From the Microbial Pathogenesis Unit, Christian de
Duve Institute of Cellular Pathology, Université catholique de
Louvain, Avenue Hippocrate 74, B-1200 Brussels and the
¶ Department of Molecular Biology, Flanders Interuniversitary
Institute for Biotechnology and University of Ghent, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium
Received for publication, February 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yersinia enterocolitica
induces apoptosis in macrophages by injecting the plasmid-encoded YopP
(YopJ in other Yersinia species). Recently it was reported
that YopP/J is a member of an ubiquitin-like protein cysteine protease
family and that the catalytic core of YopP/J is required for its
inhibition of the MAPK and NF- A number of bacterial pathogens resist the defense mechanisms of
their host by a recently discovered virulence mechanism called type III
secretion. By this mechanism, bacteria adhering at the surface of
eukaryotic cells inject proteins in the cytosol of these cells. In the
archetypal Yersinia, Yersinia pestis, the agent of bubonic plague, Yersinia pseudotuberculosis, and
Yersinia enterocolitica, the injected proteins are called
Yops. Yops are encoded by a large virulence plasmid, and their main
target seems to be the macrophage (for review see Ref. 1). One of the
Yops, called YopP in Y. enterocolitica and YopJ in Y. pseudotuberculosis and Y. pestis, causes a variety of
effects, such as suppression of tumor necrosis factor One of the earliest and most consistently observed features of
apoptosis is the activation of procaspases, a family of cysteine proteases that cleave their substrates after an aspartic acid residue
(11). Caspases are synthesized as zymogens (procaspases) that become
activated via proximity-induced autoproteolysis by interaction with
adaptor proteins or by cleavage via upstream proteases in an
intracellular cascade (12-14). Two main procaspase activation pathways
during apoptosis have been proposed: (i) the extrinsic activation of
initiator procaspases, triggered by the formation of a receptosome
complex; and (ii) the intrinsic activation of initiator procaspases,
initiated by the formation of an apoptosome complex. The extrinsic
activation of initiator procaspases is initiated by the death
domain-containing receptors of the TNF receptor superfamily (14, 15).
Binding of the ligand to the cell surface receptor leads to
trimerization (16) of the receptors and subsequently oligomerization of
their cytosolic death domains eventually leading to the recruitment of
initiator procaspases, such as procaspase-8. This complex is called the
death-inducing signaling complex (17). During this process caspase-8
can initiate cell death by directly cleaving the downstream executioner
procaspases-3, -6, and -7 (13, 18). In some cases, however, the
death-inducing signaling complex does not generate sufficient caspase-8
levels to allow efficient proteolytic activation of the downstream
executioner procaspases, and amplification of the caspase cascade by
mitochondrial-derived factors is required to kill the cell (19). A
molecular link connecting the death-inducing signaling complex
activation and mitochondria is the caspase-8-mediated cleavage of Bid,
a pro-apoptotic member of the Bcl-2 family. The C-terminal part of Bid
(tBid) translocates to the mitochondria, where it induces the release of cytochrome c (20, 21). Cytochrome c, together
with dATP/ATP, binds the apoptotic protease activating factor-1
resulting in the formation of the apoptosome complex (22), which leads
to recruitment and autoactivation of procaspase-9. In turn, caspase-9 activates procaspases-3, -6, and -7, thus initiating the caspase cascade (23). The consecutive activation of both initiator procaspases, procaspase-8 at the level of the death receptor complex and
procaspase-9 at the postmitochondrial level, then leads to sufficient
activation of the executioner procaspases resulting in cell demise.
Intrinsic activation of procaspases, triggered by environmental
insults, senescence, and developmental programs, involves the release
of cytochrome c from the mitochondrial intermembrane space
to the cytosol leading to assembly and activation of the apoptosome
complex igniting the caspase cascade. The mechanism by which this
cytochrome c release is induced is not yet clear.
In this report we describe that activation of procaspases is essential
in Y. enterocolitica YopP-mediated cell death. Moreover, YopP-mediated apoptosis initiates the cell death pathway upstream of
the cytosolic protein, Bid. Bid cleavage results in the subsequent release of cytochrome c from the mitochondria eventually
leading to the activation of procaspase-9 and the executioner
procaspases-3 and -7. The cleavage of Bid and the release of cytochrome
c can both be inhibited by broad-spectrum caspase
inhibitors, suggesting that YopP induces activation of upstream
caspases, most likely caspase-8. Finally, we provide evidence
that the protease activity of YopP is crucial for the induction of
apoptosis in macrophages by Yersinia.
Plasmids, Bacterial Strains, and Growth
Conditions--
Escherichia coli DH5
Plasmid pMSK13 is a mobilizable plasmid containing
yopPE40WT (from the pYV plasmid of Y. enterocolitica E40). Plasmid pRB16 is a derivative of pMSK13
encoding yopPE40C172T in which the catalytic cysteine is replaced by a threonine. The mutant was engineered by
site-directed mutagenesis as described by Kunkel et al. (27) using the mutation primer
5'-ACTAAAAATACCGGTTTCAGATGAGCT-3', which introduces a
PinA1 restriction site (underlined). Plasmids pMSK13 and pRB16 were used to complement the yopP knockout
mutation. For reasons of clarity, strains E40(pMSK41)(pMSK13) and
E40(pMSK41)(pRB16) will be referred to as YopPE40WT+ and
YopPE40C172T+ strains. Plasmid pGD2 was obtained by cloning
yopPA127WT, the wild type yopP gene
from strain A127, into the eukaryotic expression vector
pEF6Myc/His vector (Invitrogen, Carlsbad, CA) in frame with
the Myc and His tags, using EcoRI and NotI sites.
pCDNA1-TRADDE was described elsewhere (28).
pCDNA1-FADDE was constructed in a similar way
as pCDNA1-TRADDE by fusing the human FADDE.
pEF1-Bid was made by cloning the reverse transcriptase polymerase chain reaction fragment of full-length Bid (from mouse lung mRNA) in the
pEF1V5/His vector (Invitrogen, Carlsbad, CA).
pIKK2FLAG, an expression vector for IKK2/IKK
Bacteria were pregrown overnight in brain-heart infusion (Difco) before
infection bacteria were diluted 1/20 in fresh brain-heart infusion and
cultured under continuous shaking (110 rpm) for 120 min at room
temperature; subsequently the bacteria were induced for Yop secretion
by incubation for 30 min in a shaking water bath (110 rpm) at 37 °C.
Prior to infection bacteria were washed with RPMI 1640 (Life
Technologies, Inc.).
