From the Robert H. Lurie Comprehensive Cancer Center and the Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611
Received for publication, February 27, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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Caspases are universal effectors of
apoptosis. The mitochondrial and death receptor pathways
activate distinct apical caspases (caspase-9 and -8, respectively) that
converge on the proteolytic activation of the downstream executioner
caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24
processing intermediate (composed of its prodomain and large subunit),
which then undergoes autoproteolytic cleavage to remove the prodomain
from the active protease. Recently, several heat shock proteins have
been shown to selectively inhibit the mitochondrial apoptotic
pathway by disrupting the activation of caspase-9 downstream of
cytochrome c release. We report here that the small heat
shock protein The caspase family of cysteine proteases are critical effectors of
apoptosis that selectively cleave key proteins at aspartate residues,
thereby altering their function to promote cell death (1, 2). Caspases
are synthesized as proenzymes that are activated by trans- or
auto-proteolytic cleavage at aspartate residues. They are arranged in a
proteolytic cascade with some acting as initiators (-8, -9, and -10)
and others acting as downstream executioners (-3, -6, and-7). The
apical caspases are activated by two principal mechanisms, the
mitochondrial and death receptor pathways, that converge on the
proteolytic activation of caspase-3. In the former pathway,
mitochondria respond to a variety of stimuli including genotoxic stress
by releasing cytochrome c into the cytosol (3). Cytochrome
c then binds to Apaf-1, which oligomerizes in the presence
of ATP and recruits/activates procaspase-9; this multimeric complex is
often referred to as the apoptosome (4, 5). In the death receptor
pathway, members of the tumor necrosis factor
(TNF)1- Many of the stress stimuli that are capable of triggering apoptosis,
such as oxidative stress and heat shock, induce the synthesis of
diverse heat shock proteins (HSPs) that confer a protective effect
against a wide range of cellular stresses. Recent evidence indicates
that many HSPs are anti-apoptotic and directly inhibit caspase
activation. For instance, Hsp70 and Hsp90 bind to Apaf-1 and prevent
the recruitment of procaspase-9 to the apoptosome, thereby inhibiting
caspase-9 activation (13-15). In contrast, Hsp27, a member of the
small HSP family, has been shown to bind/sequester cytosolic cytochrome
c from the apoptosome and prevent procaspase-9 activation
(16). However, others have demonstrated that Hsp27 antagonizes
apoptosis downstream of caspase-9 activation by binding to procaspase-3
and blocking its proteolytic activation (17). These studies indicate
that some HSPs, and presumably others, confer resistance to apoptosis
by specifically inhibiting one or more components of the apoptotic machinery.
In the present report, we examined the anti-apoptotic mechanisms of
Cell Culture--
Human MDA-MB-231 breast carcinoma cells were
maintained in DMEM (Mediatech) supplemented with 10% fetal calf serum
(FCS, Life Technologies). PC-3 prostate carcinoma and Jurkat T cells
were grown in RPMI 1640 medium supplemented with 10% FCS.
Construction of FLAG-tagged cDNAs--
The full-length
Transient and Stable Transfections--
MDA-MB-231 and PC-3
cells were grown on glass coverslips and transiently transfected with 1 µg of pcDNA3-FLAG plasmid containing human Induction and Analysis of Apoptosis--
24 h after transient
transfection, cells were treated with 50 µM etoposide for
48 h (MDA-MB-231 cells) or 10 ng/ml TNF- Production of Recombinant Human Proteolytic Activation of Procaspase-9, Procaspase-3, and
Procaspase-7 in Vitro--
S-100 extracts were prepared from Jurkat T
cells as described elsewhere (3) except that cells were lysed by four
freeze-thaw cycles in a dry ice/ethanol bath. For procaspase-9
activation studies, S-100 extracts were preincubated in the absence or
presence of recombinant human Immunoprecipitation--
MDA-MB-231 cells stably expressing
FLAG-vector or FLAG-
We next examined whether Recent studies indicate that several HSPs inhibit the
mitochondrial apoptotic pathway by specifically binding to components of the cell death apparatus and disrupting the assembly of the apoptosome. Hsp70 and Hsp90 bind to Apaf-1, whereas Hsp27 binds to
cytochrome c to prevent the cytochrome
c-mediated oligomerization of Apaf-1 and subsequent
activation of procaspase-9 (13-16). We demonstrate here that the small
HSP Interestingly, the autoproteolytic maturation of capase-3 is also
inhibited by the conserved IAP family member XIAP. Like Somewhat unexpectedly, the small HSP family members In contrast to In short, B-crystallin inhibits both the mitochondrial and death
