From the Solange Gauthier Karsh Molecular Genetics
Laboratory, Children's Hospital of Eastern Ontario, Research
Institute, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada, the
§ Department of Biochemistry, Microbiology, and Immunology,
University of Ottawa, 451 Smyth Road, Ottawa K1H 8M5, Canada, and the
Department of Pathology, University of Michigan Medical School,
Ann Arbor, Michigan 48109-0602
Received for publication, December 16, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Smac/DIABLO is a mitochondrial protein that is
proteolytically processed and released during apoptosis along with
cytochrome c and other proapoptotic factors. Once in the
cytosol, Smac protein binds to inhibitors of
apoptosis (IAP) proteins and disrupts the ability of the
IAPs to inhibit caspases 3, 7, and 9. The requirement for mitochondrial
processing and release has complicated efforts to delineate the effect
of Smac overexpression and IAP inhibition on cell death processes. In
this report, we document a novel expression system using ubiquitin
fusions to express mature, biologically active Smac in the cytosol of
transfected cells. Processing of the ubiquitin-Smac fusions is
rapid and complete and generates mature Smac protein initiating
correctly with the Ala-Val-Pro-Ile tetrapeptide sequence that is
required for proper function. The biological activity of this exogenous
protein was demonstrated by its interaction with X-linked IAP, one of
the most potent of the IAPs. The presence of mature Smac was not
sufficient to trigger apoptosis of healthy cells. However, cells with
excess Smac protein were greatly sensitized to apoptotic triggers such
as etoposide exposure. Cancer cells typically display deregulated
apoptotic pathways, including Bcl2 overexpression, thereby suppressing
the release of cytochrome c and Smac. The ability to
circumvent the requirement for mitochondrial processing and release is
critical to developing Smac as a possible gene therapy payload in
cancer chemosensitization.
Virtually all known apoptosis signal pathways converge on the
caspases, a class of cysteine proteases that form a self-amplifying cascade whose activity is responsible for most of the characteristic morphological and biochemical features of apoptotic cell death (for reviews, see Refs. 1-4). The inhibitor of
apoptosis (IAP)1
genes encode a family of proteins that bind and inactivate key caspases
involved in the initiation (caspase 9) and execution (caspases 3 and 7)
of this cascade (for reviews, see Refs. 5-9). Although additional
proteins have been identified that modulate the activity of initiator
caspases, the IAPs are the only cellular proteins known to control the
effector stage of the caspase cascade. The IAPs are uniquely situated
at this central control point and have been conserved in organisms as
divergent as Drosophila, Caenorhabditis elegans,
and mammals.
The defining characteristic of an IAP is the presence of one or more
baculoviral IAP repeat (BIR)
domains. BIR domains fold into a series of four or five Several recently identified proteins negatively regulate IAP function,
including XIAP-associated factor-1
(XAF1; Ref. 13), second mitochondrial derived
activator of caspases (Smac, also known as
DIABLO; Refs. 14 and 15), and Omi (also known as HtrA2; Refs. 16-19).
Both Smac and Omi possess leader peptide sequences that target these
proteins to the intermembrane space of the mitochondria. Smac and Omi
proteins are processed and released along with cytochrome c,
apoptosis-inducing factor (AIF),
procaspases, and other proapoptotic factors when cells are triggered to
undergo apoptosis (for reviews, see Refs. 20-22). Proteolytic removal
of the mitochondrial signal peptide sequences generates novel amino
termini that are critical for interaction with the IAPs and that
disrupt IAP-caspase interactions, thereby promoting caspase activity
and apoptosis (23, 24). Remarkably these amino termini are similar to
the Drosophila proteins Grim, Reaper, Sickle, and
Hid, all of which are proapoptotic antagonists of
Drosophila IAPs (25-31). Despite the similarities at the
amino termini of these proteins, there is little or no homology
throughout the rest of the coding sequences. Furthermore, insect IAP
antagonists display this motif at their extreme amino terminus with the
methionine start codon presumably removed by endogenous amino
peptidases. Transcriptional regulation of the IAPs and their
antagonists in insect cells determines cell fate. In contrast, the
mammalian equivalents, Smac and Omi, are sequestered in an inactive
state in the mitochondrial intermembrane space until an apoptotic
stimulus has occurred.
The consequences of Smac expression have been complicated by the
absolute requirement for an alanine at the amino terminus of the mature
protein. Microinjection or transfection of Smac-like peptides from the
amino terminus of the mature protein recapitulate some but not all of
the effects of Smac (32). Transfection of plasmids encoding the
full-length Smac open reading frame results in the accumulation
of excess Smac in the mitochondrial intermembrane space. Although it
has been demonstrated that Smac overexpression sensitizes cells to
apoptotic triggers, such as UV light, death ligands, or
chemotherapeutic drugs (33-35), it is difficult to distinguish the
effects of Smac release from those caused by other proapoptotic factors
that are released concurrently. One experimental approach to resolve
this difficulty fused GFP to the mature Smac coding sequences with a
caspase 8 cleavage site used to generate the correct AVPI-initiating
mature Smac protein. TRAIL receptor engagement thus activated the
GFP-Smac protein directly without a mitochondrial requirement for
processing, sensitizing cells to TRAIL-induced killing (32).
As an alternative approach to dissecting the consequences of mature
Smac in the cytosol of cells, we have developed a novel system using
ubiquitin (Ub) fusion proteins. Ubiquitin is synthesized in cells as a
precursor polyprotein in which the Ub coding region is fused to itself
and to ribosomal protein subunits (for a review, see Ref. 36). All
cells possess ubiquitin-specific proteases, which hydrolyze the
isopeptide bond between Ub-Ub polymers or Ub-protein substrates. No
sequence requirements are present downstream of the Ub peptide, thereby
allowing the generation of any desired amino terminus for the
downstream fusion partner. We thus used this system to generate mature
Smac protein initiating with the correct AVPI amino terminus in the
cytosol of transfected cells, bypassing the normal requirement for
mitochondrial processing.
