The Small Heat Shock Protein B-Crystallin Negatively Regulates ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 19, Issue of May 11, pp. 16059 –16063, 2001 Printed in U.S.A.

The Small Heat Shock Protein ␣B-Crystallin Negatively Regulates Cytochrome c- and Caspase-8-dependent Activation of Caspase-3 by Inhibiting Its Autoproteolytic Maturation* Received for publication, February 27, 2001, and in revised form, March 22, 2001 Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.C100107200

Merideth C. Kamradt, Feng Chen, and Vincent L. Cryns‡ From the Robert H. Lurie Comprehensive Cancer Center and the Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611

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 ␣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.

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 ac-

* 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. This 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: 312-9089032; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

tivation 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-␣ 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. 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 ␣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 1 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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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

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 ␣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. Transient and Stable Transfections—MDA-MB-231 and PC-3 cells were grown on glass coverslips and transiently transfected with 1 ␮g of pcDNA3-FLAG plasmid containing human ␣B-crystallin or human Hsp27, or 1 ␮g 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 ␮g 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 ␮g/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). Induction and Analysis of Apoptosis—24 h after transient transfection, cells were treated with 50 ␮M etoposide for 48 h (MDA-MB-231 cells) or 10 ng/ml TNF-␣ and 1 ␮g/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 ␮M etoposide for 65 h or 10 ng/ml TNF-␣ and 1 ␮g/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. Production of Recombinant Human ␣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. 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 ␣B-crystallin (2–15 ␮M), Hsp27 (2–15 ␮M), or Hsp70 (StressGen Biotechnologies, 5 ␮M) 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 ␮g 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 35 S-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). Immunoprecipitation—MDA-MB-231 cells stably expressing FLAGvector or FLAG-␣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 ␮g 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

␣B-Crystallin Inhibits Etoposide- and TNF-␣-induced Apoptosis—To begin delineating the mechanisms by which ␣Bcrystallin 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 ␮M etoposide for 48 h (MDA-MB-231 cells) or 10 ng/ml TNF-␣ and 1 ␮g/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 ␣Bcrystallin, also conferred a similar degree of protection against etoposide- and TNF-␣-induced apoptosis. We also generated MDA-MB-231 clones stably expressing FLAG-tagged ␣Bcrystallin. 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. ␣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 ␮M) 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 ␮g of cytochrome c and 1 mM dATP to S-100 2

M. Kamradt and V. Cryns, unpublished results.

␣B-Crystallin Inhibits the Maturation of Caspase-3

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 ␮M etoposide for 48 h (MDA-MB-231 cells) or 10 ng/ml TNF-␣ and 1 ␮g/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, MDAMB-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 (crosshatched bars) with 50 ␮M etoposide (left panel) for 65 h or 10 ng/ml TNF-␣/1 ␮g/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).

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 ␮M concentration) only weakly inhibited the proteolytic processing of procaspase-9 compared with Hsp27 (15 ␮M) and Hsp70 (5 ␮M); 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 ␮M (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 ␣Bcrystallin is demonstrated by ␣B-crystallin’s dose-dependent reduction in the proteolytic processing of procaspase-7 (Fig. 2A,

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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 ␮M), Hsp27 (2–15 ␮M), or Hsp70 (5 ␮M), and then treated for 30 min at 37 °C with 1 ␮g 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).

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 ␮M) and Hsp70 (5 ␮M) 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. We next examined whether ␣B-crystallin inhibited the

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␣B-Crystallin Inhibits the Maturation of Caspase-3 (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. DISCUSSION

FIG. 3. ␣B-crystallin binds to the p24 partially processed caspase-3 in vivo. MDA-MB-231 cells stably expressing either FLAGvector (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.

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

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 ␣B-crystallin inhibits both the mitochondrial and death receptor apoptotic pathways by a novel mechanism: ␣Bcrystallin 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. Interestingly, the autoproteolytic maturation of capase-3 is also inhibited by the conserved IAP family member XIAP. Like ␣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 ␮M) 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. Somewhat unexpectedly, the small HSP family members ␣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

␣B-Crystallin Inhibits the Maturation of Caspase-3 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. In contrast to ␣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. In short, ␣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 ␣Bcrystallin’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. Acknowledgments—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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