receptor pathways. In S-100 cytosolic extracts treated with cytochrome
c/dATP or caspase-8,
B-crystallin inhibits the
autoproteolytic maturation of the p24 partially processed caspase-3
intermediate. In contrast, neither the closely related small heat shock
protein family member Hsp27 nor Hsp70 inhibited the maturation of the
p24 intermediate. We also demonstrate that
B-crystallin
co-immunoprecipitates with the p24 partially processed caspase-3
in vivo. Taken together, our results demonstrate
that
B-crystallin is a novel negative regulator of apoptosis that
acts distally in the conserved cell death machinery by inhibiting the
autocatalytic maturation of caspase-3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family bind to
their receptors, thereby recruiting and activating procaspase-8 via a
series of protein-protein interactions mediated by FADD (6, 7). Active
caspase-9 (the mitochondrial pathway) or caspase-8 (the death receptor
pathway) then initiate the proteolytic activation of procaspase-3 by a
multi-step mechanism. In the first step, caspase-9 or -8 cleaves
procaspase-3 at an aspartate residue between its large and small
subunits to generate a p24 intermediate (the prodomain and the large
subunit) and the p12 small subunit (8-12). Next, the prodomain is
removed from the p24 intermediate by an autoproteolytic event to
generate the p20 and p17 forms of the large subunit (8-12). Active
caspase-3 (two p17/p12 heterodimers) then induces the cell to undergo
apoptosis by proteolyzing key cellular targets.
B-crystallin, a small HSP family member related to Hsp27. Members of
the small HSP family contain a highly conserved
-crystallin domain
that is flanked by largely divergent amino- and carboxyl-terminal domains (18). They form oligomeric complexes that function as molecular
chaperones to facilitate protein folding and prevent aggregation of
denatured or misfolded proteins (18).
B-crystallin is constitutively
expressed in many tissues, and it is particularly abundant in the lens,
heart, skeletal muscle and in some cancers (18, 19). The expression of
B-crystallin is also induced by diverse cellular stresses (20).
Moreover,
B-crystallin has been shown to protect cells against
apoptosis induced by DNA-damaging agents, TNF-
, and Fas (21, 22).
However, the molecular mechanisms of
B-crystallin's anti-apoptotic
actions have not been delineated. We report here that
B-crystallin
antagonizes cytochrome c- and caspase-8-dependent activation of caspase-3 by binding to
partially processed caspase-3 and inhibiting its autoproteolytic maturation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin cDNA was PCR-amplified from human
B-crystallin
cDNA with the following oligonucleotide primers: 5'-GGCCGAATTCATGGACATCGCCATCCACCAC-3' and
5'-GGCCCTCGAGCTATTTCTTGGGGGCTG-CGG-3'. The full-length Hsp27 cDNA
was PCR-amplified from human heart cDNA
(CLONTECH) using the following primers:
5'-GGCCGAATTCATGACCGAGCGCCGCGTCCC-3' and
5'-GGCCCTCGAGTTACTTGGCGGCAGTCTCCATC-3'. The PCR products were then digested with EcoRI and XhoI and cloned into
a modified pcDNA3 vector in which the FLAG epitope was inserted
upstream of the multiple cloning site (kindly provided by Dr. J. Settleman). Sequences were confirmed by automated DNA sequencing.
B-crystallin or
human Hsp27, or 1 µg of control vector (pEGFP-N1,
CLONTECH) using LipofectAMINE Plus reagent
(Life Technologies) according to the manufacturer's instructions. For
stable transfections, MDA-MB-231 cells were transfected with 1 µg of
pcDNA3-FLAG vector or pcDNA3FLAG-
B-crystallin and
allowed to recover for 48 h; clones stably expressing these
constructs were then selected by growth in 800 µg/ml G418 (Life
Technologies) for 3 weeks. Individual G418-resistant clones were
examined for expression of
B-crystallin by immunoblotting with FLAG
M2 mAb (Sigma) as described (23).
and 1 µg/ml
cycloheximide (CHX) for 4 h (PC-3 cells). Transfected cells were
identified by GFP-fluorescence (control vector) or indirect
immunofluorescence (
B-crystallin or Hsp27) with FLAG M2 mAb as
described previously (24). Apoptosis was scored as the percentage of
transfected cells that had condensed/fragmented nuclei by staining with
Hoescht no. 33258 (Sigma) as described (24). For stable transfections,
pooled, vector-transfected cells or two clones stably expressing
B-crystallin were untreated or treated with 50 µM
etoposide for 65 h or 10 ng/ml TNF-
and 1 µg/ml CHX for
36 h; cells were then scored for apoptosis as above. At least 200 cells were counted in each experiment, and experiments were performed
in triplicate. The data is presented as the mean ± S.E.; the
significance of intergroup differences was assessed by a two-tailed,
paired Student's t test.
