From the Howard Hughes Medical Institute, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 and the § Memorial Sloan-Kettering Cancer Center, New York, New York 10021
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
"BH3 domain only" members of the BCL-2 family
including the pro-apoptotic molecule BID represent candidates to
connect with proximal signal transduction. Tumor necrosis factor Programmed cell death or apoptosis is critical for the successful
crafting of multiple lineages, the maintenance of normal tissue
homeostasis and a non-inflammatory response to toxic stimuli (1). A
distinct genetic pathway apparently shared by all multicellular organisms governs apoptosis. The BCL-2 family of proteins constitutes a
decisional checkpoint within the common portion of this pathway. Full
members of the BCL-2 family share homology in four conserved domains
designated BH1, BH2, BH3, and BH4 (2). The multidimensional NMR and
x-ray crystallographic structure of a BCL-XL monomer
indicated that BH1, 2, and 3 domains represent A divergent subset of the BCL-2 family possesses only sequence homology
to the BH3 amphipathic Recently, BAX despite possessing a hydrophobic COOH terminus has been
noted in the soluble fraction of cells as well as mitochondrial membranes (20, 21). Induced BAX expression (22) or the enforced dimerization of BAX (21) results in a downstream program of mitochondrial dysfunction as well as caspase activation. A physiologic death stimulus, the withdrawal of interleukin-3, results in the translocation of monomeric BAX from the cytosol to the mitochondria where it is a homodimerized, integral membrane protein (21). Perhaps
all pro-apoptotic BCL-2 family members will prove to have inactive
forms which undergo conformational changes as part of their activation.
The best characterized signal transduction pathways that mediate
apoptosis are the cell surface receptors of the
TNF1 family,
including CD95 (Fas/Apo-1) and CD120a (p55 TNF-R1) (23-25). Engagement
of Fas/TNF-R1 receptor leads to formation of a protein complex known as
the DISC (death-inducing signaling complex) (26-28). This complex
consists of Fas/TNF-R1, FADD (MORT1), and pro-caspase-8 (MACH/FLICE/Mch5). Once caspase-8 is recruited, it is processed and
released from the complex in active form to activate the downstream "effector" caspases (26, 29, 30). The caspase family has been
divided into three groups based upon sequence homology and substrate
specificity using a positional scanning substrate combinatorial library
(31). The specificity of caspases 2, 3, and 7 (DEXD) suggests they function at the effector phase of apoptosis. In contrast,
the optimal sequence for caspases 6, 8, and 9 ((I/L/V)EXD) resembles activation sites in the effector caspase proenzymes, arguing
they represent "initiator" caspases.
Wang and colleagues (32) described a cell-free system of apoptosis, in
which S100 extracts of untreated HeLa cells induced the activation of
caspase-3 and DNA fragmentation upon addition of dATP. Further
purification of the cytosol identified cytochrome c, which
was released from the mitochondria during hypotonic lysis of the cells.
Apaf-1, a mammalian homolog of CED-4, was a second factor isolated and
required for caspase activation (33). Recently, it has been
demonstrated that cytochrome c, Apaf-1, and caspase-9 form a
complex that initiates a downstream caspase cascade (34). In addition,
it was observed that when Xenopus egg cytosol was incubated
with isolated mitochondria, cytochrome c was released, leading to the activation of caspases and nuclear apoptosis (35). The
phenomena of cytochrome c redistribution from mitochondria to cytosol was also reported to occur in intact cells during apoptosis (36). However, the precise molecular mechanism responsible for the
release of cytochrome c from mitochondria to cytosol during apoptosis remained unknown.
TNF/CHX Treatment and Western Blot Analysis--
Cells were
treated with recombinant mouse TNF- Viability, Mitochondrial Potential, and Reactive Oxygen Species
(ROS) Measurement--
Viability was determined at designated time
points by propidium iodide dye exclusion. For mitochondrial potential
and intracellular ROS production, 5 × 105 cells were
incubated for 15 min at 37 °C with 3,3'-dihexyloxacarbocynine iodide
(DiOC6(3), 40 nM) or hydroethidine (2 µM; Molecular Probes) followed by FACScan (Becton
Dickinson) analysis.
Recombinant BID Preparation and Purification--
Murine BID was
cloned into pGEX-KG. Glutathione S-transferase-BID fusion
protein was induced in BL21DE3 by 1 mM
isopropyl-1-thio- Cleavage of BID and NH2-terminal Sequence
Analysis--
Recombinant BID (5 µg) was incubated for 2 h at
37 °C with the soluble fraction of FL5.12 cells pretreated with
TNF/CHX for 5 h. The proteins were lysed, separated by 16%
SDS-PAGE, and transferred to a polyvinylidine difluoride (Bio-Rad)
membrane. The membrane was first stained with Coomassie Blue and then
destained with 80% methanol. The desired protein bands were cut out
and subjected to NH2-terminal Edman degradation (37).