Cell Culture and Macrophage Infection--
Murine
monocyte-macrophage J774A.1 cells (ATCC TIB67) were cultured at
37 °C under 6% CO2 in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin sulfate (100 µg/ml), sodium pyruvate (1 mM), and Caspase Inhibitor Studies--
J774A.1 macrophages were
seeded at 5 × 105 cells in 24-well tissue culture
plates on glass coverslips, 15 h in advance of infection. 100 µM of the caspase peptide inhibitors (Calbiochem)
benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD.fmk),
benzyloxycarbonyl-Asp(OMe)-fluoromethylketone (B-D.fmk), acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD.cmk),
benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (zDEVD.fmk), and benzyloxycarbonyl-Ala-Ala-Asp-chloro-methylketone (zAAD.cmk) were added 2 h before infection. Infection of the
macrophages was done as described above. Immunofluorescence detection
of fragmented genomic DNA was performed using a terminal
deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end
labeling (TUNEL) assay, essentially as described by Mills et
al. (7). Cell morphology was analyzed by light microscopy.
Measurement of Caspase Activity--
Cell extracts were prepared
by lysing the cells in a buffer containing 1% Nonidet P-40, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4, 10% glycerol,
10 µg/ml leupeptin, 0.3 mM aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. Caspase activity was
determined by incubation of cell lysates (containing 40 µg of total
protein) with 50 µM of the fluorogenic substrate
acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-amc) (Peptide
Institute Inc., Osaka, Japan) in 150 µl of cell-free system buffer,
comprising 10 mM Hepes, pH 7.4, 220 mM
mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM
dithiothreitol (13). The release of fluorescent aminomethylcoumarin was
measured by fluorometry and expressed as fluorescence increase per min
( Western Analysis of YopP, Cytochrome c, Caspases, and
Bid--
For analysis of YopP, cytochrome c release, and
procaspase and Bid cleavage, macrophages were infected with Y. enterocolitica or were left untreated as a control. After 3 h, cells were harvested by centrifugation, washed with
phosphate-buffered saline (PBS), resuspended in 0.002% digitonin (RBI,
Natick, MA) PBS, and incubated for 3 min on ice. Cell membranes
and organelles were pelleted by centrifugation (15,000 × g for 5 min at 4 °C). Subsequently, 50 µg of cytosolic
protein or an equivalent of 2.5 × 104 cells were
loaded per lane on 15% polyacrylamide gels. After electrophoresis, the
gels were blotted onto nitrocellulose (BA 83; Schleicher & Schüll, Dassel, Germany), which was then probed with a rabbit
polyclonal anti-YopP antiserum, mouse monoclonal antibody to cytochrome
c (Pharmingen, San Diego, CA), polyclonal antisera raised
against recombinant murine caspases-3 or -7 (29), and polyclonal rabbit
anti-murine caspase-9 antiserum (cell signaling technology; New
England Biolabs Inc., Beverly, MA) or probed at 4 °C with a
polyclonal goat serum raised against Bid (R & D Systems, Abingdon, UK).
The anti-YopP antiserum was raised against purified YopP produced in
strain E40(pAB409)(pMSK13), affinity purified on nitrocellulose-bound
YopP, and concentrated by passing through a protein G column (Mab kit;
Amersham Pharmacia Biotech). Primary antibody binding was detected with
horseradish peroxidase-conjugated goat anti-mouse IgG, goat anti-rabbit
IgG, or rabbit anti-goat IgG (DAKO A/S, Glostrup, Danmark) for
cytochrome c, caspases and YopP, and Bid, respectively, and
visualized by enhanced chemiluminscence as described in the
manufacturer's instructions (Roche Molecular Biochemicals).
Co-immunoprecipitation--
HEK293T cells (1.5 × 106) were seeded in 9-cm Petri dishes. The next day cells
were transfected via the calcium phosphate precipitation method with 2 µg of pCDNA1-TRADDE, pCDNA1-FADDE,
pEF1-Bid, or pIKK2FLAG (coding for IKK Caspases Mediate Yersinia YopP-induced Cell
Death--
Previous results indicated that YopP/J is required for
Yersinia to induce apoptosis in macrophages (7, 8). To
determine whether procaspase activation was involved in YopP/J-mediated apoptosis, J774A.1 macrophages were pretreated with various
irreversible caspase inhibitors prior to infection with
E40(pMSK41)(pMSK13), a Y. enterocolitica yopP knockout
strain overproducing YopPE40WT (YopPE40WT+).
Ac-YVAD.cmk and zDEVD.fmk are specific inhibitors that bind to the
active site of caspase-1 and caspases-3/-7, respectively, whereas
B-D.fmk is a broad-spectrum caspase inhibitor, and zVAD.fmk is a rather
specific inhibitor of the apoptosis-related initiator caspases,
caspases-8 and -9 (30). zAAD, a granzyme B inhibitor, was used as a
control. As shown in Fig.
1A, YopP-mediated apoptosis is characterized by the induction of membrane blebbing and nuclear condensation and fragmentation. Treatment of macrophages with the
caspase inhibitors zDEVD.fmk or Ac-YVAD.cmk or the granzyme B inhibitor
zAAD.cmk prior to Y. enterocolitica infection could not
prevent YopP-mediated apoptotic cell death. Nevertheless, incubation
with zDEVD.fmk resulted in the complete inhibition of
intracellular DEVDase activity, as determined by a fluorogenic substrate assay (data not shown). In contrast, pretreatment of the
cells with the caspase inhibitors zVAD.fmk and B-D.fmk completely prevented YopP-mediated cell death. Similar results were obtained when
analyzing DNA fragmentation in these cultures by means of TUNEL
staining (Fig. 1B). Incubation of the cells with the
inhibitor alone did not alter their survival rate (data not shown).
These results suggest that YopP/J kills macrophages by activating
procaspases, but inhibition of caspase-1, -3, or -7 activity cannot
prevent the execution of apoptosis.
Yersinia-induced Apoptosis Results in Early Bid Cleavage and
Cytochrome c Release--
To determine the molecular mechanism of
Yersinia-mediated apoptosis we examined the intracellular
events taking place by means of a time kinetics experiment evaluating
procaspase activation, Bid cleavage, and cytochrome c
release. J774A.1 macrophages were infected with the wild type Y. enterocolitica A127 (pYV127) strain or with the virulence
plasmid-cured isogenic strain (pYV127 YopP Is Required for Bid Cleavage, Cytochrome c Release, and
Procaspase Activation--
To investigate which molecular events
observed during Yersinia-induced apoptosis, viz.
Bid cleavage, cytochrome c release, and procaspase
activation, were YopP-dependent we infected J774A.1 macrophages with the E40(pMSK41) strain that is YopP-deficient (YopP Disruption of the Catalytic Domain of YopP Abolishes Apoptosis
Induction by Yersinia--
Recently it was shown that the secondary
structure of YopP/J resembles that of an adenovirus protease (10).
