study.
Acknowledgements
We thank Professors Blenis and Peters for cell lines. This work was
funded by grants from the Leukaemia Research fund to A. J. S. and
the Wellcome Trust (D040800) to S. J. M. S. J. M. is a Senior
Research Fellow of the Wellcome Trust.
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