Subcellular Fractionation--
FL5.12 cells were washed once in
phosphate-buffered saline, resuspended in isotonic HIM buffer (200 mM mannitol, 70 mM sucrose, 1 mM
EGTA, 10 mM HEPES, pH 7.5) supplemented with a protease
inhibitor mixture (Sigma, added at a 1:100 dilution), and homogenized
using a Polytron homogenizer (Brinkmann Instruments) at setting 6.5 for
10 s. Nuclei and unbroken cells were separated at 120 × g for 5 min as the low speed pellet (P1). This supernatant
was centrifuged at 10,000 × g for 10 min to collect
the heavy membrane pellet (HM). This supernatant was centrifuged at
100,000 × g for 30 min to yield the light membrane
pellet (LM) and final soluble fraction (S). For subcellular
fractionation of mouse hepatocytes, cells were homogenized and
separated by differential centrifugation as described below for
preparation of mitochondria from mouse liver.
Mitochondria from Mouse Liver--
For isolation of intact
mitochondria, the liver from one mouse was minced and washed in
ice-cold HIM buffer (supplemented with 2 mg/ml de-lipidated bovine
serum albumin). The minced liver (~2 g wet weight) was gently
homogenized in 6 ml of HIM buffer in a 15-ml Wheaten Dounce glass
homogenizer using two complete up and down cycles of a glass
"B"-type pestle. The homogenate was diluted 6-fold with HIM buffer
and centrifuged at 4 °C for 10 min at 600 × g in a
Sorvall SS34 rotor. The supernatant was recovered, centrifuged at
7,000 × g for 15 min, and the pellet resuspended in
twice the original homogenate volume in HIM buffer without bovine serum
albumin. After centrifuging at 600 × g, mitochondria were recovered from the supernatant by centrifuging at 7,000 × g for 15 min. The mitochondrial pellet was suspended in 0.5 ml of MRM buffer (250 mM sucrose, 10 mM HEPES,
1 mM ATP, 5 mM sodium succinate, 0.08 mM ADP, 2 mM K2HPO4, pH
7.5) at a concentration of 1 mg of mitochondrial protein per ml, and
adjusted to 1 mM dithiothreitol just before use (38).
Protein Import--
For a standard import reaction, 60 µl of
the soluble fraction of FL5.12 cells or hepatocytes was incubated with
10 µl of mitochondria in MRM buffer (1 mg of protein/ml) at 37 °C
for 30 min. This import reaction was centrifuged at 10,000 × g for 10 min to pellet the mitochondria. Both the pellet and
the supernatant were analyzed by Western blot. For alkali extraction,
the mitochondrial pellet was resuspended in freshly prepared 0.1 M Na2CO3 (pH 11.5), and incubated
for 30 min on ice. The membranes were subsequently pelleted in an
ultracentrifuge (Beckman) at 75,000 × g for 10 min and
both the pellet and the supernatant were analyzed by Western blot.
For the BID depletion experiments, the soluble fraction of FL5.12 cells
was incubated with anti-BID Ab for 1.5 h on ice. The Ab complexes
were captured with protein A beads for 1 h and removed by
centrifugation, and the procedure was repeated. The resulting BID-depleted supernatant was used in the protein import reaction. For
the recombinant caspase experiments, the soluble fraction of FL5.12
cells was incubated with recombinant caspase-8 or -3 (1 µg/60 µl;
PharMingen) at 37 °C for 1 h and then used in the protein
import reaction.
Anti-Fas Ab Injection--
6-8-week-old (20 g) C57Bl6 mice were
injected intravenously with 5 µg of purified hamster monoclonal
antibody to mouse Fas (JO2; PharMingen) in 100 µl of 0.9% (w/v)
saline. Animals were sacrificed at the indicated times.
TNF
Western blot analysis of whole cell lysates prepared from FL5.12 cells
treated with TNF/CHX revealed that the intracellular pro-apoptotic
molecule p22 BID was cleaved to yield a major p15 and minor p13 and p11
fragments (Fig. 1B). The 2B4 T cell hybridoma which is also
killed by TNF and displays mitochondrial dysfunction (not shown) also
demonstrated the p15 fragment (Fig. 1B). Pretreatment of
cells with 50 µM zVAD-fmk markedly inhibited BID cleavage
in FL5.12 cells and to a large extent in 2B4 cells (Fig.
1B).
Identification of the Cleavage Sites in BID--
To determine the
cleavage sites in BID, recombinant murine BID (rBID) was incubated for
2 h at 37 °C with the S100 fraction of TNF/CHX-treated FL5.12
cells, S100(TNF) (Fig. 2A).
Following the reaction the mixture was size fractionated by PAGE
followed by Coomassie Blue staining. The S100(TNF) caused complete
cleavage of p22 rBID (lane 3) which was inhibited by the
inclusion of 50 µM zVAD-fmk in the reaction mixture
(lane 4). p22 rBID cleavage generated a major p15 and minor
p13 fragment. Incubation of rBID with either recombinant active
caspase-8 or caspase-3 also generated the p15 fragment (not shown).
NH2-terminal peptide sequence analysis of these fragments
revealed that p22 rBID was cleaved between amino acids
Asp59 and Gly60 to generate p15 and between
amino acids Asp75 and Ser76 to generate the p13
fragment (Fig. 2B). These fragments comigrated precisely
with the upper two fragments detected in TNF-treated cells (Fig.
1B and data not shown), arguing that intracellular BID is
also cleaved at these sites.
To determine whether the third cleavage site responsible for the less
abundant p11 seen in FL5.12 cells (Fig. 1B) was the predicted Asp98 residue we utilized a D98A mutant.