B-Crystallin and Hsp27
Proteins--
Purified recombinant
B-crystallin and Hsp27 were
produced using the Qiagen Expressionist system according to the
manufacturer's instructions. The full-length
B-crystallin cDNA
was PCR-amplified from the human
B-crystallin cDNA with the
following oligonucleotide primers:
5'-GGCCGAGCTCATGGACATCGCCATCCACCAC-3' and
5'-GGCCGGTACCCTATTTCTTGGGGGCTGCGG-3'. The full-length Hsp27 cDNA
was PCR-amplified from the human Hsp27 cDNA using the following
primers: 5'-GGCCGGATCCATGACCGAGCGCCGCGTCCC-3' and
5'-GGCCAAGCTTTTACTTGGCGGCAGTCTCCATC-3'. The PCR products were then digested with SacI and KpnI
(
B-crystallin) or BamHI and HindIII (Hsp27)
and cloned into the corresponding sites in pQE30A (Qiagen). Sequences
were confirmed by DNA sequencing. Recombinant proteins were purified
under native conditions using 250 mM imidizole to elute the
His-tagged proteins from Ni2+-NTA columns (Qiagen) and
stored at
80 °C.
B-crystallin (2-15 µM),
Hsp27 (2-15 µM), or Hsp70 (StressGen Biotechnologies, 5 µM) and 35S-labeled procaspase-9 (prepared
from procaspase-9 cDNA using the TnT T7 Quick Coupled
Transcription/Translation system (Promega) according to the
manufacturer's instructions). Caspases were activated by the addition
of 1 µg cytochrome c (Sigma)/1 mM dATP
(Amersham Pharmacia Biotech) for 30 min at 37 °C or 30 ng of
recombinant caspase-8 for 45 min at 37 °C. The reaction products
were resolved by SDS-PAGE and visualized by autoradiography as
described (23, 25). The procaspase-3 and procaspase-7 activation
studies were performed as above except that 35S-labeled
procaspase-9 was omitted, and the reaction products were analyzed by
immunoblotting with caspase-3 mAb (Transduction Laboratories) or
caspase-7 mAb (PharMingen) (23).
B-crystallin were lysed in IP lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton
X-100, 5% glycerol) at a final concentration of 1 × 106 cells/ml, and incubated on ice for 30 min. Lysates were
then centrifuged at 12000 rpm at 4 °C for 15 min. The supernatant
was incubated with protein A-agarose beads (Sigma) and 2 µg of
anti-IgG (Sigma) or anti-FLAG (Sigma) polyclonal antibodies. Complexes were immunoprecipitated overnight at 4 °C. Beads were then washed four times in IP wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 100 mM NaF) and the immunoprecipated proteins were detected by immunoblotting with
B-crystallin mAb (StressGen Biotechnologies) or caspase-3 mAb as described (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Crystallin Inhibits Etoposide- and TNF-
-induced
Apoptosis--
To begin delineating the mechanisms by which
B-crystallin inhibits apoptosis, we examined whether
ectopic expression of
B-crystallin protected cells from stimuli
that engaged the mitochondrial pathway (the DNA-damaging agent
etoposide) or the death receptor pathway (TNF-
). Human MDA-MB-231
breast cancer cells or PC-3 prostate carcinoma cells, which lack
B-crystallin (data not shown), were transiently transfected with
vector or FLAG-tagged
B-crystallin. After overnight incubation,
cells were treated with 50 µM etoposide for 48 h
(MDA-MB-231 cells) or 10 ng/ml TNF-
and 1 µg/ml CHX for 4 h
(PC-3 cells), and the percentage of transfected cells with apoptotic
nuclei was determined. As demonstrated in Fig. 1A, transient expression of
B-crystallin potently inhibited apoptosis induced by etoposide or
TNF-
. Hsp27, a small HSP family member closely related to
B-crystallin, also conferred a similar degree of protection against
etoposide- and TNF-
-induced apoptosis. We also generated MDA-MB-231
clones stably expressing FLAG-tagged
B-crystallin. As shown in Fig.