These authors identified YopP/J as a member of an ubiquitin-like
protein cysteine protease family having SUMOylase activity and showed
that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF- YopP Does Not Interact with TRADD, FADD, or Bid--
In cells in
which clustering of death receptors causes weak procaspase-8
activation, the mammalian Bid protein is a specific proximal substrate
of caspase-8 in the signaling pathway (19). YopP/J has been shown to
bind to members of the MAPK family and to IKK Previously it was shown that YopP/J is required for
Yersinia-induced apoptosis in macrophages, but the exact
mechanism of action remained unknown (7, 8). Although the signals that generally lead to apoptosis are quite diverse, the activation of
procaspases plays a central role in the initiation and execution phase
of apoptosis. The caspase family comprises three groups, the
inflammatory caspases (-1, -4, -5, and -11), the initiator caspases (2, -8, -9, and -10), and the executioner caspases (-3, -6, and -7). In
this study we wanted to determine which signaling pathways are
activated in YopP-mediated cell death. We demonstrated an early Bid
cleavage after YopP was injected into the cells by the infecting
bacteria. Bid cleavage clearly occurred before cytochrome c
release, suggesting that the truncated Bid relocalizes to the outer
mitochondrial membrane and initially causes the release of cytochrome
c (20, 21). Subsequent assembly of the cytochrome c·apoptotic protease activating
factor-1·ATP·procaspase-9 complex then initiated the
activation of procaspase-9 leading to postmitochondrial procaspase-3
and -7 activation (30, 35). This seems to be a very rapid event,
because cytochrome c release, procaspase-9 activation, and
procaspase-3 and -7 activation all occur simultaneously. Because tBid
generation becomes evident clearly before the release of cytochrome
c, our data indicate that Bid cleavage is not the result of
postmitochondrial caspase activity as described in other apoptotic
systems (31). Inhibition of the executioner caspases-3 and -7 was not
able to block YopP-mediated apoptosis. Nevertheless, pretreatment
of cells with this inhibitor completely blocked the YopP-induced
DEVDase activity, thus suggesting that the caspases-3 and -7 are
dispensable for the onset of apoptosis and merely play an executing
role in the late stage of apoptosis (36, 37). Furthermore, Ac-YVAD.cmk,
an inhibitor of caspase-1, also failed to block YopP-induced
apoptosis despite the fact that procaspase-1 becomes activated upon
Y. enterocolitica
infection,3 indicating that
caspase-1 activity is not required for Yersinia-mediated macrophage apoptosis. This finding indicates that Yersinia
activates a different cell death pathway in macrophages than
Shigella or Salmonella, two pathogens that make
use of a type III protein secretion mechanism similar to that of
Yersinia sp. Shigella and Salmonella
both produce similar apoptotic invasin proteins, named IpaB and SipB,
respectively (38). During infection these proteins are translocated to
the cell and bind to procaspase-1, resulting in its activation. In
contrast to Yersinia-mediated apoptosis, pretreatment of
macrophages with the Ac-YVAD.cmk caspase-1 inhibitor prevented the
induction of apoptosis by Shigella or Salmonella (39, 40). Furthermore, caspase-1-deficient macrophages are protected
against Shigella- or Salmonella-induced cell
death (41). When cells were pretreated with the caspase inhibitors
zVAD.fmk and B-D.fmk, YopP-mediated apoptosis could be completely
blocked. In addition, YopP-induced Bid cleavage and the subsequent
release of cytochrome c were completely blocked in the
presence of zVAD.fmk. Therefore it seems that YopP-mediated cell death
is mainly caused by the activation of initiator procaspases acting
upstream of caspases-3 and -7. Bid can be cleaved by different
proteases such as caspase-3 (31) or -8 (20, 21) or granzyme B
(42). Although processing of endogenous procaspase-8 could not be
analyzed because of the lack of anti-mouse caspase-8 antisera
with sufficient detection efficiency, procaspase-8 activation may play
a key role in YopP-mediated apoptosis because (a)
YopP-induced procaspase-3 activation occurs only after Bid had been
cleaved, and Ac-DEVD.fmk (a potent caspase-3 inhibitor) treatment could
not prevent Bid cleavage; and (b) the granzyme B inhibitor
zAAD.fmk did not affect the YopP-dependent apoptosis; in
addition, granzyme B is normally not expressed in macrophages. The
presently known activation mechanisms of pro-apoptotic initiator
procaspases include two main pathways, the death domain
receptor-initiated activation of procaspase-8 in the death-inducing signaling complex (43) and the apoptosome-mediated postmitochondrial activation of procaspase-8 by caspase-3 (32). Because the executioner procaspases became activated after Bid had been cleaved, and
caspase-3/7 inhibition could not prevent the onset of the apoptotic
program or the cleavage of Bid, it is less likely that procaspase-8
would be activated by the generation of postmitochondrial caspase
activity. IpaB or SipB can bind directly to procaspase-1, thereby
inducing procaspase-1 activation that is crucial for the induction of
apoptosis by Shigella or Salmonella infections.
Could YopP/J make use of a similar mechanism by binding to
procaspase-8? We were not able to show a clear interaction between YopP
and procaspase-8 in a yeast two hybrid
experiment.4 Another
possibility might be that YopP/J would cluster intracellular pro-apoptotic adapter molecules involved in the activation of procaspase-8 such as TRADD or FADD, thereby mimicking the
death-inducing signaling complex as formed by death receptors of the
TNF-receptor superfamily. Upon receptor activation these receptors can
recruit the adaptor molecules TRADD and/or FADD. Binding of FADD
subsequently can recruit procaspase-8 or -10, and this event leads to
proximity-induced autocleavage of these procaspases (44).
Immunoprecipitation experiments with YopP could confirm that YopP is
able to interact with IKK YopP/J is not only responsible for the induction of apoptosis, but it
also strongly interferes with macrophage signal transduction resulting
in a blockade of two major cell signaling cascades; the MAPK and the
NF- Previous studies indicated that YopP/J interacts with MKKs and IKK Taken together, our data support a model in which the YopP/J protease
activity targets both anti- and pro-apoptotic pathways in macrophages
(Fig. 7). In our model YopP/J-mediated
de-SUMOylation alters the Toll-like receptor signaling in a way
allowing procaspase-8 activation leading to Bid processing. Truncated
Bid then translocates to the mitochondria inducing the release of
cytochrome c, resulting in the assembly of the apoptosome
and the generation of activated caspase-9 and the executioner
caspases-3 and -7. Identification of the YopP/J targets will be
important to further elucidate the YopP/J signaling pathways.