Plasmid DNA Constructs
Ub-Smac--
The ubiquitin and mouse Smac/DIABLO coding regions
were fused by an overlapping PCR strategy. The Ub coding region was
amplified using the primers
5'-d-AAAAAGCTTAAAATGAGAGGCAGCCACCACCATCACCATCACATGCAGATCTTCGTG and 5'-d-CTGAGCAATAGGAACCGCACCACCTCTCAGACGCAGGAC. The Smac coding region was amplified with 5'd-CGTCTGAGAGGTGGTGCGGTTCCTATTGCTCAG and
5'-d-TACTCGAGTTAGGCGTAATCAGGGACATCGATAGGATAATCTTCACGCAGGTAGGC. Ub
and Smac PCR products were denatured at 95 °C, annealed together at
50 °C, and reamplified with the Ub forward and Smac reverse primers.
The Ub-Smac PCR product was TA cloned in PCR2.1 (Invitrogen) and
completely sequenced. The coding region was then subcloned using
HindIII and XhoI sites into a ubiquitin C
promoter-containing expression vector (courtesy of Dr. Douglas Gray,
Ottawa Regional Cancer Center, Ottawa, Canada). The final plasmid
construct encodes a 279-amino acid (aa) precursor protein consisting of
86 aa of human ubiquitin fused to aa residues 54-237 of mouse Smac
with a 9-aa carboxyl-terminal HA tag.
Ub- pGEX-XIAP-BIR1, -BIR2, and -BIR3--
Individual BIR domains of
XIAP were PCR-amplified and TA cloned using the following primers:
BIR1, 5'-d-CAGGATCCATGACTTTTAACAGTTTTGAAGG and
5'-d-GACTCGAGCTAGCTTCCCAGATAGTTTTCAAC; BIR2,
5'-d-CAGGATCCATGAGAGATCATTTTGCCTTAGAC and
5'-d-GACTCGAGCTATTCACTTCGAATATTAAGATTCCG; BIR3,
5'-d-CAGGATCCATGTCTGATGCTGTGAGTTCTG and
5'-d-GACTCGAGCTAAGTAGTTCTTACCAGACACTCCTCAAG. The XIAP BIR domain
fragments (aa 1-123, 124-240, and 241-356) were subcloned into
pGEX-4T3 using the BamHI and XhoI sites present
in the primers.
Cell Culture and Transient Transfections
Human embryonic lung (HEL) 299 cells and 293A embryonic kidney
cells were maintained at 37 °C and 5% CO2 in
Dulbecco's minimal essential medium (Invitrogen) supplemented with
10% heat-inactivated fetal calf serum (Invitrogen), penicillin, and
streptomycin (Invitrogen). Cultures were maintained in 100-mm tissue
culture grade dishes (Corning) and passaged every 72 h.
Transfections were performed using LipofectAMINE 2000TM
(Invitrogen) as outlined in the manufacturer's protocol. Plasmid DNAs
were prepared using the Qiagen MaxiprepTM kit (Qiagen)
according to the manufacturer's protocol.
Immunoprecipitation
100-mm dishes of transfected and untransfected cells were rinsed
two times in PBS. 500 µl of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl) containing protease inhibitors (10 µM aprotinin, 100 µM pepstatin A, 10 µM leupeptin, and 100 µM
phenylmethylsulfonyl fluoride) (all from Sigma) were added to each
dish, and the cell lysates were scraped into Eppendorf tubes. The
lysates were then sonicated twice for 30 s each using a probe
sonicator (Sonics and Materials, Inc.). Lysates were centrifuged at
17,000 × g for 10 min. The supernatant was removed and
placed in a fresh Eppendorf tube, and the pellet was discarded. 3 µg of anti-RIAP3 (rat XIAP) were added to the supernatant, and the samples
were incubated for 1 h on a rotating shaker at 4 °C. Following the 1-h incubation, 40 µl of a protein A-Sepharose (Amersham
Biosciences) bead slurry were added to the samples. The samples were
incubated for 1-h on a rotating shaker at 4 °C and then washed three
times in immunoprecipitation buffer. The beads were syringe-dried using a 28-gauge needle, 1× Laemmli sample buffer was added, and
samples were separated on SDS-polyacrylamide gels.
Cell Fractionation
HEL299 cells were seeded in 100-mm dishes at a density of 5 × 106 cells/dish. 24 h following Ub-Smac
transfection, the cells were washed two times in PBS, trypsinized, and
pelleted at 1, 500 × g at 4 °C. Cell pellets were
resuspended in ice-cold RSB (10 mM NaCl, 1.5 mM
MgCl2, 10 mM Tris-HCl, pH 7.5) and transferred
to a 2-ml Dounce homogenizer. After allowing the cells to swell for 7 min, the cells were broken open with several strokes of the pestle.
