From the Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia
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ABSTRACT |
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Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway. The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli. In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo. We demonstrate that the expression of pro-Nedd2 in yeast cells results in processing of the precursor. A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues. Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing. In vitro processing of pro-Nedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur. Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.
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INTRODUCTION |
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Caspases are a family of mammalian cysteine proteases that share
structural and, in some cases, functional similarity with the
Caenorhabditis elegans cell death protease CED-3 (1, 2). Caspases are produced as zymogens and require cleavage into two subunits, which constitute the active enzyme (reviewed in Refs. 3-6).
The small subunit of a caspase is derived from the carboxyl terminus of the precursor, while the large subunit originates from the
region immediately upstream of the small subunit, leaving an
amino-terminal prodomain of varying lengths in different caspases. On
the basis of prodomain size, caspases can be divided into two groups,
those with relatively long amino-terminal prodomains and those without.
It is suggested that in caspases with long prodomains, the
amino-terminal region is required for their recruitment to death
receptor complexes (7, 8). The long prodomain is also likely to serve a
function in dimerization and autoprocessing as suggested by a recent
study with interleukin-1-converting enzyme (caspase-1) (9).
Nedd2 was initially identified as a developmentally
regulated gene in the mouse central nervous system (10) and later shown to encode a cysteine protease similar to interleukin-1-converting enzyme and CED-3 (11, 12). Several lines of evidence have implicated
Nedd2 (ICH-1/caspase-2) directly or indirectly in apoptosis. Overexpression of Nedd2 induces apoptosis in various cell types (11,
12), while expression of antisense Nedd2 in FDC-P1
factor-dependent cells delays the onset of apoptosis
induced by factor withdrawal (13). Abrogation of Nedd2
expression by antisense oligonucleotides rescues PC12 cells from
apoptosis induced by nerve growth factor deprivation but not by
superoxide dismutase down-regulation (14). In addition, an
alternatively spliced form of caspase-2, which encodes a truncated
protein, has been shown to protect against cell death induced by serum
withdrawal in Rat-1 and NIH-3T3 cells, but not in all cell types (12,
15). Up-regulation of Nedd2 mRNA has been observed in
response to ischemia-induced cell death (16, 17), while down-regulation
of Nedd2 has been observed during gonadotropin-promoted follicular
survival (18). Caspase-2 activation occurs early during apoptosis
mediated by factor withdrawal,
-irradiation, etoposide treatment
(20, 21), tumor necrosis factor, and Fas
ligation.1 Caspase-2 has been
shown to associate via its prodomain with RAIDD, a death adaptor
molecule that is thought to be involved in apoptosis mediated through
death receptors via association with the death domain proteins RIP and
TRADD (19).
Previous studies have suggested that expression of
pro-Nedd22 in E. coli and mammalian cells can result in self-processing (22-24).
pro-Nedd2 expressed in E. coli shows polypeptides of 18, 13, and 12 kDa derived by cleavage at DNKD169,
DQQD333
, and EESD347
, as determined by
microsequencing (23). Since Nedd2 contains a long prodomain, it is
possible that the Nedd2 precursor (pro-Nedd2) dimerizes prior to
autoprocessing, in a manner similar to that suggested for
interleukin-1
-converting enzyme (9). In the present paper, we have
analyzed the dimerization of pro-Nedd2 using the yeast two-hybrid
assay. By mutation analysis, we have also studied the mechanism of
activation of Nedd2 in vitro and in vivo and its
functional implications in autocatalysis and cell killing.
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EXPERIMENTAL PROCEDURES |
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Yeast Two-hybrid Experiments--
The components of the yeast
two-hybrid system were purchased from CLONTECH.
Nedd2 constructs in Gal4 activation domain (AD) vector and
Gal4 DNA-binding domain (BD) vector were co-transformed into
Saccharomyces cerevisiae strain Y190, and colonies
containing both vectors were selected on SD medium lacking leucine and
tryptophan. Putative interacting Nedd2 fusion proteins were selected by
screening for -galactosidase activity in a colony lift filter assay
using 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
(X-gal). Quantitative data on positive interactions were obtained with
a liquid culture assay using
o-nitrophenyl-
-D-galactopyranoside as a
substrate.