1B, the A5 and B4 clones stably expressing
B-crystallin
were also protected against apoptosis induced by etoposide and TNF-
compared with pooled, FLAG vector-transfected cells. These findings
demonstrate unequivocally that
B-crystallin inhibits both the
mitochondrial and death receptor apoptotic pathways. In addition,
because these two pathways converge on the proteolytic activation of
procaspase-3, they suggest that
B-crystallin may negatively regulate
apoptosis by inhibiting caspase-3 activation and/or other downstream
events shared by both pathways.
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Fig. 1.
Transient and stable expression of
B-crystallin inhibits etoposide- and
TNF-
-induced apoptosis. A,
human breast carcinoma MDA-MB-231 cells (left panel) or
prostate carcinoma PC-3 cells (right panel) were transiently
transfected with vector, FLAG-tagged
B-crystallin, or FLAG-tagged
Hsp27 cDNAs. After overnight incubation, cells were treated with 50 µM etoposide for 48 h (MDA-MB-231 cells) or 10 ng/ml
TNF-
and 1 µg/ml CHX for 4 h (PC-3 cells), and the percentage
of transfected cells with apoptotic nuclear morphology was
determined as detailed under "Experimental Procedures."
B, MDA-MB-231 cells stably expressing pcDNA3-FLAG vector
or pcDNA3FLAG-
B-crystallin were made as detailed under
"Experimental Procedures." The expression of
B-crystallin was
confirmed by immunoblotting (two independent clones expressing
B-crystallin, A5 and B4, are shown). Cells were untreated
(open bars) or treated (cross-hatched bars) with
50 µM etoposide (left panel) for 65 h or
10 ng/ml TNF-
/1 µg/ml CHX (right panel) for 36 h
and scored for apoptosis as detailed under "Experimental
Procedures." In both A and B, the data
represent the mean ± S.E. of three independent experiments (*,
p < 0.01).
B-Crystallin Inhibits the Autoproteolytic Maturation of the p24
Partially Processed Caspase-3--
We first examined the ability of
recombinant
B-crystallin to inhibit the cytochrome
c-dependent activation of procaspase-9 and
procaspase-3 in Jurkat S-100 cytosolic extracts. Importantly, the range
of concentrations of
B-crystallin (2-15 µM) used are consistent with those observed in a variety of cancer cell lines, either constitutively or in response to heat
shock.2 As shown in Fig.
2A (upper panel),
the addition of 1 µg of cytochrome c and 1 mM
dATP to S-100 extracts induced the cleavage of 35S-labeled
procaspase-9 to its well characterized p35 proteolytic product
(indicated by an arrow).
B-Crystallin (even at 15 µM concentration) only weakly inhibited the proteolytic
processing of procaspase-9 compared with Hsp27 (15 µM)
and Hsp70 (5 µM); the latter observation is consistent
with the reported ability of Hsp27 and Hsp70 to disrupt apoptosome
assembly and prevent procaspase-9 activation (13, 15, 16). As
demonstrated in Fig. 2A, middle panel, the
addition of cytochrome c and dATP to S-100 extracts also
triggered the cleavage of procaspase-3 into its p20 and p17 forms of
the large subunit (indicated by arrows); the caspase-3 mAb
used in these studies does not detect the small p12 subunit. However,
the addition of
B-crystallin led to the dose-dependent
accumulation of the p24 partially processed caspase-3 (prodomain plus
the large subunit) with a corresponding reduction in the amount of
fully processed caspase-3, particularly p17. The p24 processing
intermediate was observed at concentrations of
B-crystallin as low
as 2 µM (on prolonged exposure of the immunoblot, data
not shown). Because
B-crystallin is similar in size to the p24
partially processed caspase-3, we demonstrated that the caspase-3 mAb
does not cross-react with
B-crystallin (Fig. 2C). The
functional relevance of the caspase-3 maturation defect caused by
B-crystallin is demonstrated by
B-crystallin's dose-dependent reduction in the proteolytic processing of
procaspase-7 (Fig. 2A, lower panel), a downstream
target of caspase-3. In contrast, neither Hsp27 nor Hsp70 led to the
accumulation of the p24 intermediate in cytochrome
c/dATP-treated S-100 extracts (even with prolonged exposure
of immunoblots). Instead, both Hsp27 (10-15 µM) and
Hsp70 (5 µM) inhibited the initial caspase-9 cleavage of
procaspase-3, again consistent with their previously reported
inhibition of caspase-9 activation by cytochrome c (13, 15,
16). Because the maturation of the p24 partially processed caspase-3
requires the autocatalytic removal of its prodomain (8-12), these
findings indicate that the principal mechanism by which
B-crystallin
inhibits the cytochrome c-dependent
activation of caspase-3 is by blocking its autoproteolytic
maturation.