One-half volume 2.5× MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 7.5) was immediately added to give a final
concentration of 1× MS, and the homogenate was mixed by inversion. The
homogenate was then transferred to an Eppendorf tube and centrifuged at
1,300 × g for 5 min to remove nuclei and large
membrane fragments. The supernatant was centrifuged one more time at
1,300 × g and then transferred to a clean Eppendorf tube. The supernatant was centrifuged at 17,000 × g
for 15 min to obtain a mitochondrial pellet. The mitochondrial pellet
was washed in 1× MS buffer, and the 17,000 × g
sedimentation was repeated. The mitochondrial pellet was resuspended in
a small volume of TE (10 mM Tris-HCl, 1 mM
EDTA). Following the first 17,000 × g centrifugation,
the supernatant was processed further by ultracentrifugation (Beckman
TL-100) at 100,000 × g for 1 h at 4 °C. From
this centrifugation the cytosolic fraction (supernatant) and light
membrane fraction (pellet) were obtained. The pellet was resuspended in
a small volume of TE buffer. To all samples, an appropriate volume of 4× Laemmli sample buffer was added to give a final 1×
concentration. Samples were boiled 5 min prior to electrophoresis on
SDS-polyacrylamide gels.
GST Fusion Purification and Pull-downs
Overnight cultures of pGEX-XIAP-BIR1, -BIR2, and -BIR3 were
diluted 1:10 in fresh medium and allowed to grow for 1 h
prior to induction. 50 µM zinc acetate was then added to
the cultures along with 0.1 mM
isopropyl-1-thio- Cell lysates for pull-down analysis were prepared by lysing 100-mm
dishes of untransfected, Ub-Smac-, and Ub- Cell Death Assays
HeLa cells were plated and transfected simultaneously in 96-well
dishes. Plasmid DNA (320 ng/well) was diluted in 25 µl/well Opti-MEM
(Invitrogen) and dispensed in 96-well plates. 0.4 µl/well LipofectAMINE 2000 was diluted in 25 µl/well OptiMEM and added to
each well. HeLa cells were trypsinized, counted, and adjusted to 5 × 105 cells/ml, and 100 µl of cell suspension were added
to each well. At 24 h post-transfection, the medium was
replaced with Dulbecco's minimal essential medium, 10% fetal calf
serum containing etoposide (Sigma) at varying doses in triplicate. Cell
viability was determined 48 h postexposure to etoposide using
WST-1 (Roche Molecular Biochemicals).
SDS-PAGE and Western Blotting
Protein samples in 1× sample buffer were electrophoresed on
12% polyacrylamide gels and electroblotted onto
ImmobilonTM-P polyvinylidene difluoride membrane
(Millipore) at 15 V for 30 min using a semidry electrotransfer
apparatus (Hoefer Semiphor). Blots were immediately placed into 5%
skim milk in PBS, 0.1% Triton X-100 (PBST) and incubated overnight.
This was followed by a 1-h incubation in primary antibody diluted in
2% skim milk in PBST. Blots were washed 3 × 5 min in 2% skim
milk in PBST and then incubated for 1 h in horseradish peroxidase
(HRP)-conjugated secondary antibody diluted in 2% skim milk, PBST.
Antibody binding was detected by enhanced chemiluminescence (ECL)
(Amersham Biosciences) using Kodak film.
Immunofluorescence Microscopy
Cells grown on coverslips were rinsed briefly in PBS
and then fixed in 4% paraformaldehyde for 5 min. The coverslips were then rinsed 3 × 5 min in PBS, and the cells were permeabilized in
0.2% Triton X-100 (Pierce) for 20 min. The coverslips were again
rinsed 3 × 5 min in PBS. Immunofluorescence labeling was performed sequentially at room temperature. All antibody incubations lasted 45 min with 3 × 5-min washes in PBS between each
application of antibody. Following the incubation of the secondary
antibody, cells were washed 3 × 5 min in PBS and then
counterstained with 1 mg/ml Hoescht (Molecular Probes) diluted 1:5,000
to visualize nuclei. Cells were then washed for 30 s in PBS and
mounted in Vectashield mounting medium (Vector). Immunolabeled samples
were visualized using a Zeiss Axiophot epifluorescence microscope
equipped with a 100-watt mercury arc lamp. Images were digitally
recorded with an AxioCam CCD camera using AxioVision version 3.1 software.
Antibodies
Anti-HSP70, a mouse monoclonal IgG specific for mitochondrial
heat shock protein 70 (Affinity Bioreagents Inc.), was used at 1:100
for immunofluorescence labeling and 1:200 for Western blotting. Rat
monoclonal anti-HA (Roche Molecular Biochemicals) was used at 1:500 for
Western blotting. Mouse monoclonal anti-Smac/DIABLO (Zymed
Laboratories Inc.) was used at 1:100 for immunofluorescence labeling and 1:500 for Western blotting. 5 µl of rabbit polyclonal anti-RIAP3 were used for immunoprecipitation. Goat polyclonal anti-mouse IgG F(ab')2 conjugated to Alexa 488 (Molecular Probes) and goat polyclonal anti-mouse IgG
F(ab')2 conjugated to indocarbocyanine (CY3; Jackson
Immunoresearch Laboratories, Inc.) were both used at 1:400 for
immunofluorescence microscopy. For Western blotting a goat anti-mouse
IgG conjugated to HRP (Bio-Rad) was used at 1:5,000. Goat anti-rat IgG
conjugated to HRP (Amersham Biosciences) was used at 1:2,000 for
Western blotting.
Ubiquitin-Smac Protein Is Rapidly and Completely Processed to Yield
Mature Smac--
To bypass the mitochondrial requirement for Smac
processing, we developed a ubiquitin fusion system. As shown
schematically in Fig. 1A, the
coding region for human ubiquitin (76 codons) was fused in-frame to the
184 codons corresponding to processed, mature mouse Smac/DIABLO. In
addition, a HA tag was added to the carboxyl terminus to allow
distinction between the transgene-encoded and the endogenous Smac
proteins. Cleavage of the 31.4-kDa precursor protein by the endogenous
ubiquitin-specific proteases was predicted to occur immediately
following the carboxyl-terminal Gly-Gly dipeptide of ubiquitin,
generating the correct Ala-Val-Pro-Ile amino terminus of processed,
cytosolic Smac.