B pathways. Here we analyzed the
YopP/J-induced apoptotic signaling pathway. YopP-mediated cell death
could be inhibited by addition of the zVAD caspase inhibitor,
but not by DEVD or YVAD. Generation of truncated Bid (tBid) was the
first apoptosis-related event that we observed. The subsequent
translocation of tBid to the mitochondria induced the release of
cytochrome c, leading to the activation of procaspase-9 and
the executioner procaspases-3 and -7. Inhibition of the
postmitochondrial executioner caspases-3 and -7 did not affect Bid
cleavage. Bid cleavage could not be observed in a
yopP-deficient Y. enterocolitica strain,
showing that this event requires YopP. Disruption of the catalytic core
of YopP abolished the rapid generation of tBid, thereby hampering
induction of apoptosis by Y. enterocolitica. This finding
supports the idea that YopP/J induces apoptosis by directly acting on
cell death pathways, rather than being the mere consequence of gene
induction inhibition in combination with microbial stimulation of the macrophage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF-
)1 and interleukin-8
production, as the result of blockade of the activation of
mitogen-activated protein kinase (MAPK) kinases (MKKs), MAPK,
and nuclear factor
B (NF-
B) (2-6) and finally induction of
apoptosis in macrophages (7, 8). YopJ-induced apoptosis by Y. pseudotuberculosis has also been observed during an experimental
mouse infection thereby showing that apoptosis plays a role in the
establishment of a systemic infection (9). YopP/J interacts with IKK
and MKKs, and recently it has been suggested that YopP/J belongs to a
family of cysteine proteases related to the ubiquitin-like protein
proteases (6, 10). Ubiquitin-like protein proteases cleave the C
terminus of an 11-kDa small ubiquitin-related modifier SUMO-1.
The protease YopJ was shown to reduce the cellular concentration of
SUMO-1-conjugated proteins in an overexpression experiment; however, no
direct substrate of YopJ was yet identified. Mutation of the YopJ
catalytic cysteine results in the loss of its protease activity and
hampers its capacity to inhibit NF-
B and MKK activation. It has been
suggested that YopP/J-induced apoptosis is due to its capability of
inhibiting the activation of NF-
B (5). However, the exact mechanism
by which YopP/J induces apoptosis is not yet known.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used for
standard manipulations. E. coli SM10
pir+ (24)
was used to deliver mobile plasmids in Y. enterocolitica. Y. enterocolitica E40(pYV40) is a wild type, low virulence
strain from serotype O:9 (25). Y. enterocolitica E40(pMSK41)
is a yopP knockout (yopP23 allele) of
E40(pYV40) (7). E40(pAB409) is a multiknockout mutant of E40(pYV40)
unable to produce YopH, -O, -P, -E, -M, and -B (2). Y. enterocolitica A127(pYV127) is a wild type, high virulence
strain from serotype O:8 (2, 26).
, was a
generous gift from Drs. J. Schmitt and R. de Martin (University of
Vienna, Vienna, Austria).
-mercaptoethanol (2 × 10
5
M). Unless otherwise indicated, macrophages were seeded in
medium without antibiotics at a density of
105/cm2 15 h before infection. Macrophages
were infected with Y. enterocolitica grown under conditions
for moderate Yop induction at 37 °C (see above) with a multiplicity
of infection (m.o.i.) of 50.
F/min). (Cytoflor; PerSeptive Biosystems, Cambridge, MA).
), in the absence
or presence of 2 µg of pEF6M/H-YopPA127WT.
Empty vector was added to a total amount of 5 µg of DNA for each
transfection. 24 h after transfection, cells were lysed in 50 mM Hepes, pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 0.1 mM aprotinin, and 1 mM leupeptin,
and lysates were incubated for 5 h with 2.4 µg of
anti-Myc-tagged antibody (Roche Diagnostics, Basel, Switzerland). Immune complexes were incubated for 1 h with protein A-Trisacryl beads, which were subsequently washed five times with lysis buffer. Coprecipitating proteins were separated by SDS polyacrylamide gel
electrophoresis and analyzed by Western blotting with horseradish peroxidase-coupled anti-E-tagged antibody (Amersham Pharmacia Biotech),
anti-Bid antibody followed by horseradish peroxidase-coupled anti-goat
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or
anti-FLAG-tagged antibody (Sigma) followed by horseradish
peroxidase-coupled anti-mouse antibody (Amersham Pharmacia Biotech),
depending on the plasmid transfected. Protein bands were visualized
using enhanced chemiluminscence as described in the manufacturer's
instructions (Roche Molecular Biochemicals).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (89K):
[in a new window]
Fig. 1.
Y. enterocolitica YopP-induced
apoptosis of J774A.1 macrophages is inhibited by broad-spectrum caspase
inhibitors. J774A.1 macrophages were left untreated or pretreated
with 100 µM of the tetrapeptide caspase inhibitors
Ac-YVAD.cmk (caspase-1 inhibitor), zDEVD.fmk (caspases-3 and -7 inhibitor), zAAD.cmk (granzyme B inhibitor), and zVAD.fmk or B-D.fmk
(broad-spectrum caspase inhibitors) 2 h before infection with a
YopP overproducing (YopPE40WT+) Y. enterocolitica strain at a m.o.i. of 50. Non-infected J774A.1
cells were used as a control. A, microscopic images were
taken 3 h after infection to analyze apoptotic cell morphology.
B, were fixed after 6 h of infection and TUNEL-stained,
and fluorescent images were taken to analyze the effect of the caspase
inhibitors on DNA fragmentation.
), and at different
time intervals cytosolic fractions of these cultures were prepared.
Non-infected cells served as a control. YopP could be detected in the
macrophage cytosol in substantial amounts as soon as 40 min after
co-incubation of cells with bacteria (Fig.
2A). The first
apoptosis-related intracellular event that we were able to monitor was
Bid cleavage (Fig. 2B). Bid does not contain a transmembrane
domain and is located in the cytosol. Cytosolic Bid was cleaved to a
13-kDa fragment (tBid), and the concentration of tBid gradually
increased from 60 min on to 80 min after infection of the macrophages.
Bid protein is a specific proximal substrate of caspase-8 in the
signaling pathway to apoptosis (20, 21). We were not able to analyze
endogenous procaspase-8 expression and/or activation in J774A.1 cells
because of the lack of anti-murine caspase-8 antibodies with
sufficient detection efficiency. It has been described that the
C-terminal fragment of Bid translocates from the cytosol to the
mitochondrial membrane, a process that finally leads to the release of
cytochrome c (20, 21). Cytochrome c release
became evident 20 min after the generation of tBid in the cell (Fig.
2B). The release of cytochrome c results in a
rapid assembly of the apoptosome complex as witnessed by the processing
of procaspase-9 to its enzymatically active p35/p37 subunits,
eventually leading to the activation of the downstream executioner
procaspases-3 and -7 (Fig. 2C). This activation of the
apoptotic cascade finally resulted in the total demise of the
macrophage resulting in the loss of the cell membrane integrity (Fig.