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Fig. 2.
B-crystallin inhibits the
autoproteolytic maturation of caspase-3 in vitro.
A,
B-crystallin inhibits the cytochrome
c-dependent maturation of the p24 partially
processed caspase-3. Jurkat S-100 extracts were preincubated in the
absence or presence of recombinant
B-crystallin (2-15
µM), Hsp27 (2-15 µM), or Hsp70 (5 µM), and then treated for 30 min at 37 °C with 1 µg
cytochrome c and 1 mM dATP. For the caspase-9
activation studies (upper panel), 35S-labeled
procaspase-9 was added to extracts before adding cytochrome
c/dATP. Procaspase-9 and its mature p35 proteolytic product
are indicated. For the caspase-3 (middle panel) and
caspase-7 (lower panel) activation studies, the endogenous
caspase-3 or -7 in the S-100 extracts was analyzed by immunoblotting.
Procaspase-3, the partially processed p24 intermediate (prodomain plus
the large subunit) and the p20 and p17 forms of the large subunit are
all indicated (middle panel). Procaspase-7 and its
proteolytically processed p31 and p17 forms are indicated (lower
panel). B,
B-crystallin inhibits the
caspase-8-dependent maturation of the p24 partially
processed caspase-3. Caspase-3 activation was studied as in
A except that S-100 extracts were activated by treatment
with 30 ng of recombinant caspase-8 for 45 min at 37 °C.
C, the caspase-3 mAb does not cross-react with
B-crystallin. 20 ng of purified, recombinant
B-crystallin was
analyzed by immunoblotting with caspase-3 mAb (left panel)
or
B-crystallin mAb (right panel).
B-crystallin inhibited the
caspase-8-dependent activation of procaspase-3 in S-100
extracts. In this system, procaspase-3 is directly cleaved by caspase-8
to generate the p24 intermediate, and the prodomain is subsequently removed by autoproteolytic cleavage to produce the p20/p17 forms of the
large subunit (8-12). As demonstrated in Fig. 2B, treatment of S-100 extracts with active caspase-8 led to the cleavage of procaspase-3 to p20 and p17. However, the addition of
B-crystallin led to the dramatic dose-dependent accumulation of the p24
processing intermediate and a concomitant reduction in the amount of
fully processed caspase-3, especially p17. In contrast, adding Hsp27 or
Hsp70 did not lead to the accumulation of the p24 intermediate. Moreover, neither Hsp27 nor Hsp70 substantially inhibited the cleavage
of procaspase-3 by caspase-8. Although
B-crystallin did not inhibit
capase-7 activation in S-100 extracts treated with caspase-8 (data not
shown), caspase-8 can directly cleave procaspase-7; hence, caspase-3
activation is not necessary for procaspase-7 processing in this system
(8). Taken together, these observations indicate that
B-crystallin
negatively regulates the cytochrome c- and
caspase-8-dependent activation of caspase-3 by inhibiting
the autoproteolytic maturation of its p24 intermediate.
B-Crystallin Binds to the p24 Partially Processed
Caspase-3 in Vivo--
To determine whether
B-crystallin binds to
caspase-3 in vivo, we immunoprecipitated
B-crystallin
from cells stably expressing FLAG-tagged
B-crystallin. Whole cell
lysates derived from FLAG-vector- or FLAG-
B-crystallin-expressing
cells were immunoprecipitated with FLAG mAb. As shown in Fig.
3 (middle panel),
B-crystallin co-immunoprecipitated with the p24 partially processed
caspase-3 (indicated by an arrow) in cells stably expressing
FLAG-tagged
B-crystallin; this interaction was not observed in
FLAG-vector-transfected cells. Importantly, the caspase-3 processing
intermediate that co-immunoprecipitated with
B-crystallin is the
identical size as the p24 partially processed caspase-3 observed in
cytosolic extracts treated with cytochrome c/dATP or
caspase-8 in the presence of
B-crystallin (data not shown). Although
the p24 intermediate is far less abundant than procaspase-3 in whole
cell lysates (Fig. 3, lower panel), it abundantly
co-immunoprecipitated with
B-crystallin in the absence of any
detectable interaction between
B-crystallin and procaspase-3,
thereby underscoring the specificity of the interaction between
B-crystallin and partially processed caspase-3 in vivo.