Plasmid transfection in HeLa cells resulted in the rapid and complete
processing of the expressed Ub-Smac fusion protein. Unprocessed Ub-Smac
(31.4-kDa) protein was not detectable by Western blot with either
anti-Smac (Fig. 1B) or anti-HA (data not shown). The
endogenous Smac protein was also visible by Western blot, present in
both the unprocessed, mitochondrial precursor form and the mature
cytosolic form (Fig. 1B, lane 2). In transfected cells, the processing of Ub-Smac was predicted to generate a 21.6-kDa mature Smac protein, slightly larger than the endogenous mature human
Smac (due to the presence of the HA tag) but smaller than the
mitochondrial Smac precursor protein.
To determine whether the processed Smac protein was capable of binding
to XIAP protein, we performed GST pull-down analysis. Protein lysates
were prepared from Ub-Smac-transfected cells, allowed to bind to
recombinant, glutathione bead-bound XIAP protein fragments, and washed
extensively. Western blot analysis with anti-Smac (Fig. 1B)
or anti-HA (not shown) demonstrated that the transfected Smac protein
bound to XIAP BIR3 and BIR2 but not to BIR1. Although GST pull-down
analysis is not quantitative, there was an apparent and consistently
observed enhanced XIAP BIR3 binding relative to BIR2 as has been
described in the literature (37). We next confirmed interaction between
XIAP and Smac in vivo. Endogenous XIAP protein was
immunoprecipitated with rabbit polyclonal anti-RIAP3 antibody. Western
blot analysis was then carried out with anti-HA. As seen in Fig.
1C, processed Ub-Smac bound to XIAP.
Smac Protein Derived from the Ub-Smac Precursor Protein Is
Cytoplasmic--
The principal advantage of this system is the ability
to express mature Smac in the cytoplasm. To confirm that the expressed Smac resides in the cytoplasm, we performed immunofluorescence microscopy. Anti-Smac immunofluorescence microscopy of untransfected cells shows a punctate staining pattern with some generalized cytoplasmic staining (Fig.
2A). We interpreted this as
mitochondrial localization (as previously reported; Refs. 14 and
15) with some leakage of Smac into the cytoplasm. An antibody to
mitochondrial HSP70 was then used to illustrate the mitochondrial
staining pattern as seen in Fig. 2B. Labeling of
Ub-Smac-transfected cells with anti-Smac antibody showed a clearly
cytoplasmic staining pattern with relatively little mitochondrial
labeling due to the endogenous Smac protein (Fig. 2C).
Unfortunately the commercially available anti-HA antibodies do not
function well in immunofluorescence microscopy, which would have
allowed us to distinguish the Ub-Smac-derived protein from the
endogenous Smac. Confirmation that the Ub-Smac-derived, mature Smac was
localized to the cytoplasm was obtained by cell fractionation in which
mitochondrial, cytoplasmic, and light membrane (endoplasmic reticulum
and Golgi) fractions were prepared. As seen in Fig. 2D,
mitochondrial HSP70 partitioned cleanly in the mitochondrial fraction,
whereas the Smac protein (detected with the anti-HA antibody) localized
exclusively in the cytoplasmic fraction.
Smac Function Is Dependent on the Amino-terminal AVPI
Tetrapeptide--
We next examined the contribution of the
Ala-Val-Pro-Ile amino-terminal tetrapeptide of mature Smac to the
Smac-XIAP interaction. Site-directed mutagenesis was performed on the
Ub-Smac plasmid such that the 12 nucleotides encoding the first four
amino acids of mature Smac were deleted. HeLa cells were transfected
with Ub-Smac and Ub-Smac-
We examined the consequences of cytosolic, mature Smac expression on
cell viability in both HeLa and 293A cells. No decrease in cell
viability was observed in either the short (24 h post-transfection) or
longer term (5 days post-transfection) as assessed relative to reporter
gene-transfected cells (LacZ or GFP; Fig.
4). We therefore determined whether
mature Smac predisposes cells to undergo apoptosis using etoposide as a
trigger. Cells were transfected in 96-well plates. At 24 h
post-transfection, etoposide was added to triplicate samples in doses
ranging from 5 to 500 µM. Viability was evaluated by
colorimetric assay based on the cleavage of the tetrazolium salt WST-1
by mitochondrial dehydrogenases in viable cells as well as by cell
count using trypan blue exclusion. As seen in Fig.
5, control plasmids expressing GFP or
LacZ had no effect on cell viability. In contrast, cytosolic Smac
greatly sensitized cells to etoposide-mediated killing at all exposure
levels. Deletion of the Ala-Val-Pro-Ile tetrapeptide sequence in
Ub-Smac- Cancer continues to be one of the leading causes of morbidity and
mortality in western countries. It is now widely accepted that
deregulated apoptosis is a fundamental characteristic of cancer cells
(for a review, see Ref. 38), necessary for the suppression of normal
cell death that would occur due to deregulated proto-oncogene
expression. Furthermore, cancer cells encounter severe environmental
stresses including reduced oxygen levels in solid tumor masses, loss of
trophic factor support, and detachment from the extracellular matrix,
any of which would trigger apoptosis in a normal cell. Finally
chemotherapeutic drugs and radiation therapy act by triggering
apoptosis, and treatment failure may be in part due to further
mutations in critical cell death pathways. Indeed virtually all
components of the endogenous and exogenous apoptosis pathways are
mutated or inactivated in at least some cancers (for a review, see Ref.