35S-Labeled in vitro translated BID and
BID(D98A) were incubated with S100(TNF) from FL5.12 cells and analyzed
by SDS-PAGE. Cleavage of wild type BID generated the three cleavage
products seen in vivo, whereas cleavage of BID(D89A)
generated only the p15 and p13 fragments (not shown). Taken together,
BID is cleaved after three Asp residues located at positions 59, 75, or
98 generating three fragments (p15, p13, and p11) (Fig.
2B).
TNF/CHX Treatment Leads to Accumulation of p15 BID in Mitochondria
as an Integral Membrane Protein and Release of Cytochrome c--
To
assess the location of intracellular BID, we disrupted FL5.12 cells
using isotonic lysis conditions which kept mitochondria intact with a
retained outer membrane. A substantial portion of p22 BID was
consistently in the soluble S100 fraction (S) representing the cytosol
as well as the mitochondria-enriched HM fraction as documented by the
mitochondrial markers (cytochrome c, intermembrane space;
cytochrome c oxidase, inner membrane) (Fig.
3A, lanes 1-4). The low speed
pellet (P1) comprised of residual whole cells, nuclei, and some
mitochondria, also displays BID.
At 5 h following TNF/CHX treatment the p15 BID fragment was often
still present in the cytosol but was predominantly in the mitochondrial
HM fraction (Fig. 3A, lanes 5-8). By 7 h p15 BID was
almost exclusively in the mitochondria. The p13 and p11 minor fragments
were associated exclusively with the mitochondrial fraction (not
shown). In addition, following the TNF/CHX death stimulus, most of the
cytochrome c was released from the mitochondrial HM fraction, found either in the S100 fraction or presumably as part of
membrane fragments in the LM fraction (Fig. 3A, middle
panel).
To assess the membrane association of p22 BID and its cleavage
products, the mitochondria (HM fraction) from TNF/CHX-treated FL5.12
cells were incubated in hypotonic buffer, alkaline buffer, or in high
salt. The mitochondrial pellet (P) was separated from the supernatant
(S) by high speed centrifugation. p22 BID was sensitive to all three
treatments (>50% found in the supernatant (Fig. 3B));
whereas, p15, p13, and p11 were markedly resistant to these treatments
(Fig. 3B) indicative of an integral membrane position.
Cytosolic p15 BID Targets Mouse Liver Mitochondria While p22 BID
Does Not--
To assess whether the p15 BID fragment can target
mitochondria, the cytosol of FL5.12 cells 5 h after TNF/CHX
treatment, S100(TNF) (Fig. 3C, lane 2) was incubated with
purified, intact mitochondria from mouse liver (Fig. 3C, lane
1) in a standard protein import reaction. At 37 °C >90% of
p15 BID but <10% of p22 BID targeted mitochondria (Fig. 3C,
lanes 3 and 4). Moreover, the targeted p15 but not p22
was resistant to alkali extraction (lanes 9-12), indicating
that p15 BID was now an integral membrane protein. Targeting of p15 BID
did not occur at 4 °C (lanes 5 and 6).
Moreover, the inclusion of zVAD-fmk in the reaction did not inhibit
targeting of pre-existing p15 (lanes 7 and 8)
arguing that this event does not require an additional caspase cleavage
at the mitochondria.
Targeting of Cytosolic p15 BID to Mitochondria Is Required for the
Release of Cytochrome c--
The cleavage of cytosolic BID by
TNF-induced caspases and the targeting of p15 BID to the mitochondria
represents an attractive correlate with the mitochondrial
dysfunction/cytochrome c release. We next wished to
determine if the targeting of p15 to mitochondria is in and of itself
required for the release of cytochrome c. When the cytosol
of TNF/CHX-treated cells, S100(TNF), was incubated with mouse liver
mitochondria for 30 min at 37 °C, p15 BID-targeted mitochondria and
up to ~50% of cytochrome c was released into the
supernatant (Fig. 4A, lanes 1 and 2). p15 BID was resistant to alkali extraction whereas
as expected cytochrome c was not (lanes 3 and
4). Strikingly, depletion of p15 BID from the S100(TNF) by
an anti-BID Ab which eliminated p15 targeting also prevented the
release of cytochrome c (lanes 5-8). Depleting
BAX from the activated S100 fraction by using a polyclonal anti-BAX Ab
(651) did not inhibit cytochrome c release from mitochondria
(data not shown).
BID Is the Required Substrate of Recombinant Caspases Responsible
for the Release of Cytochrome c--
We next asked whether BID is also
a required substrate that must be cleaved by caspases in order for
cytochrome c to be released. In this paradigm, a soluble
fraction (S100) from untreated FL5.12 cells was preincubated with
recombinant caspase-8 or caspase-3 (rCas-8 and -3) and then added to
mouse liver mitochondria. When either the S100 fraction or recombinant
caspases were incubated separately with mitochondria there was no
release of cytochrome c (Fig. 4B, lanes 2 and
3, and data not shown). However, addition of active rCas-8
to the S100-generated p15 BID (lanes 6 and 7), while addition of rCas-3 generated both p15 and p11 BID (lanes 10 and 11). In both instances, the BID fragments
targeted mitochondria as integral membrane proteins (lanes 8 and 9, and data not shown) and ~50% of cytochrome
c was released (lower panel, lanes 6, 7, 10, and
11). Addition of zVAD-fmk after BID cleavage, prior to exposing mitochondria to S100(rCas-8) did not inhibit the release of
cytochrome c (data not shown). Once again, immunodepletion of p15 BID from the S100(rCas-8) prevented the release of cytochrome c when this activated cytosol was added to mitochondria
(Fig. 4C, lanes 3 and 4).