These findings suggest that
B-crystallin antagonizes the
autocatalytic processing of caspase-3 by binding to and inhibiting the
partially processed protease.
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Fig. 3.
B-crystallin binds to the p24
partially processed caspase-3 in vivo. MDA-MB-231 cells
stably expressing either FLAG-vector (left panel) or
FLAG-tagged
B-crystallin (right panel) were lysed and
immunoprecipitated with IgG or FLAG antibodies as detailed under
"Experimental Procedures." Whole cell lysates and each of the
immunoprecipitated complexes were then analyzed by immunoblotting with
B-crystallin (upper panel) or caspase-3 (middle
panel) mAbs. Procaspase-3 and the p24 partially processed
caspase-3 are indicated. For the caspase-3 immunoblot, the molecular
mass of markers in kDa is indicated at the left of the
panel. The lower panel shows an independent
experiment in which a whole cell lysate and FLAG-immunoprecipitate from
FLAG-
B-crystallin-expressing cells were immunoblotted with caspase-3
mAb.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-crystallin inhibits both the mitochondrial and death receptor
apoptotic pathways by a novel mechanism:
B-crystallin binds to
caspase-3 that has been partially processed by caspase-9 or caspase-8
cleavage (p24) and inhibits the autoproteolytic removal of its
prodomain to produce its large subunit. Because this autocatalytic
maturation is required for caspase-3 activation by both the
mitochondrial and death receptor pathways, the inhibition of caspase-3
maturation by
B-crystallin is a parsimonious strategy to inhibit
both pathways. In contrast, Hsp70 inhibits only the mitochondrial
pathway and may even sensitize cells to Fas-induced apoptosis (13, 15,
27). Importantly, neither Hsp27 nor Hsp70 had any effect on the
autoproteolytic maturation of caspase-3. These observations indicate
unambiguously that
B-crystallin inhibits apoptosis by a novel
mechanism that is distinct from that of other HSPs examined to date.
B-crystallin, XIAP does not inhibit the initial cleavage of
procaspase-3 by caspase-8 in cytosolic extracts, but it binds to the
p24 partially processed caspase-3 and inhibits its autoproteolytic
maturation (12). Although
B-crystallin is less potent in this
respect, its inhibition of caspase-3's autoproteolytic maturation is
observed at physiologically relevant concentrations (2-15
µM) as determined by quantitation of
B-crystallin
levels in cancer cell lines.2 Indeed, we have likely
underestimated the concentration of
B-crystallin in some tissues.
For instance,
-crystallin (a protein composed of
B-crystallin and
A-crystallin) accounts for 40% of the soluble protein in the lens,
and
B-crystallin constitutes as much as 5% of the total protein in
striated muscle (18, 19). However, XIAP is also a potent
inhibitor/substrate of active caspases-3 and -7 (12, 26) whereas
B-crystallin is not cleaved by caspases (data not presented). Hence,
the molecular mechanism by which
B-crystallin inhibits the
autoproteolytic maturation of capase-3 is likely to be different from
that of XIAP.
B-crystallin and
Hsp27 inhibit apoptosis by largely distinct mechanisms. In our studies,
Hsp27 clearly disrupts the cytochrome
c-dependent activation of procaspase-9, as
others have reported (16), whereas
B-crystallin only weakly inhibits
this event. Moreover, Hsp27 does not inhibit the autoproteolytic
maturation of caspase-3. Although
B-crystallin and Hsp27 share
~40% amino acid identity, most of the identical residues are found
within their respective
-crystallin domains; the amino and carboxyl
termini are largely divergent (18). Hence, the different anti-apoptotic
mechanisms of these closely related small HSPs are likely to be the
result of their distinct amino and/or carboxyl termini. Although we and others (21, 22) have observed that Hsp27, like
B-crystallin, inhibits both the mitochondrial and death receptor apoptotic pathways, the mechanism(s) by which Hsp27 inhibits the latter is unclear. In our
studies, Hsp27 did not inhibit the caspase-8-dependent activation of procaspase-3, suggesting that Hsp27 inhibits death receptor apoptosis upstream of this step (perhaps by interfering with
procaspase-8 activation). Nevertheless, our findings provide unequivocal evidence that
B-crystallin and Hsp27 inhibit apoptosis by distinct mechanisms.