39). Numerous strategies are under investigation to reestablish normal
apoptosis in cancer cells, thereby triggering outright apoptosis or
sensitization to treatment.
The IAPs control the activity of strategic caspases involved in the
initiation and execution phase of apoptosis and are frequently overexpressed in cancer cell lines and primary biopsy tumor samples (40-43). IAP-mediated caspase inhibition contributes to
chemotherapeutic drug and radiation resistance. Interestingly, while
caspase 8 and 10 gene inactivation has been documented (44, 45), no cases of effector caspase gene deletion or silencing have been reported, suggesting that these caspases play indispensable roles in
cell processes in addition to apoptosis (for a review, see Ref. 46). If
one presupposes that caspases 3 and 7 remain functional, albeit latent,
in all cancer cells, then the IAPs constitute the final point of
deregulation that cancer cells can utilize to suppress apoptosis. It is
therefore reasonable to propose that strategies that interfere with IAP
expression or function will find utility in increasing the
susceptibility of cancer cells to drug and radiation therapies.
Smac-based peptide therapy was recently demonstrated to increase the
efficacy of TRAIL ligand in triggering tumor regression in a mouse
xenograft model of malignant glioma, illustrating the therapeutic
potential of IAP antagonists (47).
Cancer cells frequently overexpress antiapoptotic Bcl2 family members
or delete/inactivate proapoptotic Bcl2 family members, thereby
suppressing cell death by blocking the release of apoptogenic factors
such as cytochrome c and Smac. Expression strategies that bypass the mitochondrial control point are advantageous both for the
precise dissection of the contributions of various apoptotic factors
and potentially in the development of cancer gene therapy payloads.
Previous efforts to characterize cytoplasmic Smac have used either
microinjection of recombinant protein (48) or plasmid expression
vectors encoding the Smac cDNA lacking the mitochondrial targeting
sequence (34, 47). Presumably some degree of initiation methionine
removal occurs, thereby generating the necessary amino-terminal alanine, but the extent of this processing has not been reported. In
this report, we characterize a unique expression system that generates
mature, cytoplasmic Smac protein without requiring mitochondrial permeabilization. We demonstrated that the mature Smac protein derived
from the ubiquitin fusion protein interacts with XIAP and is localized
to the cytoplasm of the cell. Finally we demonstrated that
Smac-mediated IAP inhibition is not sufficient to trigger outright cell
death but did, however, sensitize cells to apoptotic triggers.
The mechanism of action of Smac is still controversial. Splice isoforms
of Smac (Smac-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices and
a three-stranded
-sheet with a single zinc ion coordinated by
conserved cysteine and histidine residues (5). There are currently
eight members of the mammalian IAP family of which XIAP, cIAP1, and
cIAP2 form an obvious subgroup, each possessing three BIR domains and a
carboxyl-terminal RING zinc finger motif. The RING finger domain
possesses ubiquitin ligase activity, catalyzing autoubiquitination as
well as ubiquitination of caspase 3 and other IAP-interacting proteins
(10-12).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
AVPI-Smac--
Primers
5'-d-CGTCTGAGAGGTGGTGCTCAGAAATCGGAGCC and
5'-d-GGCTCCGATTTCTGAGCACCACCTCTCAGACG were used to delete the
AVPI codons of Ub-Smac using the QuikChangeTM Mutagenesis
kit (Stratagene) according to the manufacturer's instructions. The
Ub-Smac plasmid was used as a template for PCR, the product was TA
cloned, sequenced, and subcloned into the ubiquitin promoter expression
vector to generate Ub-
AVPI-Smac.
-D-galactopyranoside, and the cultures
were induced at 28 °C for 2.5 h. Bacteria were pelleted and
resuspended in STE buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA) containing protease
inhibitors (10 µM aprotinin, 100 µM
pepstatin A, 10 µM leupeptin, and 100 µM
phenylmethylsulfonyl fluoride). Dithiothreitol was added to 1 mM, and the pellet was vortexed before adding 20 mg/ml
lysozyme (Roche Molecular Biochemicals). The suspension was then
incubated on ice for 15 min. A 1/50 volume of 10%
taurocholic acid (Calbiochem) was added along with 0.1 mM
DNase (Sigma) and 0.1 mM MgCl2., and the
suspension was incubated on ice for 10 min, sonicated 3 × 20 s using a probe sonicator with an amplitude of 20%, and then
centrifuged at 14,000 × g for 15 min. The supernatant
was filtered through cheesecloth, and a 1/50 volume of
20% Triton X-100 (Sigma) was added. Glutathione-Sepharose bead slurry
(Amersham Biosciences) was added to the lysate and incubated at 4 °C
for 1 h on a rotating platform. Bead-protein complexes were
collected and washed four times in NETN buffer (2 M
Tris-HCl, pH 8.0, 4 M NaCl, 20% Triton X-100, 0.5 M EDTA). The bead complexes were then stored in 200 µl of
NETN buffer and 50 µM zinc acetate until further use. GST
fusion proteins were quantified by SDS-PAGE using bovine serum albumin
as a protein standard prior to performing pull-down analysis.
AVPI-Smac-transfected 293A
cells in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 10 mM EDTA) and
protease inhibitors. Lysates were collected in Eppendorf tubes,
incubated on ice for 20 min, and centrifuged at 17,000 × g for 5 min. For pull-down analysis, 100 µg of total cell
lysate and 2.5 µg of GST fusion protein were mixed in an Eppendorf
tube, and 500 µl of lysis buffer were added to allow for adequate
mixing on a rotating shaker. After a 2-h incubation at 4 °C, the
protein-bead complexes were washed three times in ice-cold lysis
buffer. Beads were syringe-dried and resuspended in a small volume of
1× sample buffer prior to SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
The ubiquitin-Smac expression system.