Construction of Two-hybrid Vectors--
Wild-type
Nedd2 plasmid MSN2.4 carrying the entire coding region of
Nedd2 and a Cys320 Gly Nedd2 mutant have
been described previously (11). The coding region of Nedd2
was amplified from pMSN2.4 by 25-cycle PCR using Pfu
polymerase (Stratagene) and the following oligonucleotide primers with
BamHI restriction sites: primer A,
5'-CGCGGATCCACAGGAGGAGCAGGATTTTG; primer B,
5'-CGCGGATCCAACGTGGGTGGGTAGCC (regions complementary to the
Nedd2 sequence are underlined). The amplified product was cloned in frame to both the Gal4BD in the yeast expression vector pAS2.1 (Gal4BDWT) and the Gal4AD in pACT2 (Gal4ADWT). A Nedd2 Cys320
Gly mutant was amplified similarly using primers
A and B and cloned in the two yeast vectors to generate Gal4BDC320 and
Gal4ADC320. To generate Nedd2 minus prodomain (MPD) constructs,
wild-type and Cys320 mutant plasmids were used as a
template for PCR using primers B and C (primer C,
5'-CGCGGATCCATAATGGTGATGGTCCT). Resulting products were
cloned into the BamHI site of pAS2.1 and pACT2 to generate Gal4BDMPD, Gal4BDMPDC320, Gal4ADMPD, and Gal4ADMPDC320 vectors.
In Vitro Proteolysis Experiments-- Recombinant active caspase-3 was generated as described previously (26). For mouse caspase-2 expression, the PCR-amplified coding region of Nedd2 was cloned into the pET32 vector (Novagen), and recombinant enzyme was expressed according to instructions provided by Novagen. Wild-type and mutant Nedd2 cDNAs cloned into pBluescript vector (Stratagene) were used as a template for the production of [35S]methionine-labeled protein using a coupled transcription/translation kit (Promega). 2.5-5 µl of translated product was incubated with recombinant enzyme for 3 h at 37 °C, electrophoresed on SDS-polyacrylamide gels, transferred to PolyScreen polyvinylidene difluoride membrane (DuPont), and exposed to x-ray film.
Transient Transfection--
For expression in mammalian cells,
wild-type and mutant Nedd2 cDNAs were cloned into the
pCXN2 vector (27). For cell death assays, NIH-3T3 cells were plated at
2.5 × 105/well in 6-well dishes the day before
transfection. For each well, we used 2 µg of the Nedd2
expression plasmid mixed with 0.5 µg of the -galactosidase
expression plasmid (pEF-
gal). All transfections were carried out
using Superfect transfection reagent (Qiagen) according to the
manufacturer's protocol. Cells were fixed and stained with X-gal
18-24 h post-transfection as described previously (11). For
immunoblotting experiments, cells plated at a density of
106/60-mm dish were transfected with 5 µg of the
Nedd2 expression constructs and harvested 12 h after
transfection.
Immunoblotting-- Transiently transfected NIH-3T3 cell pellets were boiled in SDS-polyacrylamide gel electrophoresis buffer prior to electrophoresis. Nedd2 constructs fused to the Gal4AD in pACT2 were transformed into S. cerevisiae Y190, and colonies were selected in medium lacking Leu. Yeast extracts were prepared using the trichloroacetic acid protein extraction method following the CLONTECH protocol. Proteins were separated by SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membrane. Immunoblotting was carried out using a 1:500 dilution of an anti-caspase-2 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), which detects the precursor and the p14 and p12 subunits of the human Nedd2 protein (20), or a Gal4AD antibody (CLONTECH) at a 1:10,000 dilution. After probing with an appropriate horseradish peroxidase-coupled antibody, signals were detected by ECL (Amersham Corp.).
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RESULTS |
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Dimerization and Processing of pro-Nedd2 in Yeast--
Since
pro-Nedd2 can undergo processing when expressed in E. coli
and mammalian cells (22, 23), we used a functionally inactive pro-Nedd2
mutant in which the catalytic Cys320 residue was replaced
by a Gly residue for our protein-protein interaction studies using the
yeast two-hybrid system. This mutant is unable to induce apoptosis in
mammalian cells, presumably due to a lack of autoprocessing activity
(11, 28). Nedd2 constructs were cloned into both the Gal4BD and Gal4AD
vectors (Fig. 1) and transformed into
S. cerevisiae. Analysis of Nedd2 protein expression in yeast
showed that the 51-kDa wild-type Nedd2 fused to 19-kDa Gal4AD was
present as a band of ~56 kDa, suggesting that autoprocessing occurred
from the C terminus removing a ~14-kDa fragment (Fig. 2). The Nedd2 Cys320 mutant
was expressed mostly as full-length precursor protein (~70 kDa),
suggesting that the inability of this mutant to induce apoptosis in
mammalian cells is due to a lack of autoprocessing activity. Following
cotransformation, no interaction between the BD and AD wild-type
pro-Nedd2 was observed, suggesting that processing occurs rapidly upon
dimerization and that full-length Nedd2 precursor is required for
stable dimerization to take place. Indeed, the two full-length
Cys320 mutant fusion proteins were able to interact, as
evident by the activation of the LacZ reporter gene and
-galactosidase activity, measured by X-gal staining of colonies (not
shown) and colorimetric assays (Table I).