B-crystallin, two other HSPs have been shown to
promote, rather than inhibit, caspase-3 maturation (28, 29). Hsp60,
Hsp10, and procaspase-3 form a multimeric complex in the mitochondria
of intact cells. In cytosolic extracts, Hsp60 and Hsp10 promote the
proteolytic activation of procaspase-3 by caspase-8 and -9 in an
ATP-dependent fashion, suggesting that their chaperone
activity enhances the sensitivity of procaspase-3 to proteolytic
cleavage by apical caspases. Together with our observation that
B-crystallin inhibits the autocatalytic maturation of caspase-3,
these findings indicate that the proteolytic maturation of caspase-3 is
an exquisitely regulated event.
B-crystallin is a novel negative regulator of apoptosis
that acts distally in the conserved cell death apparatus (downstream of
any previously reported HSP) by disrupting the autoproteolytic
maturation of caspase-3. Given the abundance of
B-crystallin in the
lens and in muscle,
B-crystallin is likely to play a particularly
important role in regulating apoptosis in these tissues. Indeed, the
recent observation that a missense mutation in
B-crystallin (R120G)
causes a familial syndrome characterized by cataracts and generalized
myopathy underscores the importance of
B-crystallin in the lens and
in muscle (30). Interestingly, the differentiation of lens epithelial
cells into lens fibers is accomplished by an atypical apoptotic
mechanism that leads to the removal of the nucleus and other organelles
from terminally differentiated lens fiber cells that survive for the
lifetime of the organism (31, 32). Because
B-crystallin is
abundantly expressed in lens fiber cells, it is tempting to speculate
that its presence prevents the completion of the apoptotic program and
promotes the long-term survival of these cells. In addition, our
findings clearly raise a number of important questions. For instance, what roles do oligomerization and/or chaperone function play
in
B-crystallin's anti-apoptotic actions? Although oligomerization of Hsp27 is necessary for its anti-apoptotic actions (33), the role of
oligomerization in
B-crystallin's anti-apoptotic actions has not
been studied. These and other issues will be examined in future studies
using mutants of
B-crystallin that are impaired in one or more of
these biochemical properties.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Talanian for providing active caspase-8 and Drs. Junying Yuan, Honglin Li, and Marcus Peter for their critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant from the Muscular Dystrophy Association (to V. L. C.), by National Institutes of Health Grants NS31957 (to V. L. C.) and 5T32-CA70085 (to M. C. K.), by institutional research grants to Northwestern University from the Howard Hughes Medical Institute (to V. L. C.), and by the Elizabeth Boughton Trust (to V. L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Endocrinology, Tarry 15-755, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-0644; Fax: 312-908-9032; E-mail: v-cryns@northwestern.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.C100107200
2 M. Kamradt and V. Cryns, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; HSP, heat shock protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CHX, cycloheximide; PCR, polymerase chain reaction, mAb, monoclonal antibody; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; IP, immunoprecipitate; FADD, Fas-associated death domain protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Cryns, V. L.,
and Yuan, J.
(1998)
Genes Dev.
12,
1551-1570 |
2. |
Thornberry, N. A.,
and Lazebnik, Y.
(1998)
Science
281,
1312-1316 |
3. | Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Cell 86, 147-157[Medline] [Order article via Infotrieve] |
4. | Zou, H., Henzel, W., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[Medline] [Order article via Infotrieve] |
5. | Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S. (1998) Mol. Cell 1, 949-957[Medline] [Order article via Infotrieve] |
6. | Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve] |
7. | Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[Medline] [Order article via Infotrieve] |
8. |
Fernandes-Alnemri, T.,
Armstrong, R. C.,
Krebs, J.,
Srinivasula, S. M.,
Wang, L.,
Bullrich, F.,
Fritz, L. C.,
Trapani, J. A.,
Tomaselli, K. J.,
Litwack, G.,
and Alnemri, E. S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7464-7469 |
9. | Martin, S. J., Amarante-Mendes, G. P., Shi, L., Chuang, T.-H., Casiano, C. A., O'Brien, G. A., Fitzgerald, P., Tan, E. M., Bokoch, G. M., Greenberg, A. H., and Green, D. M. (1996) EMBO J. 15, 2407-2416[Abstract] |
10. | Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve] |
11. |
Han, Z.,
Hendrickson, E. A.,
Bremner, T. A.,
and Wyche, J. H.
(1997)
J. Biol. Chem.
272,
13432-13436 |
12. |
Deveraux, Q. L.,
Roy, N.,
Stennicke, H. R.,
Van Arsdale, T.,
Zhou, Q.,
Srinivasula, S. M.,
Alnemri, E. S.,
Salvesen, G. S.,
and Reed, J. C.
(1998)
EMBO J.