A, schematic diagram of the Ub-Smac expression vector. The
cleavage site at the end of the ubiquitin peptide sequence, which
generates the correct amino terminus corresponding to mature Smac
released from the mitochondria during apoptosis, is indicated.
B, Western blot analysis with anti-Smac antibody. Endogenous
Smac is present in two forms, one form possessing the 55-amino acid
mitochondrial targeting peptide (mt. Smac) and cytosolic
Smac in which this leader peptide has been removed (cyto
Smac). C, co-immunoprecipitation of Smac-transfected
cell lysates using anti-RIAP3. Western blot analysis using the anti-HA
antibody shows overexpressed Ub-Smac present in cell lysates
(left panel). Following immunoprecipitation with anti-RIAP3,
Western blot analysis shows that Ub-Smac co-immunoprecipitated with
XIAP (right panel).
View larger version (45K):
[in a new window]
Fig. 2.
Ub-Smac expression generates cytoplasmic,
mature Smac protein. Immunofluorescence labeling of endogenous
Smac in untransfected HEL299 cells (A) and mitochondrial
HSP70 (B) and overexpressed Ub-Smac in transfected HEL299
cells (C). Each micrograph shows the merged image of both
the 4,6-diamidino-2-phenylindole and the immunostaining. The
bar represents 100 µm. D, cell fractionation of
Ub-Smac-transfected HEL299 cells. Western blot analysis using
anti-HSP70 (upper panel) is a control for the cell
fractionation technique and shows mitochondrial protein HSP70 localized
to the mitochondrial fraction. Western blot analysis using anti-HA
shows overexpressed Ub-Smac localized to the cytosolic fraction
(lower panel). Pre-spin lysate shows
the presence of HSP70 and Ub-Smac prior to the fractionation
procedure.
AVPI expression plasmids, and expression was confirmed by both anti-Smac (not shown) and anti-HA Western blot
(Fig. 3). Pull-down analysis was
performed with GST-XIAP-BIR1, -BIR2, and -BIR3 recombinant proteins. As
seen in Fig. 3, Smac bound to XIAP BIR3 most strongly, to a lesser
extent to BIR2, and not to BIR1. In contrast, the Smac-
AVPI protein
failed to bind to any of the recombinant XIAP BIR domains.
View larger version (31K):
[in a new window]
Fig. 3.
Smac interaction with XIAP is dependent on
the AVPI tetrapeptide sequence. GST pull-down analysis was used to
demonstrate that Ub-Smac protein binds to XIAP BIR3 > BIR2 and
not to BIR1. In contrast, Ub- AVPI-Smac does not bind to any of the
recombinant XIAP BIR domains (right panel). Western blot
analysis using anti-HA antibody shows that both overexpressed Ub-Smac
and Ub-
AVPI-Smac were present in cell lysates prior to performing
the GST pull-down analysis (left panel).
AVPI-transfected cells completely abrogated the effect of
Smac overexpression (Fig. 5).
View larger version (22K):
[in a new window]
Fig. 4.
Ub-Smac expression does not kill cells.
Triplicate samples of HeLa cells transfected with plasmids encoding
LacZ, Ub-Smac, or Ub- AVPI-Smac were harvested, and viable cell
counts were determined by trypan blue exclusion at 24 h and 5 days
post-transfection. Transfection efficiency was estimated at greater
than 90% based on
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
staining.
View larger version (20K):
[in a new window]
Fig. 5.
Ub-Smac expression sensitizes HeLa cells to
etoposide-mediated apoptosis. HeLa cells were transfected with the
indicated plasmids and exposed to etoposide in doses ranging from 5 to
500 µM. Survival was assessed by WST-1 colorimetric assay
and expressed as percentage of survival relative to cells exposed to 0 µM etoposide. eGFP, enhanced
GFP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, Smac-
, and Smac-
) have been identified that
lack the mitochondrial targeting sequence and the IAP binding motif and
localize to the cytoplasm. Furthermore, Smac-
and additional deletion mutants were reported to promote apoptosis as efficiently as
full-length Smac (49). These results were interpreted as an indication
that the primary means by which Smac triggers apoptosis is via its
carboxyl-terminal
-helical bundle domain and that IAP binding is a
secondary effect. In contrast, several studies have suggested that the
amino-terminal seven amino acids of Smac are sufficient to sensitize
cells to apoptotic triggers (32, 34, 47). Our studies directly address
the contribution of the AVPI tetrapeptide. Site-directed mutagenesis
was used to delete this sequence. We saw no binding of the Smac-
AVPI
protein either in vitro using GST fusion pull-downs or
in vivo by co-immunoprecipitation analysis (data not shown).
Furthermore, in our assays, the deletion of the IAP recognition motif
completely abrogated sensitization to etoposide-mediated killing. Taken
together, our studies suggest that the primary mechanism of the
proapoptotic activity of Smac is via the inhibition of IAP function.
Finally the ubiquitin fusion method is readily amenable for the study
of other non-methionine-initiating proteins, such as Omi, and was
recently used in the characterization of the Jafrac2 protein, a
Drosophila IAP antagonist that is processed and released
from the endoplasmic reticulum (50).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jenny Ho, Sandra Hurley, and Natasha Schokman for excellent technical assistance and the staff and students of the Molecular Genetics Laboratories and Aegera Oncology for helpful discussions. In particular we wish to single out Dr. Eric LaCasse and Charles Lefebvre for advice. The ubiquitin C promoter plasmid was a kind gift from Dr. Douglas Gray (Ottawa Regional Cancer Center).