Cotransformation of the two parent vectors or either vector carrying
the Cys320 mutant did not show any activation of the
reporter genes. Since the prodomain in interleukin-1
-converting
enzyme has been shown to be required for dimerization of precursor (9),
we analyzed the role of the Nedd2 prodomain in dimerization. When
prodomain constructs in AD and BD plasmids were cotransformed into
S. cerevisiae, strong activation of the reporter gene was
observed, suggesting that the prodomain region can homodimerize
readily. Furthermore, both wild-type Nedd2 and Cys320
constructs lacking the prodomain were unable to interact with the
full-length Cys320 mutant (Table I), indicating that the
prodomain region is essential for dimerization of pro-Nedd2. We also
assessed whether the prodomain alone was sufficient to mediate
dimerization with the Cys320 mutant. No reporter gene
activity was evident when the prodomain construct was cotransformed
with the Cys320 mutant, suggesting that although the
prodomain is required for interaction between precursor molecules, it
is not sufficient.
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Aspartate Mutants Affect Dimerization and Processing of pro-Nedd2-- Since wild-type pro-Nedd2 was unable to homodimerize, we further examined whether processing of this caspase affects dimerization. Since the processing of caspases occurs by cleavage of the precursor at the C terminus of Asp residues, we used Nedd2 constructs in which Asp residues at which cleavage is reported to occur (Asp169, Asp333, and Asp347) had been replaced with either Gly or Ala residues by site-directed mutagenesis. In addition, we used three other Asp mutants (Asp135, Asp326, and Asp330) since these Asp residues lie in the vicinity of the above cleavage sites and have been shown to abolish the proapoptotic activity of Nedd2 (24). When cotransformed with the Cys320 mutant, Asp135, Asp169, and Asp347 failed to show any activation of the reporter gene, while Asp326, Asp330, and Asp333 were able to dimerize with Cys320 (Table II). Except for the Asp135 mutant, in all cases, the ability to dimerize appeared to correlate with the inability to undergo in vivo autoproteolytic processing. The Asp326 mutant blocked the removal of p14, an intermediate for the p12 subunit, and was able to dimerize with Cys320, indicating that residues in p14 are required for dimerization (Fig. 2). Asp330 and Asp333 mutants were also poorly processed in yeast (data not shown) and mammalian cells (see below) and were able to dimerize with Cys320 (Table II). Asp347, cleavage at which results in the generation of p12 (23), was processed in S. cerevisiae, presumably by cleavage at Asp333, removing the p14 subunit (Fig. 2). Similarly, the Asp169 mutant showed a smaller product than expected due to cleavage at the C-terminal Asp residues. Although the Asp135 mutant was not well processed in S. cerevisiae, it failed to interact with Cys320. Asp135 was also unable to homodimerize or heterodimerize with all other Asp mutants or the prodomain (Table II).