2D). None of the apoptotic events took place in non-infected cells or cells treated with the plasmid-cured strain
(pYV127
). Taken together, our results indicate that
Yersinia infection results in a rapid generation of tBid
that will translocate to the mitochondria and induce the release of
cytochrome c leading to the assembly of the apoptosome
resulting in the activation of the executioner procaspases.
View larger version (35K):
[in a new window]
Fig. 2.
Time kinetic analysis of YopP translocation,
Bid cleavage, cytochrome c release, and procaspase
activation during Y. enterocolitica-induced apoptosis
of macrophages. J774A.1 macrophages were infected with a
plasmid-cured A127 strain (pYV127 ) or with the wild type
A127 strain (pYV127) at a m.o.i. of 50. Non-infected (NI)
J774A.1 cells were used as a control. At different time intervals
cytosolic fractions of J774A.1 cells were prepared by digitonin lysis,
and analyzed by polyacrylamide gel electrophoresis and Western
blotting, making use of anti-YopP (A), anti-Bid
(B), anti-cytochrome c (B), or
anti-caspase-9, -3, and -7 (C) antibodies. As a control for
cell death, the percentage of cells that lost plasma membrane integrity
(D), determined by propidium iodide (PI) uptake,
was analyzed.
) and with the E40(pMSK41)(pMSK13) strain that
overproduces YopPE40WT (YopPE40WT+). Our data
demonstrate that Bid cleavage is YopP-dependent, because
macrophages infected with the YopP
strain, expressing all
other Yops, only contained the full-length Bid protein of 22 kDa as
uninfected macrophages do, in contrast to the observed Bid cleavage
when cells were infected with the YopPE40WT+ strain (Fig.
3A). As expected the
downstream effects of Bid cleavage, such as mitochondrial cytochrome
c release (Fig. 3A) and subsequent procaspase-3
and -7 activation, also did not occur when the YopP
strain was used for infection (Fig. 3B). The absence of
procaspase activation upon infection with the YopP
strain
was confirmed by measuring the DEVDase activity in the respective cell
lysates (Fig. 3C). In addition, cleavage of Bid and
subsequent cytochrome c release could be prevented by
pretreatment of the macrophages with the caspase inhibitor zVAD.fmk
(Fig. 3A). To exclude the possibility that Bid cleavage is
the result of a feedback loop, catalyzed by postmitochondrial
procaspase-3 activation (32), we analyzed the levels of tBid in
zDEVD.fmk pretreated and control macrophages infected with the
YopPE40WT+ strain. Hence, if Bid cleavage would indeed be
the upstream trigger of the YopP-induced apoptotic signaling cascade as
a result of initiator procaspase-8 activation, it should not be
affected by pretreatment with the zDEVD.fmk inhibitor. As expected from
the results obtained in the kinetics experiment (Fig. 2), caspase-3/-7
inhibition did not change the amount of tBid generated upon
YopPE40WT+ infection (Fig.
4A). As a control we show that
zDEVD.fmk addition prior to Yersinia infection
prevented the generation of DEVDase activity (Fig. 4B).
These data indicate that YopP in some way activates an upstream caspase
activity, most likely caspase-8, resulting in the cleavage of Bid.
View larger version (20K):
[in a new window]
Fig. 3.
YopP is necessary for the induction of Bid
cleavage, cytochrome c release, and procaspase-3 and
-7 activation during Y. enterocolitica-induced
apoptosis of macrophages. J774A.1 were infected for 3 h with
Y. enterocolitica YopP and
YopPE40WT+ strains at a m.o.i. of 50 with or without
pretreatment of the caspase inhibitor zVAD.fmk (100 µM).
Equivalent amounts of proteins were analyzed by Western blot making use
of anti-Bid (A), anti-cytochrome c antibodies
(A), or anti-caspases-3 or -7 antibodies (B).
Open and closed arrows indicate cleaved and
uncleaved proteins, respectively. Caspase activity in cell extracts was
measured making use of the Ac-DEVD aminomethyl coumarin
fluorogenic substrate, as described under "Experimental Procedures"
(C). NI, non-infected.
View larger version (24K):
[in a new window]
Fig. 4.
YopP-induced Bid cleavage is not the result
of a caspase-3-dependent feedback loop. Control and
zDEVD.fmk (100 µM) pretreated J774A.1 macrophages were
infected with the YopPE40WT+ strain, and 2.5 h after
infection cells were lysed. A, cell lysates were analyzed by
polyacrylamide gel electrophoresis and Western blotting, making use of
an anti-Bid antiserum. B. activity was determined by
incubation of cell lysates with the fluorogenic substrate Ac-DEVD.amc,
as described under "Experimental Procedures." NI,
non-infected.
B pathways. Alignment of the catalytic domains of Y. enterocolitica
YopPA127,2
Y. pestis YopJKIM5, and Y. pseudotuberculosis YopJYPIII with the human SUMO
proteases SUSP1 and SENP1 (33, 34) indicates that the residues of the
catalytic triade (His, Asp/Glu, and Cys) are conserved in YopP/J (Fig.
5A). To analyze whether the
YopP cysteine protease activity is required for induction of apoptosis, we mutated the conserved catalytic cysteine in YopP, and a
YopP-deficient Y. enterocolitica strain was complemented
with wild type YopP (YopPE40WT+) or mutated YopP
(YopPE40C172T+). Then macrophages were infected with the
different Yersinia strains generated, and the effect of the
YopP catalytic cysteine mutant on the apoptosis induction by YopP was
analyzed. The Y. enterocolitica E40 strain is less virulent
and therefore results in a slower onset of apoptosis as compared with
the A127 strain. Mutation of the YopP catalytic cysteine abolished
completely the ability of Y. enterocolitica to induce
apoptosis in the infected macrophages (Fig. 5). Microscopic evaluation
of the infected cultures indicated clear induction of apoptosis,
characterized by the typical hallmarks as membrane blebbing and nuclear
fragmentation, when the macrophages were co-incubated with the
YopPE40WT+ strain (Fig. 5B). In contrast, the
YopPE40C172T+-infected cells looked similar to
YopP
-infected cultures, showing a non-apoptotic
morphology. Importantly, we demonstrated that both the wild type and
the mutant YopP were translocated equally well to the macrophages upon
infection with the respective Yersinia strains (Fig.
5C), but only infection with the YopPE40WT+
strain induced Bid processing (Fig. 5D). These observations
were in agreement with the fact that cytosolic caspase activity was
only generated in the YopPE40WT+-infected cultures (Fig.
5E). Overexpression of YopPA127WT and
YopPA127C172T in HEK293T cells confirmed that the catalytic
cysteine is required for inhibition of NF-
B activation (data not
shown), as shown previously (10). These data indicate that YopP/J
protease activity is required both for the inhibition of NF-
B
signaling and the induction of apoptosis by Yersinia.