![]() |
FOOTNOTES |
---|
* This work was supported by funds from the Canadian Institutes of Health Research (CIHR) (to P. L. and R. G. K.), the National Cancer Institute of Canada (to R. G. K.), and the Howard Hughes Medical Institute (to R. G. 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.
¶ Recipient of a CIHR postgraduate scholarship.
** A CIHR New Investigator. To whom correspondence should be addressed: Children's Hospital of Eastern Ontario, Research Inst., Rm. R308, 401 Smyth Rd., Ottawa K1H 8L1, Canada. Tel.: 613-738-3927; Fax: 613-738-4833; E-mail: peter@mgcheo.med.uottawa.ca.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.C200695200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IAP, inhibitor of apoptosis; XIAP, X-linked IAP; Smac, second mitochondrial derived activator of caspases; BIR, baculoviral IAP repeat; Ub, ubiquitin; GFP, green fluorescent protein; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; aa, amino acid(s); HA, hemagglutinin; HEL, human embryonic lung; PBS, phosphate-buffered saline; RIAP3, rat XIAP; GST, glutathione S-transferase; HSP70, heat shock protein 70; HRP, horseradish peroxidase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Krepela, E. (2001) Neoplasma 48, 332-349[Medline] [Order article via Infotrieve] |
2. | Creagh, E. M., and Martin, S. J. (2001) Biochem. Soc. Trans. 29, 696-702[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zimmermann, K. C., Bonzon, C., and Green, D. R. (2001) Pharmacol. Ther. 92, 57-70[CrossRef][Medline] [Order article via Infotrieve] |
4. | Salvesen, G. S. (2002) Cell Death Differ. 9, 3-5[CrossRef][Medline] [Order article via Infotrieve] |
5. | Shi, Y. (2002) Mol. Cell 9, 459-470[Medline] [Order article via Infotrieve] |
6. | Salvesen, G. S., and Duckett, C. S. (2002) Nat. Rev. Mol. Cell. Biol. 3, 401-410[CrossRef][Medline] [Order article via Infotrieve] |
7. | Deveraux, Q. L., Schendel, S. L., and Reed, J. C. (2001) Cardiol. Clin. 19, 57-74[Medline] [Order article via Infotrieve] |
8. | Stennicke, H. R., Ryan, C. A., and Salvesen, G. S. (2002) Trends Biochem. Sci. 27, 94-101[CrossRef][Medline] [Order article via Infotrieve] |
9. | Goyal, L. (2001) Cell 104, 805-808[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Yang, Y.,
Fang, S.,
Jensen, J. P.,
Weissman, A. M.,
and Ashwell, J. D.
(2000)
Science
288,
874-877 |
11. |
Suzuki, Y.,
Nakabayashi, Y.,
and Takahashi, R.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
8662-8667 |
12. |
MacFarlane, M.,
Merrison, W.,
Bratton, S. B.,
and Cohen, G. M.
(2002)
J. Biol. Chem.
277,
36611-36616 |
13. | Liston, P., Fong, W. G., Kelly, N. L., Toji, S., Miyazaki, T., Conte, D., Tamai, K., Craig, C. G., McBurney, M. W., and Korneluk, R. G. (2001) Nat. Cell Biol. 3, 128-133[CrossRef][Medline] [Order article via Infotrieve] |
14. | Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve] |
15. | Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve] |
16. | Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R. (2001) Mol Cell. 8, 613-621[Medline] [Order article via Infotrieve] |
17. |
Hegde, R.,
Srinivasula, S. M.,
Zhang, Z.,
Wassell, R.,
Mukattash, R.,
Cilenti, L.,
DuBois, G.,
Lazebnik, Y.,
Zervos, A. S.,
Fernandes-Alnemri, T.,
and Alnemri, E. S.
(2002)
J. Biol. Chem.
277,
432-438 |
18. |
Verhagen, A. M.,
Silke, J.,
Ekert, P. G.,
Pakusch, M.,
Kaufmann, H.,
Connolly, L. M.,
Day, C. L.,
Tikoo, A.,
Burke, R.,
Wrobel, C.,
Moritz, R. L.,
Simpson, R. J.,
and Vaux, D. L.
(2002)
J. Biol. Chem.
277,
445-454 |
19. |
Martins, L. M.,
Iaccarino, I.,
Tenev, T.,
Gschmeissner, S.,
Totty, N. F.,
Lemoine, N. R.,
Savopoulos, J.,
Gray, C. W.,
Creasy, C. L.,
Dingwall, C.,
and Downward, J.
(2002)
J. Biol. Chem.
277,
439-444 |
20. | Ferri, K. F., and Kroemer, G. (2001) Bioessays 23, 111-115[CrossRef][Medline] [Order article via Infotrieve] |
21. | Martinou, J.-C., and Green, D. G. (2001) Nat. Rev. Mol. Cell. Biol. 2, 63-67[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ravagnan, L., Roumier, T., and Kroemer, G. (2002) J. Cell. Physiol. 192, 131-137[CrossRef][Medline] [Order article via Infotrieve] |
23. | Wu, G., Chai, J., Suber, T. L., Wu, J. W., Du, C., Wang, X., and Shi, Y. (2000) Nature 408, 1008-1012[CrossRef][Medline] [Order article via Infotrieve] |
24. | Srinivasula, S. M., Hegde, R., Saleh, A., Datta, P., Shiozaki, E., Chai, J., Lee, R. A., Robbins, P. D., Fernandes-Alnemri, T., Shi, Y., and Alnemri, E. S. (2001) Nature 410, 112-116[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Song, Z.,
Guan, B.,
Bergman, A.,
Nicholson, D. W.,
Thornberry, N. A.,
Peterson, E. P.,
and Steller, H.