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In Vitro Processing of pro-Nedd2 by Recombinant Caspases-- We and others have previously shown that pro-Nedd2 can be cleaved into two subunits in vitro by several caspases including caspase-2 and caspase-3 (22, 23). To ascertain whether the mutations in various Asp residues can affect the cleavage of the precursor, mutant proteins were subjected to cleavage by recombinant Nedd2 (Fig. 3). While the wild-type Nedd2 and Asp347 mutants were efficiently processed by recombinant Nedd2, as apparent by the disappearance of the 51-kDa pro-Nedd2 and the appearance of smaller polypeptides representing intermediates and subunits, Asp135 and Asp326 only showed partial cleavage, and most of the precursor remained intact (Fig. 3B). Mutations at Asp330 and Asp333 suppressed the release of p14. Asp169 showed significant processing, but the cleavage profile of this mutant was significantly altered from the wild-type pro-Nedd2. Since CPP32 (caspase-3) has previously been shown to mediate the cleavage of pro-Nedd2 (22, 23), we subjected various Asp mutants to proteolysis by recombinant CPP32 (Fig. 3C). Interestingly, Asp135 and Asp326 mutants, which are poorly processed by Nedd2, were efficiently cleaved by CPP32. CPP32 cleavage of Asp330 and Asp333 (P4 and P1 positions in the D330QQD333 sequence in pro-Nedd2) mutants generated a fragment larger than 14 kDa, suggesting that CPP32 cleaves pro-Nedd2 at Asp333. These results suggest that the mechanisms of processing of pro-Nedd2 by caspase-2 and caspase-3 are different and that the two caspases may have different preferences for various cleavage sites in the precursor molecule.
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In Vivo Processing and Apoptotic Activity of Nedd2 Mutants-- The first biological assays to show a role for Nedd2 in apoptosis utilized pro-Nedd2 overexpression in transiently transfected cells (11, 12). These experiments clearly showed that overexpression of pro-Nedd2 induced apoptosis, but it was not clear how the cell death was mediated. We and others have suggested that the overexpression of caspases can result in autoprocessing (11, 12, 24, 28), perhaps in a concentration-dependent manner, but it has not been firmly established in the transient transfection system, despite the fact that these assays are now widely used. We analyzed the death-inducing activities and processing of the various Nedd2 mutants in transiently transfected NIH-3T3 fibroblasts (Figs. 4 and 5). Consistent with the yeast experiments and in vitro Nedd2 processing data above, mutations at Asp169 and Asp347 retained their apoptotic activity, while substitutions at Cys320, Asp135, Asp326, and Asp333 completely abolished the death-inducing activity of pro-Nedd2. Asp330 retained some apoptotic activity (~45%). We also analyzed the in vivo processing of various mutants using an antibody against the small subunit of caspase-2. NIH-3T3 cells were transfected with various constructs and harvested 12 h later. Immunoblotting of extracts from these cells showed that wild-type pro-Nedd2 and the Asp347 mutant were efficiently processed in vivo; however, in the Asp347 mutant, p14 but not p12 was released, indicating that cleavage at Asp347 generates the mature p12 subunit. Asp330 and Asp333 showed partial processing, with release of the prodomain generating a p18 + p14 intermediate that is further processed to some extent in the case of Asp330 to generate p14 and p12 subunits. Asp135 and Asp326 mutants were not processed (Fig. 5), which is consistent with their inability to induce apoptosis. Some processing of Cys320 mutant was also evident. Since this mutant is catalytically inert, we believe that partial processing of Cys320 in transfected cells may be mediated by other endogenous proteases/caspases rather than autocatalysis.
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DISCUSSION |
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In this paper, we show that pro-Nedd2 is able to dimerize in
S. cerevisiae. Dimerization of pro-Nedd2 only occurred in
mutants that lacked the ability to autoprocess, and removal of either the prodomain or the C terminus abolished this interaction. This is
consistent with the assumption that dimerization occurs prior to the
processing of pro-Nedd2. Preceding this study, only
interleukin-1-converting enzyme had been shown to be able to form
dimers in vivo (9), but it is likely that all caspases
containing long amino-terminal prodomains are capable of forming
homodimers. This would be in keeping with the processing of many other
enzymes unrelated to caspases, where the prodomain region is required
for optimal folding and maturation of the active enzyme (29).
Interestingly, the prodomain alone, devoid of the rest of the sequence,
was able to homodimerize. However, the prodomain did not heterodimerize with the Cys320 pro-Nedd2, suggesting that the
prodomain-prodomain interaction is important but not sufficient to
stabilize the pro-Nedd2 dimer. No such studies have been carried out
with interleukin-1
-converting enzyme, but a similar scenario can be
envisaged with other caspases with long prodomain regions.
Since wild-type Nedd2 was unable to form stable dimers in yeast, we analyzed various Asp mutants that are known to affect the processing of Nedd2. Mutations of Asp326, Asp330, or Asp333 restored the ability of pro-Nedd2 to form dimers in S. cerevisiae. None of these mutants showed appreciable processing when expressed in S. cerevisiae. When analyzed in transient cell killing experiments, Asp326 and Asp333 were unable to induce apoptosis in NIH-3T3 cells, while Asp330 showed reduced (~45%) activity compared with wild-type Nedd2. In in vitro proteolysis assays, Asp326 was poorly cleaved by recombinant active Nedd2, while Asp330 and Asp333 showed partial processing. This partial cleavability of pro-Nedd2, both in vitro and in NIH-3T3 cells, is consistent with the relative dimerization potential of these mutants; mutants that do not undergo processing efficiently form dimers more effectively.