View larger version (62K):
[in a new window]
Fig. 5.
The YopP catalytic cysteine is
necessary for the induction of apoptosis during Y. enterocolitica infection of macrophages. A,
sequence alignment of the conserved putative catalytic domains of
Y. enterocolitica YopPA127,2
Y. pestis YopJKIM5 (Accession number
NP047569), and Y. pseudotuberculosis
YopJYPIII (Accession number AAA68488) with the human SUMO
proteases hSUSP1 (Accession number AF196304) and hSENP1 (Accession
number AF149770) (33, 34). Arrowheads indicate the amino
acid residues of the catalytic triad (His, Asp/Glu, and Cys), and
shading denotes identical amino acid residues. J774A.1 were infected
with Y. enterocolitica YopP ,
YopPE40WT+, and YopPE40C172T+ strains at a
m.o.i. of 50. Non-infected (NI) J774A.1 cells were used as a
control. B, light microscopic images were taken 3 h
after infection to analyze apoptotic cell morphology. At different time
intervals cytosolic fractions of J774A.1 cells were prepared by
digitonin lysis and analyzed by polyacrylamide gel electrophoresis and
Western blotting using anti-YopP (C) and anti-Bid
(D) antibodies. Open and closed arrows
indicate cleaved and uncleaved proteins, respectively. E,
caspase activity in cell extracts was measured making use of the
Ac-DEVD aminomethyl coumarin fluorogenic substrate, as described under
"Experimental Procedures."
and to interfere with
these signaling pathways. The mechanism by which YopP/J induces
apoptosis is not yet completely clear, but our data suggest an
implication of caspase-dependent cleavage of Bid. A
possible hypothesis could be that YopP/J clusters pro-apoptotic signaling molecules thereby starting the cell death machinery. For this
reason we analyzed whether YopP could bind directly to pro-apoptotic
signal transduction molecules such as TRADD, FADD, or Bid. Therefore,
we overexpressed these molecules in HEK293T cells, together with
YopPA127WT, and performed an immunoprecipitation experiment. FADD, TRADD, and Bid could not be
co-immunoprecipitated with YopPA127WT (Fig.
6), indicating that YopP is not
able to cluster these pro-apoptotic mediators thereby initiating an
apoptotic cascade. As a positive control, we could co-immunoprecipitate IKK
with YopPA127WT (6). Additionally,
YopPA127WT overexpression did not lead to Bid processing
(data not shown), indicating that Bid is not a direct substrate for the
YopP protease.
View larger version (39K):
[in a new window]
Fig. 6.
YopP does not interact with
TRADD, FADD, and Bid. HEK293T cells were transiently transfected
with the expression plasmids pCDNA1-TRADDE,
pCDNA1-FADDE, pEF1-Bid, or pIKK2FLAG, in
the absence or presence of 2 µg of
pEF6M/H-YopPA127WT, as described under
"Experimental Procedures." Cultures were lysed 20 h after
transfection, and YopP was immunoprecipitated with anti-Myc-tagged
antibodies. Co-immunoprecipitation of TRADD, FADD, Bid, or IKK2
was detected via Western blot analysis with anti-E-, anti-Bid-, or
anti-FLAG-tagged antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(6) but did not provide evidence for
binding of YopP with TRADD or FADD. Hence, the trigger that leads to
YopP/J-induced procaspase activation required for Bid cleavage remains
elusive. Given the observation that YopP is a protease we also
considered whether Bid could be a direct substrate for YopP.
Overexpression of YopP in HEK293T cells did not lead to processing of
Bid or apoptosis.5
Overexpression of wild type YopP, but not of YopPA127C172T,
resulted in the inhibition of NF-
B activation indicating that YopP
has enzymatic activity upon overexpression in HEK293 cells.
B pathway are both involved in transcriptional gene control
(2-5). Indeed, the production of proinflammatory cytokines would be
disadvantageous for the extracellular lifestyle of Yersinia.
It has been shown that YopP/J inhibits these pathways by interacting
directly with IKK
and several MKKs, thereby preventing their
activation (6). Recently it was suggested that the YopP/J-induced apoptosis of macrophages could merely result from its inhibition of
NF-
B activation in combination with lipopolysaccharide (LPS) (5,
45). In contrast to macrophages, other cell types (fibroblasts and HeLa
cells) do not undergo apoptotic cell death upon Yersinia infection. Nevertheless, YopP/J is translocated efficiently in HeLa
cells (4),6 resulting in
inhibition of NF-
B activation and interleukin-8 cytokine production
(4, 5). YopP/J exerts its effect on HeLa cells, but this does not lead
to the induction of programmed cell death, suggesting the absence of a
signaling pathway in this cell type when compared with macrophages. As
shown for Yersinia and E. coli, LPS can trigger
apoptosis when NF-
B activation is inhibited (5, 46). LPS binds the
CD14 receptor, a receptor only present on monocytes and macrophages.
The LPS signal is transduced across the plasma membrane by the
toll-like receptor 4 (TLR-4), which associates with CD14 to form the
LPS receptor complex (47). Recently it was shown that another class of
microbial surface molecules, the bacterial lipoproteins (BLPs), induced
apoptosis in macrophages by binding TLR-2 (48). Furthermore, epithelial cell lines transfected with TLR-2 underwent bacterial
lipoprotein-mediated apoptosis. Altogether these studies suggest that
bacteria-mediated apoptosis requires the presence of Toll-like
receptors at the cell surface. During Yersinia infection,
LPS and/or BLPs and YopP might both be required to fulfill the process
of apoptosis. In addition it was suggested that the BLP-stimulated
TLR-2-triggered apoptosis occurs via a pathway involving MyD88, FADD,
and caspase-8 (49). These data indicate that the early Bid cleavage
observed in our experiments could well be because of activation of the initiator procaspase-8 by TLR-2 signaling. In this context, YopP/J would only be required to inhibit gene activation thereby preventing the up-regulation of anti-apoptotic proteins in the macrophage. This
hypothesis would imply that infection of macrophages with the
YopPE40C172T+ mutant strain would not affect the early Bid
processing, already significant 20 min after YopP injection in the
macrophage cytosol upon infection. Because we show here that Bid
cleavage is impaired upon YopPE40C172T+ infection, we
speculate that YopP/J induces apoptosis by a direct action on cell
death signaling pathways.
and prevents their phosphorylation. However, MKK SUMOylation was not
observed, and data on IKK
SUMOylation are not available; hence the
targets for YopP/J de-SUMOylation remain to be identified. MKKs could
serve as shuttles to escort YopP/J to the signaling complex where it
would affect critical SUMO-1-conjugated proteins. It has been observed
that the NF-
B signaling molecules such as IKK
can be recruited to
the TNF·receptor signaling complex (50); a similar recruitment event
could be valid for the Toll-like receptors. Hence, it should be
considered that YopP/J may directly affect Toll-like receptor signaling
in this way causing apoptotic signaling in macrophages.