BCL-XL, BCL-2 Does Not Prevent TNF
Intracellular p22 BID was cleaved in FL5.12-BCL-XL cells
treated with TNF/CHX, and by 5 h following treatment the p15 BID fragment while often still present in the cytosol was predominantly in
the mitochondrial HM fraction (Fig. 5B, lanes 5-8). The p13 minor fragment was associated exclusively with the mitochondrial fraction (Fig. 5B). However, the translocation of p15 BID to
mitochondria was not accompanied by a detectable release of cytochrome
c (Fig. 5B, lower panel).
To assess the extent of membrane association, the mitochondria (HM
fraction) from TNF/CHX-treated FL5.12-BCL-XL cells were incubated in hypotonic buffer, alkaline buffer, or in high salt. The
mitochondrial pellet (P) was separated from the supernatant (S) by high
speed centrifugation. p22 BID was sensitive to all three treatments
(>50% found in the supernatant (data not shown); whereas, p15 and p13
were markedly resistant to these treatments indicative of an integral
membrane position. Interestingly, the p11 BID minor fragment detected
in FL5.12 parental cells (Fig. 3B) was not detected in
FL5.12-BCL-XL cells (Fig. 5B and data not shown).
In Vivo: Anti-Fas Ab Injection Results in Accumulation of p15 BID
in the Cytosol of Hepatocytes and Its Subsequent Translocation to
Mitochondria--
To assess the involvement of BID in the TNF/Fas
death pathway in vivo, mice were injected with anti-Fas Ab
which results in massive hepatocyte cell death. To determine the
subcellular location of BID, we disrupted hepatocytes using isotonic
lysis conditions which kept their mitochondria intact with a retained
outer membrane. The p22 BID in normal, untreated hepatocytes was
predominantly in the cytosolic (S) fraction (Fig.
6A, lanes 1 and 2).
However, by 1 h following anti-Fas Ab injection p15 BID appeared
in the soluble S100 fraction (S) (Fig. 6A, lanes 3 and
4). Of note by 3 h following Ab injection p15 was
associated exclusively with the mitochondrial fraction (HM) (Fig.
6A, lanes 5 and 6). In addition, the p15 but not
p22 BID from the liver cytosol of mice treated for 1 h with
anti-Fas Ab was capable of targeting mitochondria in vitro
(Fig. 6B). Moreover, that same cytosol that possessed p15 at
1 h post-treatment released cytochrome c from
mitochondria (Fig. 6C, lanes 3 and 4). However,
the cytosolic fraction from hepatocytes 3 h post-treatment, which
no longer had p15 BID present (Fig. 6A, lane 5), did not
release cytochrome c substantially (Fig. 6C, lanes
5 and 6).
These data indicate that the TNF and Fas death signal pathways
converge at BID, a shared pro-apoptotic effector belonging to the BH3
domain only subset of the BCL-2 family. Our studies suggest a model in
which cytosolic p22 BID represents an inactive conformation of the
molecule that is proteolytically cleaved to generate an active p15 BID
(Fig. 7). In retrospect, this may account for the greater protection by caspase inhibitors of BID-induced death
(10) compared with BAX-induced death (22). The p15 conformation rather
selectively targets mitochondria where it resides as an integral
membrane protein responsible for the release of cytochrome c
(Fig. 7). A subpopulation of the full-length p22 BID is also strongly
associated with the mitochondrial membrane (Fig. 3B), suggesting that a cleavage-independent pathway for BID activation may
also exist. Caspase-8 presumably directly cleaves BID following its own
activation by TNF-R/Fas engagement as caspase-8 prefers the
Asp59 site of BID. Removal of the NH2 terminus
would retain and potentially expose the predicted amphipathic (TNF
) treatment induced a caspase-mediated cleavage of cytosolic,
inactive p22 BID at internal Asp sites to yield a major p15 and minor
p13 and p11 fragments. p15 BID translocates to mitochondria as an
integral membrane protein. p15 BID within cytosol targeted normal
mitochondria and released cytochrome c. Immunodepletion of
p15 BID prevents cytochrome c release. In vivo,
anti-Fas Ab results in the appearance of p15 BID in the cytosol of
hepatocytes which translocates to mitochondria where it releases
cytochrome c. Addition of activated caspase-8 to normal
cytosol generates p15 BID which is also required in this system for
release of cytochrome c. In the presence of BCL-XL/BCL-2, TNF
still induced BID cleavage and p15 BID
became an integral mitochondrial membrane protein. However,
BCL-XL/BCL-2 prevented the release of cytochrome
c, yet other aspects of mitochondrial dysfunction still
transpired and cells died nonetheless. Thus, while BID appears to be
required for the release of cytochrome c in the TNF death
pathway, the release of cytochrome c may not be required
for cell death.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
helices in close
proximity which create a hydrophobic pocket presumably involved in
interactions with other BCL-2 family members (3). Intriguingly, the
BCL-2 family possesses pro-apoptotic (BAX, BAK, and BOK) as well as anti-apoptotic (BCL-2, BCL-XL, BCL-W, MCL-1, and A1)
molecules (2). The ratio of anti- to pro-apoptotic molecules such as BCL-2/BAX determines the response to a death signal (4). A striking
characteristic of many BCL-2 family members is their propensity to form
homo- and heterodimers (5, 6). The NMR analysis of a
BCL-XL/BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-XL pocket
and a BH3 amphipathic
-helical peptide from BAK (7). Deletions
within BAK (8) and an extensive mutational analysis of BAX (9) argues
that the BH3 domain serves as a minimal "death domain" critical for
both dimerization and killing.