17,
2215-2223 |
13. | Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000) Nat. Cell Biol. 2, 469-475[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Pandey, P.,
Saleh, A.,
Nakazawa, A.,
Kumar, S.,
Srinivasula, S. M.,
Kumar, V.,
Weichselbaum, R.,
Nalin, C.,
Alnemri, E. S.,
Kufe, D.,
and Kharbanda, S.
(2000)
EMBO J.
19,
4310-4322 |
15. | Saleh, A., Srinivasula, S. M., Balkir, L., Robbins, P. D., and Alnemri, E. S. (2000) Nat. Cell Biol. 2, 476-483[CrossRef][Medline] [Order article via Infotrieve] |
16. | Bruey, J. M., Ducasse, C., Bonniaud, P., Ravagnan, L., Susin, S. A., Diaz-Latoud, C., Gurbuxani, S., Arrigo, A. P., Kroemer, G., Solary, E., and Garrido, C. (2000) Nat. Cell Biol. 2, 645-652[CrossRef][Medline] [Order article via Infotrieve] |
17. | Pandey, P., Farber, R., Nakazawa, A., Kumar, S., Bharti, A., Nalin, C., Weichselbaum, R., Kufe, D., and Kharbanda, S. (2000) Oncogene 19, 1975-1981[CrossRef][Medline] [Order article via Infotrieve] |
18. | Clark, J. I., and Muchowski, P. J. (2000) Curr. Opin. Struct. Biol. 10, 52-59[CrossRef][Medline] [Order article via Infotrieve] |
19. | Kato, K., Shinohara, H., Kurobe, N., Inaguma, Y., Shimizu, K., and Ohshima, K. (1991) Biochim. Biophys. Acta 1074, 201-208[Medline] [Order article via Infotrieve] |
20. | Klemenz, R., Frohli, E., Steiger, R. H., Schafer, R., and Aoyama, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3652-3656[Abstract] |
21. | Mehlen, P., Kretz-Remy, C., Preville, X., and Arrigo, A. P. (1996) EMBO J. 15, 2695-2706[Abstract] |
22. |
Mehlen, P.,
Schulze-Osthoff, K.,
and Arrigo, A. P.
(1996)
J. Biol. Chem.
271,
16510-16514 |
23. |
Cryns, V. L.,
Bergeron, L.,
Zhu, H.,
Li, H.,
and Yuan, J.
(1996)
J. Biol. Chem.
271,
31277-31282 |
24. | Byun, Y., Chen, F., Chang, R., Trivedi, M., Green, K., and Cryns, V. (2001) Cell Death Differ. In press |
25. |
Cryns, V.,
Byun, Y.,
Rana, A.,
Mellor, H.,
Lustig, K.,
Ghanem, L.,
Parker, P.,
Kirschner, M.,
and Yuan, J.
(1997)
J. Biol. Chem.
272,
29449-29453 |
26. | Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 388, 300-304[CrossRef][Medline] [Order article via Infotrieve] |
27. | Liossis, S. N., Ding, X. Z., Kiang, J. G., and Tsokos, G. C. (1997) J. Immunol. 158, 5668-5675[Abstract] |
28. |
Xanthoudakis, S.,
Roy, S.,
Rasper, D.,
Hennessey, T.,
Aubin, Y.,
Cassady, R.,
Tawa, P.,
Ruel, R.,
Rosen, A.,
and Nicholson, D. W.
(1999)
EMBO J.
18,
2049-2056 |
29. |
Samali, A.,
Cai, J.,
Zhivotovsky, B.,
Jones, D. P.,
and Orrenius, S.
(1999)
EMBO J.
18,
2040-2048 |
30. | Vicart, P., Caron, A., Guicheney, P., Li, Z., Prévost, M.-C., Faure, A., Chateau, D., Chapon, F., Tomé, F., Dupret, J.-M., Paulin, D., and Fardeau, M. (1998) Nat. Genet. 20, 92-95[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Ishizaki, Y.,
Jacobson, M. D.,
and Raff, M. C.
(1998)
J. Cell Biol.
140,
153-158 |
32. |
Bassnett, S.,
and Mataic, D.
(1997)
J. Cell Biol.
137,
37-49 |
33. |
Rogalla, T.,
Ehrnsperger, M.,
Preville, X.,
Kotlyarov, A.,
Lutsch, G.,
Ducasse, C.,
Paul, C.,
Wieske, M.,
Arrigo, A. P.,
Buchner, J.,
and Gaestel, M.
(1999)
J. Biol. Chem.
274,
18947-18956 |