View larger version (29K):
[in a new window]
Fig. 7.
Model showing the effects of YopP on the
intracellular signal transduction pathways in macrophages upon
infection. YopP/J of Y. enterocolitica, which has
been suggested to be an ubiquitin-like protein protease, has been shown
to decrease both the cellular concentration of SUMO-1-conjugated
proteins and the levels of free SUMO-1 (10). YopP/J is responsible for
the down-regulation of the inflammatory response, by binding to and
preventing the activation of members of the MKK family and of IKK
(6). By blocking both these pathways YopP/J efficiently shuts down
multiple kinase cascades and cytokine induction required by the host
cell to respond to a bacterial infection. YopP is also responsible for
the induction of apoptosis of macrophages (7, 8). Here we demonstrate
that this involves the apoptotic signaling cascade upstream of Bid,
probably by interference with a signaling pathway triggered from the
TLRs. YopP-mediated de-SUMOylation may directly act on the TLR
signaling pathway leading to caspase-8-mediated Bid processing, rather
than being the mere consequence of the inhibition of NF-
B
activation, which would result in the down-regulation of several
anti-apoptotic genes, as was previously suggested (5, 45). The cysteine
protease activity of YopP/J is necessary for both the down-regulation
of the inflammatory response and the induction of apoptosis of
macrophages (see Ref. 10 and this work). However, exactly how the
YopP-de-SUMOylating activity is interrelated with the inhibition of the
MKKs and IKK
and the induction toward apoptosis awaits further
research. APAF-1, apoptotic protease activating factor-1;
DD, death domain; IRAK, IL-1 receptor-associated kinase;
MyD88, myeloid differentiation factor 88.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank B. Foultier for the sequence of yopPA127.
![]() |
FOOTNOTES |
---|
* This work was supported by the Belgian Fonds National de la Recherche Scientifique Médicale (convention 3.4595.97), the Direction Générale de la Recherche Scientifique-Communauté Française de Belgique (Action de Recherche Concertée 99/04-236), the Interuniversitaire Attractiepolen (number P4/26), European Community-Research Technology and Development Grant QLRT-199-00739, and the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (number 3G000601). C. G. was a recipient of a Marie-Curie fellowship from the European Union (contract ERBFMBICT972047).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.
§ Contributed equally to this work.
Present address: Division of Cell Biology, Cancer Inst.,
Plesmanstraat 121, 1066 CX Amsterdam, The Netherlands.
** Present address: Laboratoire de Bacteriologie Moléculaire, Université Libre de Bruxelles, Route de lennik 808, CP614, 1070 Bruxelles, Belgium.
¶¶ Present address: UMR1582, Institut Gustave Roussy, 94805 Villejuif, France.
To whom correspondence should be addressed. Tel.:
32-2-764-7449; Fax: 32-2-764-7498; E-mail:
cornelis@mipa.ucl.ac.be.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M101573200
2 B. Foultier and G. C., unpublished results.
3 G. D. and G. C., unpublished results.
4 M. V. G. and P. V., unpublished results.
5 G. D. and G. C., unpublished results.
6 M.-P. S. and G. C., unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor necrosis factor-
;
cmk, chloromethylketone;
fmk, fluoromethylketone;
Ac-YVAD, acetyl-Tyr-Val-Ala-Asp;
B-D, benzyloxycarbonyl-Asp(OMe);
BLP(s), bacterial lipoprotein(s);
FADD, Fas-associated death domain;
FADDE, Fas-associated death
domain C-terminal coupled to an E tag;
IKK, I
B
kinase kinase;
LPS, lipopolysaccharide;
MAPK, mitogen-activated protein kinase;
MKK(s), MAPK kinase(s);
m.o.i., multiplicity of infection;
NF-
B, nuclear factor
B;
TRADDE, TNF
receptor-associated death domain C-terminal coupled to an E tag;
TRADD, TNF receptor-associated death domain;
TLR, toll-like receptor;
zAAD, benzyloxycarbonyl-Ala-Ala-Asp;
zDEVD, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe);
zVAD, benzyloxycarbonyl-Val-Ala-Asp(OMe);
tBid, truncated Bid;
WT, wild type;
TUNEL, terminal deoxyribonucleotidyl transferase-mediated
dUTP-digoxigenin nick-end labeling;
SUMO-1, small ubiquitin-related
modifier-1.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Cornelis, G. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8778-8783 |
2. |
Boland, A.,
and Cornelis, G. R.
(1998)
Infect. Immun.
66,
1878-1884 |
3. | Palmer, L. E., Hobbie, S., Galan, J. E., and Bliska, J. B. (1998) Mol. Microbiol. 27, 953-965[CrossRef][Medline] [Order article via Infotrieve] |
4. | Schesser, K., Spiik, A. K., Dukuzumuremyi, J. M., Neurath, M. F., Pettersson, S., and Wolf-Watz, H. (1998) Mol. Microbiol. 28, 1067-1079[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Ruckdeschel, K.,
Harb, S.,
Roggenkamp, A.,
Hornef, M.,
Zumbihl, R.,
Kohler, S.,
Heesemann, J.,
and Rouot, B.
(1998)
J. Exp. Med.
187,
1069-1079 |
6. |
Orth, K.,
Palmer, L. E.,
Bao, Z. Q.,
Stewart, S.,
Rudolph, A. E.,
Bliska, J. B.,
and Dixon, J. E.
(1999)
Science
285,
1920-1923 |
7. |
Mills, S. D.,
Boland, A.,
Sory, M. P.,
van der Smissen, P.,
Kerbourch, C.,
Finlay, B. B.,
and Cornelis, G. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12638-12643 |
8. |
Monack, D. M.,
Mecsas, J.,
Ghori, N.,
and Falkow, S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10385-10390 |
9. |
Monack, D. M.,
Mecsas, J.,
Bouley, D.,
and Falkow, S.
(1998)
J. Exp. Med.
188,
2127-2137 |
10. |
Orth, K.,
Xu, Z.,
Mudgett, M. B.,
Bao, Z. Q.,
Palmer, L. E.,
Bliska, J. B.,
Mangel, W. F.,
Staskawicz, B.,
and Dixon, J. E.