Based on microsequencing of the active enzyme subunits resulting from
expression of pro-Nedd2 in E. coli, previous work has defined the cleavage sites in pro-Nedd2 as DNKD169,
DQQD333
, and EESD347
(23). Our results
suggest that to generate the small subunit of the mature enzyme, the
first cleavage probably occurs at Asp333 followed by
further cleavage at Asp347. Although cleavage at
Asp347 generates the mature p12 subunit, it is not crucial
for catalytic activity of Nedd2, since the Asp347 mutant
can undergo processing and retains proapoptotic activity similar to
wild-type Nedd2. In immunoblot analysis of Nedd2 following Fas antibody
and etoposide treatment of Jurkat cells, a product of about 14 kDa is
always evident during early stages, which appears to be the precursor
for p12 that becomes apparent during later stages of
apoptosis.1 Mutation at Asp169, at which the
cleavage is known to occur to generate the p18 subunit by removing the
prodomain (23), also had no effect on cell killing, suggesting that
removal of prodomain is not essential to generate active enzyme.
Using a Semliki Forest virus expression system, Allet et al. (24) showed that the Asp135 mutant is unable to be processed in infected Chinese hamster ovary cells. This mutant was also unable to induce cell death when microinjected into sympathetic neurons (24). Our results are consistent with these earlier studies. Since the Asp135 mutant was not well cleaved by recombinant Nedd2, even at Asp residues in the C-terminal region of pro-Nedd2, it appears that the Asp135 residue is important in maintaining the overall structure of the precursor molecule, and in the mutant this structure is disrupted in such a way that other cleavage sites are no longer accessible to caspase cleavage. This is further supported by our data showing that despite the fact that Asp135 is not processed, unlike Cys320, it is unable to homodimerize or to heterodimerize with Cys320 in S. cerevisiae. As expected, the Asp135 mutant also failed to induce apoptosis when overexpressed in mammalian cells. Mutation of Asp326 also inhibited processing and cell killing activity of Nedd2, suggesting a situation similar to Asp135.
Our results clearly suggest that pro-Nedd2 can homodimerize prior to processing. We propose that homodimerization of the precursor molecules is important for its autoprocessing and proapoptotic activity and is distinct from oligomerization of subunits that constitute the active enzyme. Our data also indicate that autoprocessing occurs in a concentration-dependent manner and that killing of cells by overexpression of pro-Nedd2 is mainly due to autoactivation. ProNedd2 is widely expressed in many cell types without having any adverse effect (11, 12, 15). There is some evidence for transcriptional activation of Nedd2 preceding cell death in various systems (16-18). However, recruitment through adaptors, such as RAIDD (19), may also be important in achieving optimum precursor concentration in specific cellular compartments, promoting autocleavage. Since pro-Nedd2 can also be cleaved into active subunits by downstream caspases, such as caspase-3, once these caspases are activated, a cascade event would ensure that all precursor is rapidly processed in cells undergoing apoptosis, thus amplifying the apoptotic response.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Bernard Allet for providing the Asp135, Asp326, Asp330, and Asp333 mutants and to Dr. Jun-ichi Miyazaki for the pCXN2 mammalian expression vector.
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FOOTNOTES |
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* This work was supported by a Wellcome Trust Senior Research Fellowship and National Health and Medical Research Council of Australia Grant 960532 (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Hanson Centre for
Cancer Research, Institute of Medical and Veterinary Science, PO Box
14, Rundle Mall, Adelaide, SA 5000, Australia. Fax: 61-8-82223139 or
61-8-82223162; E-mail: sharad.kumar{at}imvs.sa.gov.au.
1 N. L. Harvey, A. J. Butt, and S. Kumar, unpublished data.
2
The abbreviations used are: pro-Nedd2, the
51-kDa precursor of Nedd2; AD, activation domain; DB, DNA-binding
domain; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; MPD,
Nedd2 minus pro- domain; PCR, polymerase chain reaction.
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REFERENCES |
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