helical domain. These "BH3 domain only"
members include the mammalian BID, BAD, BIK, BIM, BLK, and HRK and
EGL-1 of Caenorhabditis elegans. Of note, all of these
molecules are pro-apoptotic lending credence to the thesis that BH3
represents a minimal death domain (10-16). Where examined these BH3
domain only molecules are capable of heterodimerizing with classic
BCL-2 family members. Mutagenesis of the BH3 domain of BID (10) and BAD
(17) indicated that BH3 was essential for these interactions as well as
the killing activity. Several of these molecules, BID and BAD lack the
typical hydrophobic COOH-terminal sequence that is found in most BCL-2
family members, which for BCL-2 has been shown to function as a signal
anchor segment required for its targeting mitochondria (18). Consistent
with this BID and BAD have cytosolic as well as membrane based
localizations (10, 19). These characteristics suggested that BID and
BAD may represent death ligands, sensors that receive death signals in
the cytosol and translocate to membranes where they interact with
membrane bound, classic BCL-2 members which serve as "receptors" (10). This was supported by the demonstration that cells in response to
the survival factor interleukin-3, inactivated BAD by phosphorylation.
This has the dual impact of dictating BAD's location as well as its
binding partners. Phosphorylated BAD is sequestered in the cytosol
bound to 14-3-3; whereas, only the active non-phosphorylated BAD
heterodimerized with BCL-XL or BCL-2 at membrane sites to
prevent cell death (19). Moreover, the demonstration that egl-1
regulates all the developmental deaths in C. elegans and
maps upstream to ced-9 (16) argues that BH3 domain only molecules are
evolutionarily conserved components of a central death pathway. This
constellation suggests that such molecules are candidates to
interconnect proximal signal transduction pathways with the distal
death effector mechanisms based at intracellular membranes.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(1 ng/ml; Sigma) and
cycloheximide (1 µg/ml; Sigma), and lysed at the indicated times in
50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton
X-100 supplemented with a protease inhibitor mixture (Sigma, added at a
1:100 dilution). Lysates were separated by SDS-PAGE, and transferred to
a polyvinylidine difluoride (Bio-Rad) membrane. The membrane was first
blocked with 5% milk for 1 h, followed by incubation with primary
and secondary antibodies for 1 h each, and finally developed with enhanced chemiluminescence (Amersham). A rabbit anti-mouse BID polyclonal antibody (10) was used at 1:1000 dilution, anti-cytochrome c monoclonal antibody (PharMingen) was used at 1:500
dilution, and anti-cytochrome c oxidase subunit IV antibody
was used at 1:1000 dilution. The horseradish peroxidase-conjugated
secondary antibodies (Caltag) were used at 1:2000 dilution.
-D-galactopyranoside. The bacterial
pellet was resuspended in lysis buffer (1% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol in
phosphate-buffered saline) supplemented with a protease inhibitor
mixture (Sigma, added at a 1:100 dilution), and sonicated. After
centrifugation at 10,000 × g for 20 min, the
supernatant was applied to glutathione-agarose beads (Sigma). The beads
were washed with buffer and treated with 10 units of thrombin per
original liter. Cleaved BID was eluted from beads and the cleavage
reaction was terminated by adding 50 µg/ml
N
-p-tosyl-L-lysine
chloromethyl ketone. To remove the glutathione S-transferase
protein and incompletely cleaved fusion proteins, the preparation was
further purified on a Mono-Q column and the proteins were eluted with a
NaCl gradient.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/Cycloheximide Treatment of FL5.12 Results in Mitochondrial
Dysfunction, Caspase-mediated Cleavage of BID, and Cell Death--
As
part of a survey of the effects of death stimuli on the subcellular
localization and post-translational modification of BCL-2 family
members, we examined the response of the early hematopoietic cell line
FL5.12 to TNF
. Most non-transformed cells are resistant to TNF
unless treated with a protein synthesis inhibitor (e.g. cycloheximide) which presumably eliminates a short half-life survival molecule (39). Treatment of FL5.12 with a combination of
TNF
/cycloheximide (TNF/CHX) resulted in a rapid reduction in the
mitochondrial transmembrane potential (
m) as assessed by
the cationic, lipophilic dye dihexyloxacarbocynine iodide
(DiOC6(3)) (Fig.
1A). The production of ROS
such as superoxide as measured by hydroethidine and cell death as
determined by propidium iodide dye exclusion, followed closely (Fig.
1A). Both the mitochondrial dysfunction and cell death were
blocked by pretreatment with the broad caspase inhibitor, zVAD-fmk
(Fig. 1A).
View larger version (25K):
[in a new window]
Fig. 1.
TNF /CHX treatment of cells results in
mitochondrial dysfunction and caspase-mediated cleavage of BID.
A, TNF
/CHX-induced death and mitochondrial dysfunction in
FL5.12 cells. Cells were treated with TNF
(1 ng/ml) and CHX (1 µg/ml) in the presence or absence of 50 µM zVAD-fmk.