(2000)
Science
290,
1594-1597 |
11. | Cohen, G. M. (1997) Biochem. J. 326, 1-16[Medline] [Order article via Infotrieve] |
12. | Stennicke, H. R., and Salvesen, G. S. (1999) Cell Death Differ. 6, 1054-1059[CrossRef][Medline] [Order article via Infotrieve] |
13. | Van de Craen, M., Declercq, W., Van den Brande, I., Fiers, W., and Vandenabeele, P. (1999) Cell Death Differ. 6, 1117-1124[CrossRef][Medline] [Order article via Infotrieve] |
14. | Wallach, D., Varfolomeev, E. E., Malinin, N. L., Goltsev, Y. V., Kovalenko, A. V., and Boldin, M. P. (1999) Annu. Rev. Immunol. 17, 331-367[CrossRef][Medline] [Order article via Infotrieve] |
15. | Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., and Peter, M. E. (1998) Eur. J. Biochem. 254, 439-459[Abstract] |
16. | Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H., and Lesslauer, W. (1993) Cell 73, 431-445[Medline] [Order article via Infotrieve] |
17. | Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P. H., and Peter, M. E. (1995) EMBO J. 14, 5579-5588[Abstract] |
18. |
Muzio, M.,
Salvesen, G. S.,
and Dixit, V. M.
(1997)
J. Biol. Chem.
272,
2952-2956 |
19. |
Scaffidi, C.,
Fulda, S.,
Srinivasan, A.,
Friesen, C.,
Li, F.,
Tomaselli, K. J.,
Debatin, K. M.,
Krammer, P. H.,
and Peter, M. E.
(1998)
EMBO J.
17,
1675-1687 |
20. | Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve] |
21. | Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[Medline] [Order article via Infotrieve] |
22. |
Cain, K.,
Brown, D. G.,
Langlais, C.,
and Cohen, G. M.
(1999)
J. Biol. Chem.
274,
22686-22692 |
23. | Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[Medline] [Order article via Infotrieve] |
24. | Miller, V. L., and Mekalanos, J. J. (1988) J. Bacteriol. 170, 2575-2583[Medline] [Order article via Infotrieve] |
25. | Sory, M. P., Boland, A., Lambermont, I., and Cornelis, G. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11998-12002[Abstract] |
26. | Ichinohe, H., Yoshioka, M., Fukushima, H., Kaneko, S., and Maruyama, T. (1991) J. Clin. Microbiol. 29, 846-847[Medline] [Order article via Infotrieve] |
27. | Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve] |
28. | Carpentier, I., and Beyaert, R. (1999) FEBS Lett. 460, 246-250[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Vercammen, D.,
Brouckaert, G.,
Denecker, G.,
Van de Craen, M.,
Declercq, W.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
188,
919-930 |
30. | Ekert, P. G., Silke, J., and Vaux, D. L. (1999) Cell Death Differ. 6, 1081-1086[CrossRef][Medline] [Order article via Infotrieve] |
31. | Slee, E. A., Keogh, S. A., and Martin, S. J. (2000) Cell Death Differ. 7, 556-565[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Slee, E. A.,
Harte, M. T.,
Kluck, R. M.,
Wolf, B. B.,
Casiano, C. A.,
Newmeyer, D. D.,
Wang, H. G.,
Reed, J. C.,
Nicholson, D. W.,
Alnemri, E. S.,
Green, D. R.,
and Martin, S. J.
(1999)
J. Cell Biol.
144,
281-292 |
33. |
Kim, K. I.,
Baek, S. H.,
Jeon, Y. J.,
Nishimori, S.,
Suzuki, T.,
Uchida, S.,
Shimbara, N.,
Saitoh, H.,
Tanaka, K.,
and Chung, C. H.
(2000)
J. Biol. Chem.
275,
14102-14106 |
34. |
Gong, L.,
Millas, S.,
Maul, G. G.,
and Yeh, E. T.
(2000)
J. Biol. Chem.
275,
3355-3359 |
35. | Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Cell 86, 147-157[Medline] [Order article via Infotrieve] |
36. | Nicholson, D. W., and Thornberry, N. A. (1997) Trends Biochem. Sci. 22, 299-306[CrossRef][Medline] [Order article via Infotrieve] |
37. | Reed, J. C. (1997) Cell 91, 559-562[Medline] [Order article via Infotrieve] |
38. | Navarre, W. W., and Zychlinsky, A. (2000) Cell. Microbiol. 2, 265-273[CrossRef][Medline] [Order article via Infotrieve] |
39. | Chen, Y., Smith, M. R., Thirumalai, K., and Zychlinsky, A. (1996) EMBO J. 15, 3853-3860[Abstract] |
40. |
Hersh, D.,
Monack, D. M.,
Smith, M. R.,
Ghori, N.,
Falkow, S.,
and Zychlinsky, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2396-2401 |
41. |
Hilbi, H.,
Moss, J. E.,
Hersh, D.,
Chen, Y.,
Arondel, J.,
Banerjee, S.,
Flavell, R. A.,
Yuan, J.,
Sansonetti, P. J.,
and Zychlinsky, A.
(1998)
J. Biol. Chem.
273,
32895-32900 |
42. |
Heibein, J. A.,
Goping, I. S.,
Barry, M.,
Pinkoski, M. J.,
Shore, G. C.,
Green, D. R.,
and Bleackley, R. C.
(2000)
J. Exp. Med.
192,
1391-1402 |
43. |
Medema, J. P.,
Scaffidi, C.,
Kischkel, F. C.,
Shevchenko, A.,
Mann, M.,
Krammer, P. H.,
and Peter, M. E.
(1997)
EMBO J.
16,
2794-2804 |
44. |
Muzio, M.,
Stockwell, B. R.,
Stennicke, H. R.,
Salvesen, G. S.,
and Dixit, V. M.
(1998)
J. Biol. Chem.
273,
2926-2930 |
45. |
Ruckdeschel, K.,
Mannel, O.,
Richter, K.,
Jacobi, C.,
Trülzsch, K.,
Rouot, B.,
and Heesemann, J.
(2001)
J. Immunol.
166,
1823-1831 |
46. | Buscher, D., Hipskind, R. A., Krautwald, S., Reimann, T., and Baccarini, M. (1995) Mol. Cell. Biol. 15, 466-475[Abstract] |
47. |
Poltorak, A.,
He, X.,
Smirnova, I.,
Liu, M. Y.,
Huffel, C. V.,
Du, X.,
Birdwell, D.,
Alejos, E.,
Silva, M.,
Galanos, C.,
Freudenberg, M.,
Ricciardi-Castagnoli, P.,
Layton, B.,
and Beutler, B.
(1998)
Science
282,
2085-2088 |
48. |
Aliprantis, A. O.,
Yang, R. B.,
Mark, M. R.,
Suggett, S.,
Devaux, B.,
Radolf, J. D.,
Klimpel, G. R.,
Godowski, P.,
and Zychlinsky, A.
(1999)
Science
285,
736-739 |
49. |
Aliprantis, A. O.,
Yang, R. B.,
Weiss, D. S.,
Godowski, P.,
and Zychlinsky, A.
(2000)
EMBO J.
19,
3325-3336 |
50. | Zhang, S. Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000) Immunity 12, 301-311[Medline] [Order article via Infotrieve] |