(2000)
Mol. Cell. Biol.
20,
2907-2914 |
26. | Bangs, P., Franc, N., and White, K. (2000) Cell Death Differ. 7, 1027-1034[CrossRef][Medline] [Order article via Infotrieve] |
27. | Christich, A., Kauppila, S., Chen, P., Sogame, N., Ho, S. I., and Abrams, J. M. (2002) Curr. Biol. 12, 137-140[CrossRef][Medline] [Order article via Infotrieve] |
28. | Wing, J. P., Karres, J. S., Ogdahl, J. L., Zhou, L., Schwartz, L. M., and Nambu, J. R. (2002) Curr. Biol. 12, 131-135[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Wright, C. W.,
and Clem, R. J.
(2002)
J. Biol. Chem.
277,
2454-2462 |
30. | Holley, C. L., Olson, M. R., Colon-Ramos, D. A, and Kornbluth, S. (2002) Nat. Cell Biol. 4, 439-444[CrossRef][Medline] [Order article via Infotrieve] |
31. | Srinivasula, S. M., Datta, P., Kobayashi, M., Wu, J. W., Fujioka, M., Hegde, R., Zhang, Z., Mukattash, R., Fernandes-Alnemri, T., Shi, Y., Jaynes, J. B., and Alnemri, E. S. (2002) Curr. Biol. 12, 125-130[CrossRef][Medline] [Order article via Infotrieve] |
32. | Srinivasula, S. M., Datta, P., Fan, X. J., Fernandes-Alnemri, T., Huang, Z., and Alnemri, E. S. (2000) Biol. Chem. 275, 36152-36157[CrossRef] |
33. |
Li, S.,
Zhao, Y., He, X.,
Kim, T.-H.,
Kuharsky, D. K.,
Rabinowich, H.,
Chen, J., Du, C.,
and Yin, X.-M.
(2002)
J. Biol. Chem.
277,
26912-26920 |
34. |
Guo, F.,
Nimmanapalli, R.,
Paranawithana, S.,
Wittman, S.,
Griffin, D.,
Bali, P.,
O'Bryan, E.,
Fumero, C.,
Wang, H. G.,
and Bhalla, K.
(2002)
Blood
99,
3419-3426 |
35. |
Ekert, P. G.,
Silke, J.,
Hawkins, C. J.,
Verhagen, A. M.,
and Vaux, D. L.
(2001)
J. Cell Biol.
152,
483-490 |
36. |
Glickman, M. H.,
and Ciechanover, A.
(2002)
Physiol. Rev.
82,
373-428 |
37. | Liu, Z., Sun, C., Olejniczak, E. T., Meadows, R. P., Betz, S. F., Oost, T., Herrmann, J., Wu, J. C., and Fesik, S. W. (2000) Nature 408, 1004-1008[CrossRef][Medline] [Order article via Infotrieve] |
38. | Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57-70[Medline] [Order article via Infotrieve] |
39. | Igney, F. H., and Krammer, P. H. (2002) Nat. Rev. Cancer 2, 277-288[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Tamm, I.,
Kornblau, S. M.,
Segall, H.,
Krajewski, S.,
Welsh, K.,
Kitada, S.,
Scudiero, D. A.,
Tudor, G.,
Qui, Y. H.,
Monks, A.,
Andreeff, M.,
and Reed, J. C.
(2000)
Clin. Cancer Res.
6,
1796-1803 |
41. | Hofmann, H. S., Simm, A., Hammer, A., Silber, R. E., and Bartling, B. (2002) J. Cancer Res. Clin. Oncol. 128, 554-560[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Gordon, G. J.,
Appasani, K.,
Parcells, J. P.,
Mukhopadhyay, N. K.,
Jaklitsch, M. T.,
Richards, W. G.,
Sugarbaker, D. J.,
and Bueno, R.
(2002)
Carcinogenesis
23,
1017-1024 |
43. | McEleny, K. R., Watson, R. W., Coffey, R. N., O'Neill, A. J., and Fitzpatrick, J. M. (2002) Prostate 51, 133-140[CrossRef][Medline] [Order article via Infotrieve] |
44. | Teitz, T., Wei, T., Valentine, M. B., Vanin, E. F., Grenet, J., Valentine, V. A., Behm, F. G., Look, A. T., Lahti, J. M, and Kidd, V. J. (2000) Nat. Med. 6, 529-535[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Harada, K.,
Toyooka, S.,
Shivapurkar, N.,
Maitra, A.,
Reddy, J. L.,
Matta, H.,
Miyajima, K.,
Timmons, C. F.,
Tomlinson, G. E.,
Mastrangelo, D.,
Hay, R. J.,
Chaudhary, P. M.,
and Gazdar, A. F.
(2002)
Cancer Res.
62,
5897-5901 |
46. | Los, M., Stroh, C., Janicke, R. U., Engels, I. H., and Schulze-Osthoff, K. (2001) Trends Immunol. 22, 31-34[CrossRef][Medline] [Order article via Infotrieve] |
47. | Fulda, S., Wick, W., Weller, M., and Debatin, K. M. (2002) Nat. Med. 8, 808-815[Medline] [Order article via Infotrieve] |
48. |
Deshmukh, M., Du, C.,
Wang, X.,
and Johnson, E. M.. Jr.
(2002)
J. Neurosci.
22,
8018-8027 |
49. | Roberts, D. L., Merrison, W., MacFarlane, M., and Cohen, G. M. (2001) Cell Biol. 153, 221-228[CrossRef] |
50. |
Tenev, T.,
Zachariou, A.,
Wilson, R.,
Paul, A.,
and Meier, P.
(2002)
EMBO J.
21,
5118-5129 |