Viability, mitochondrial membrane potential (
m), and ROS
production were determined at designated time points. Viability was
determined by propidium iodide (PI) dye exclusion,
m was assessed by DiOC6(3), and ROS
production was assessed by conversion of hydroethidine to ethidium
(HE). B, TNF
/CHX treatment results in cleavage
of BID in cells that is inhibited by zVAD. FL5.12 or 2B4 cells were
treated with TNF
/CHX in the presence (lanes 3 and
6) or absence (lanes 2 and 5) of 50 µM zVAD-fmk. Whole cell lysates were prepared at the
indicated time points, fractionated by polyacrylamide gel
electrophoresis (SDS-PAGE) and analyzed by Western blot with anti-BID
Ab.
View larger version (89K):
[in a new window]
Fig. 2.
Sequence determination of the cleavage sites
in BID. A, recombinant murine BID (5 µg) was
incubated for 2 h at 37 °C with the soluble fraction of FL5.12
cells 5 h after treatment with TNF /CHX, S100(TNF). The reaction
was performed in the presence (lane 4) or absence
(lane 3) of 50 µM zVAD-fmk, separated by 16%
SDS-PAGE and stained with Coomassie Blue. Lane 1,
recombinant murine BID. Lane 2, S100(TNF). p15 and p13
denote the major cleavage products present in lane 3. The
NH2 terminus of these products was determined by
NH2-terminal sequence analysis. B, summary of
cleavage sites in murine BID. p11 cleavage site was identified by the
inability of a D98A mutant BID to be cleaved to p11.
View larger version (36K):
[in a new window]
Fig. 3.
TNF /CHX treatment leads to accumulation of
p15 BID in mitochondria and release of cytochrome c.
A, subcellular distribution of BID and cytochrome
c following TNF
/CHX treatment. FL5.12 cells non-treated
(lanes 1-4) or 5 h after treatment with TNF
/CHX
(lanes 5-8) were suspended in isotonic buffer, homogenized,
and separated into soluble fraction (S), light membrane
fraction (LM), heavy membrane fraction (HM), and
low speed pellet (P1) by differential centrifugation. The
fractions were analyzed by Western blot with anti-BID Ab,
anti-cytochrome c mAb (Cyt c; PharMingen), and
anti-cytochrome c oxidase subunit IV (Cyt oxi).
The P1 pellet contains residual whole cells, nuclei, and mitochondria.
The HM fraction is enriched for intact mitochondria. The LM fraction
contains the endoplasmic reticulum and plasma membrane, and the soluble
(S) fraction represents the cytosol. B, BID cleavage
products in mitochondria are resistant to alkali and salt extraction.
Mitochondria (HM fractions) were prepared from FL5.12 cells treated
with TNF
/CHX, incubated in 20 mM HEPES/hypotonic buffer
(Hypo, lanes 1 and 2) or in 0.1 M
Na2CO3, pH 11.5 (Alkali, lanes 3 and
4), or in 0.5 M NaCl (NaCl, lanes 5 and 6), and centrifuged at 200,000 × g for
10 min to yield mitochondrial pellet (P) and supernatant
(S). The fractions were analyzed by Western blot with
anti-BID Ab. C, cytosolic p15 BID targets mouse liver
mitochondria but full-length p22 BID does not. The soluble fraction
from FL5.12 cells pretreated with TNF
/CHX, S100(TNF) (lane
2) was incubated with purified, intact mitochondria from mouse
liver (Mito) (lane 1) in a standard protein
import reaction in a volume of 60 µl for 30 min at 37 °C
(lanes 3, 4, and 7-12) or 4 °C (lanes
5 and 6). 50 µM zVAD-fmk was added to the
import reaction in lanes 7 and 8. At the end of
the reaction, mitochondria were recovered by centrifugation and the
mitochondrial pellet (P) and supernatant (S) were
analyzed by Western blot with anti-BID Ab (left panel) or
after extraction of the mitochondrial membrane with 0.1 M
Na2CO3, pH 11.5 (Alkali, lanes 11 and 12).
View larger version (31K):
[in a new window]
Fig. 4.
Targeting of cytosolic p15 BID to mouse liver
mitochondria is required to release cytochrome c.
A, the soluble fraction of FL5.12 cells treated with
TNF /CHX and depleted of p15 BID fails to release cytochrome
c. Purified, intact mitochondria from mouse liver were
incubated for 30 min at 37 °C with the soluble fraction of FL5.12
cells pretreated with TNF
/CHX, S100(TNF), which had (lanes
5-8) or had not (lanes 1-4) been immunodepleted of
p15 BID using anti-BID Ab. At the end of the reaction, mitochondria
were recovered by centrifugation and the mitochondrial pellet
(P) and supernatant (S) were analyzed by Western
blot with anti-BID Ab (upper panel)/anti-cytochrome
c mAb (lower panel) or after extraction of the
mitochondrial pellets with 0.1 M
Na2CO3 (pH 11.5) (Alkali, lanes 3, 4, 7, and 8). B, cytosolic p15 BID generated by
recombinant caspase cleavage targets mitochondria and results in
release of cytochrome c. Purified, intact mitochondria from
mouse liver were incubated for 30 min at 37 °C with the soluble
fraction (S100) from untreated FL5.12 cells (lanes 2-5) or
that S100 which had been incubated with 1 µg of recombinant caspase-8
for 1 h, S100 (rCas-8) (lanes 6-9) or 1 µg of recombinant caspase-3, S100(rCas3) (lanes 10 and
11). At the end of the reaction, the mitochondrial pellet
(P) and supernatant (S) were analyzed as in
A. C, the cytosolic fraction of FL5.12 cells incubated with
recombinant caspase-8 when immunodepleted of p15 BID fails to release
cytochrome c from mitochondria. Purified, intact
mitochondria from mouse liver were incubated with the S100(rCas-8) as
in B (lanes 1 and 2) or this
S100(rCas-8) immunodepleted of p15 BID using anti-BID Ab (lanes
3 and 4). At the end of the reaction, the mitochondrial
pellet (P) and supernatant (S) were analyzed as
in A. p15 BID depleted by the first and second
immunoprecipitation of the S100(rCas-8) is shown in lanes 5 and 6.
-induced
Translocation of p15 BID to Mitochondria, But Does Interfere with the
Release of Cytochrome c--
To assess the role of BCL-XL,
BCL-2 in the TNF death pathway, we asked whether these anti-apoptotic
molecules would protect FL5.12 cells from TNF/CHX, whether p15 BID is
generated and translocates to mitochondria, and whether cytochrome
c is released. Treatment of FL5.12-BCL-XL cells
with TNF/CHX resulted in rapid reduction in
m, production
of ROS and cell death (Fig.
5A). Both the mitochondrial
dysfunction and cell death were blocked by pretreatment with the
caspase inhibitor, zVAD-fmk (Fig. 5A). Similar results were
obtained with FL5.12-BCL-2 cells (data not shown).
View larger version (26K):
[in a new window]
Fig. 5.
BCL-XL,BCL-2 does not prevent
TNF -induced translocation of p15 BID to mitochondria, but does
prevent cytochrome c release. A, TNF
/CHX
induces death and mitochondrial dysfunction in
FL5.12-BCL-XL cells. The experimental procedure was
identical to that in Fig. 1A. B, subcellular distribution of
BID and cytochrome c in FL5.12-BCL-XL cells
following TNF
/CHX treatment. The experimental procedure was
identical to that in Fig. 3A.
View larger version (19K):
[in a new window]
Fig. 6.
In vivo: anti-Fas Ab injection results
in accumulation of p15 BID in the cytosol of hepatocytes and its
subsequent translocation to mitochondria. A, livers
from untreated mice (lanes 1 and 2) or mice
treated with anti-Fas Ab (JO2; PharMingen) after 1 h (lanes
3 and 4) or 3 h (lanes 5 and
6) were suspended in isotonic buffer, homogenized, and
separated by differential centrifugation into soluble fraction
(S) representing the cytosol and heavy membrane fraction
(HM) enriched for intact mitochondria as confirmed by
cytochrome c Ab. There is relatively little cytochrome
c within the soluble fraction reflecting degradation of
released cytochrome c. Fractions were analyzed by Western
blot with anti-BID Ab. B, p15 BID from the cytosol of
anti-Fas Ab-treated hepatocytes targets mitochondria. The soluble
fraction from liver 1 h after intravenous administration of
anti-Fas Ab, S100( -Fas/1 h), was added to purified, intact
mitochondria from liver of non-treated mice for 30 min at 37 °C. At
the end of the reaction, the mitochondrial pellet (P) and
supernatant (S) were analyzed as in Fig. 4A. C,
mouse liver cytosol with p15 BID (1-h post-anti-Fas Ab injection)
releases cytochrome c from mitochondria. Purified, intact
mitochondria from liver of non-treated mice were incubated for 30 min
at 37 °C with the S100 fraction of liver from untreated mice
(lanes 1 and 2), or mice treated with anti-Fas Ab
after 1 h (lanes 3 and 4) or 3 h
(lanes 5 and 6). At the end of the reaction, the
mitochondrial pellet (P) and supernatant (S) were
analyzed by Western blot with anti-cytochrome c mAb.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
helix, BH3, on the active COOH-terminal p15 fragment. This proteolytic
cleavage may alter an inert, intramolecular folded BID or alternatively
release BID from a tethering chaperone-like molecule. Immunodepletion
of p15 BID from cytosols activated by either TNF-R engagement or
caspase-8 addition indicates that p15 BID is requisite for the release
of cytochrome c from mitochondria.
View larger version (28K):
[in a new window]
Fig. 7.
Model of BID cleavage and translocation
following TNF-R1/Fas engagement.
The rapid movement of p15 BID from cytosol to mitochondrial membrane suggests a specific mechanism of targeting. One possibility is a ligand-receptor model (10) in which BH3 of p15 BID binds to membrane bound BCL-2 or BAX which serve as receptors. Alternatively, other BID-protein or BID-lipid pathways may regulate targeting. However, such an association would be a transient intermediate in that the vast majority of p15 BID at mitochondria is an alkaline-resistant integral membrane protein. p15 BID might co-integrate with such partner proteins or insert itself. But why would p15 BID be of such singular importance for the release of cytochrome c? BAX, BCL-2, and BCL-XL are able to form distinct ion conductive pores in artificial membranes (40-43). p15 BID might regulate such channels, form a hybrid channel, or polymerize to form a channel itself. A cascade of disturbed ion homeostasis and altered transmembrane potential might ensue and could result in mitochondrial swelling and ultimate rupture of the more taut outer mitochondrial membrane, which has been proposed as a mechanism of cytochrome c release (44). Alternatively, p15 BID with or without partner proteins might constitute a pore for the selective passage of cytochrome c which has been proposed for diphtheria toxin B fragment and the A fragment (45).
Intracellular p22 BID was cleaved at three internal Asp sites:
Asp59, Asp75, and Asp98
(Fig. 2). Of note the minor fragments of p13 and p11 resulting from
cleavage at Asp75 and Asp98,
respectively, are only detected in the mitochondria. While caspase-8 prefers the Asp59 site, other caspases perhaps at the level
of mitochondria may be responsible for the p13 and p11 fragments. The
p11 fragment was not observed in mitochondria protected by
BCL-XL lending support to this thesis. The
LQTD recognition motif for the predominant p15 fragment
is an atypical site for initiator caspases (6, 8, 9, (I/L/V)EXD). The DEMD
motif which was
recognized by recombinant caspase-3 is a classic site for effector
caspases (2, 3, 7 (DEXD)). Of note all three recognition
sites are well conserved between mouse and human BID (Fig. 2). Caspase
cleavage of BCL-2 (46) and BCL-XL (47) have been reported
which convert them from anti- to pro-apoptotic molecules. Thus, caspase
cleavage of the BCL-2 members may represent a feed forward loop to
ensure cell death.
The observation that BCL-2, BCL-XL can in certain settings interfere with TNF/Fas-mediated cell death suggests the existence of regulatory steps beyond the activation of caspase-8 (48-51). Recently, a cell-free system has indicated that caspase-8 activates a mitochondria dependent pathway that involves cytochrome c as well as a mitochondria independent pathway (52). Moreover, a displacement model holds that BID could participate in an additional augmentation loop. BID binding to BCL-XL could release Apaf-1 making it available to interact with cytochrome c, following its release by BID, which would activate caspase-9 and subsequently caspase-3.
However, the use of inhibitors that eliminate measurable caspase
activity indicates that in certain deaths a downstream program of
mitochondrial dysfunction characterized by altered m and
production of ROS still runs (21, 22, 53). This may be of particular
relevance for TNF-induced death which has been noted to be influenced
by anti-oxidants (54) and potentially the profound hemorrhagic necrosis
of the liver that follows in vivo administration of anti-Fas
Ab (55).
Recently, two other groups have also noted the cleavage of BID by
purifying a factor that released cytochrome c (56) or screening for substrates cleaved by caspase-8 (57). However, we differ
from the conclusions drawn in those papers in that we noted that while
the presence of BCL-XL/BCL-2 prevented the detectable release of cytochrome c, the cells still died with similar
kinetics. As observed for FL5.12 cells, the presence of BCL-2 or
BCL-XL does not prevent TNF/Fas killing in most cell types
(51, 58). This response is similar to what has been proposed as a type
I cell (51). Hepatocytes may represent a type II cell in that a Bcl-2
transgene has been reported to protect mice from anti-Fas Ab-induced
apoptosis (59). The findings in FL5.12 cells contrast with the capacity
of BCL-XL, BCL-2 to save these cells after interleukin-3 deprivation, by preventing BAX dimerization and translocation from
cytosol to mitochondria (21). Conversely, TNF treatment of the same
FL5.12-BCL-XL cells still results in cleavage and translocation of p15 BID to mitochondria where it resides as an integral membrane protein. Despite the established importance of p15
BID in releasing cytochrome c, the presence of
BCL-XL prevented its detectable release. While we cannot
exclude the release of a small percentage of cytochrome c
(60), nevertheless the other parameters of mitochondrial dysfunction
including m and ROS production still transpired to the
same extent and the cells died in a comparable time course. Thus, BID
appears to be strategic in this pathway serving as the lynch pin that
connects the initiator caspase to cytochrome c release.
However, the robust release of cytochrome c does not appear
to be required for Fas/TNF-R1-induced cell death.
Pro-apoptotic BCL-2 members are demonstrating distinct interconnections
with death and survival signaling pathways. BID is integral for
TNF-R1/Fas release of cytochrome c. BAX is singularly required for nerve growth factor deprivation death in neurons despite
their expression of multiple pro-apoptotic members (61). BAD is
inactivated by phosphorylation following interleukin-3 survival factor
(19). This suggests that selected pro-apoptotic BCL-2 members will
reside in distinct signal transduction pathways and in their activated
conformation constitute death effectors operative at intracellular
organelles, especially mitochondria.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank G. Shore for advice on protein import, J. Hare for anti-cytochrome c oxidase antibodies, and Mary Pichler for preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* 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.
Supported by a fellowship from European Moleculary Biology Organization.
¶ To whom correspondence should be addressed: Smith 758, Dana-Farber Cancer Institute, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115. Tel.: 617-632-6404; Fax: 617-632-6401; E-mail: stanley_korsmeyer{at}dfci.harvard.edu.
The abbreviations used are: TNF, tumor necrosis factor; CHX, cycloheximide; ROS, reactive oxygen species; PAGE, polyacrylamide gel electrophoresis; HM, heavy membrane; LM, light membrane; Ab, antibody; rBID, recombinant BID; DiOC6((3), 3,3'-dihexyloxacarbocynine iodide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|