Autocatalytic Processing of HtrA2/Omi Is Essential for Induction of Caspase-dependent Cell Death through Antagonizing XIAP*

A mature form of nuclear-encoded mitochondrial serine protease HtrA2/Omi is pivotal in regulating apoptotic cell death; however, the underlying mechanism of the processing event of HtrA2/Omi and its relevant biological function remain to be clarified. Here, we describe that HtrA2/Omi is autocatalytically processed to the 36-kDa protein fragment, which is required for the cytochrome c-dependent caspase activation along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP, eventually promoting apoptotic cell death. We have shown that the autocatalytic processing of HtrA2/Omi occurs via an intermolecular event, demonstrated by incubating an in vitro translated HtrA2/Omi (S306A) mutant with the enzymatically active glutathione S-transferase-HtrA2/Omi protein. Using N-terminal amino acid sequencing and mutational analysis, we identified that the autocatalytic cleavage site is the carboxyl side of alanine 133 of HtrA2/Omi, resulting in exposure of an inhibitor of apoptosis protein binding motif in its N terminus. Our study provides evidence that the autocatalytic processing of HtrA2/Omi is crucial for regulating HtrA2/Omi-mediated apoptotic cell death.

HtrA 1 (high temperature requirement A), also known as DegP, was first described as a periplasmic protein in Escherichia coli (1,2). HtrA acts as a serine protease at high temperatures and as a chaperone at low temperatures (3)(4)(5). Most studies have shown that HtrA serine proteases exhibit endo-proteolytic activity by cleaving misfolded proteins or other cellular proteins as well as autocatalytic processing activity by mediating self-catalytic processing of HtrA serine proteases (4 -6).
Mammalian homologues of HtrA, human HtrA (huHtrA1/ L56), huHtrA2/Omi, and huHtrA3, have recently been identified (7)(8)(9)(10). huHtrA1/L56, which is the human homologue of the E. coli HtrA, was previously identified as a protein repressed in SV40-transformed fibroblasts and differentially expressed in osteoarthritic cartilage (9). The second mammalian homologue of HtrA, human HtrA2 (also known as Omi), was first identified as a protein that interacts with Mxi2, an alternatively spliced form of p38 stress-activated kinase (SAPK) (8). HtrA2 mRNAs are expressed ubiquitously in various tissues and cell types (8,10). The full-length HtrA2 protein, which encodes 458 amino acid residues, consists of a mitochondrial target sequence in its N-terminal region, a putative transmembrane (TM) domain, an inhibitor of apoptosis protein (IAP) binding motif (IBM), a single C-terminal PDZ (post-synaptic density, discs large and zonula occludens) domain that mediates protein-protein interactions (11)(12)(13), and a conserved catalytic domain of serine proteases that contains the His 198 , Asp 228 and Ser 306 catalytic triad and GNS 306 GGPL motif in its conserved active site (8,14).
Recently, HtrA2 has shown an intriguing function, contributing both to caspase-dependent and caspase-independent cell death (15)(16)(17)(18)(19). HtrA2 antagonizes inhibitory effect of XIAP (X-linked inhibitor of apoptosis) on caspases through interaction with the IBM of HtrA2. Moreover, HtrA2 is involved in caspase-independent cell death through regulating its serine protease activity; however, the molecular mechanism in this pathway remains to be elucidated. HtrA2 is predominantly localized to the mitochondria, from which a mature form is released into the cytosol in response to apoptotic stimuli (14 -18, 20, 21). The N-terminal 133 amino acid residues seem to be processed in the mature HtrA2 protein (15)(16)(17)(18)22). Processing of HtrA2 exposes an internal tetrapeptide (AVPS) reaper-like motif or an IBM in its N terminus, which is conserved in Smac/DIABLO (Smac/direct IAP-binding protein with low pI) and the Drosophila death proteins Reaper, Grim, Hid, and Sickle (14,(23)(24)(25)(26)(27)(28)(29)(30). Mature HtrA2 is involved in regulating apoptotic induction along with neutralizing XIAP-mediated inhibition of caspases through interaction with XIAP in the cytosol in response to the apoptotic stimuli (15,(31)(32)(33)(34). These previous studies indicate that the processing of the HtrA2 precursor to the mature form is indispensable for apoptotic regulation. Nonetheless, to date no in-depth studies have examined whether HtrA2 undergoes autocatalytic processing to generate the mature form or whether it is cleaved by other mitochondrial proteases (19,22,33,35). Here, we present a detailed investigation of HtrA2 autocatalytic processing, occurring through an intermolecular mechanism that was observed in an in vitro cleavage reaction in which proteolytically inactive HtrA2 served as a substrate for catalytically active HtrA2. In addition, we demonstrate that the mature HtrA2 generated by the autocatalytic processing can promote caspase activation by inhibiting the XIAP activity, inducing apoptotic cell death.
Construction of Vectors for Expression of Recombinant HtrA2 and the S306A Mutant-The N-terminally truncated HtrA2 constructs, ⌬129 (truncation of amino acid residues 1-129) and ⌬133 (truncation of amino acid residues 1-133), were generated by PCR amplification with Pfu DNA polymerase (Stratagene) from the pcDNA-HtrA2-FLAG as a template (37). The resulting PCR-amplified fragments were subcloned into the pGEX-4T (Amersham Biosciences) vector carrying an N-terminal GST epitope tag for purification of proteins expressed in E. coli.
The QuikChange site-directed mutagenesis kit (Stratagene) was used according to manufacturer's instructions to generate a proteolytically inactive mutant (S306A) and a putative cleavage site mutant (R/R) of HtrA2. The resulting HtrA2 (S306A) and (R/R) mutants contained an alanine substitution at amino acid residue 306 and an arginine substitution at both 132 and 133 amino acid residues, respectively. Details of all plasmid constructs and all primer sequences are available upon request. The sequence integrity of all plasmid constructs was verified by DNA sequencing with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems).
Purification of Recombinant HtrA2 Expressed in E. coli-Expression of GST-HtrA2 fusion proteins in BL21 cells was induced with 0.1 mM isopropyl-1-thio-␤-D-galactoside as described previously (36,37). The cultures were collected by centrifugation, and the bacterial pellets were resuspended in lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 0.5% Nonidet P-40 (pH 8.0)) containing 7 mM dithiothreitol (DTT) and 2 g/ml protease inhibitors aprotinin and leupeptin. The GST-HtrA2 protein was purified from crude cell lysates under non-denaturing conditions by selective binding to glutathione-Sepharose 4B beads (Amersham Biosciences) as described previously (38 -40). The bead-bound GST-HtrA2 proteins were resuspended in 1ϫ SDS loading buffer and resolved by 15% SDS-PAGE. For purification of GST-HtrA2, the protein-bound beads were resuspended in 100 l of elution buffer (50 mM Tris-HCl (pH 7.6), 20 mM KCl, 1 mM DTT) containing 5 mM reduced glutathione. The GST-HtrA2 protein was eluted from glutathione beads by incubation for 5 min at 37°C. Protein purity and concentrations were estimated by comparison with bovine serum albumin of known concentration in SDS-PAGE followed by staining of the gel with Coomassie Brilliant Blue dye.
For purification of HtrA2, the HtrA2 protein-bound beads were resuspended in 100 l of cleavage buffer (50 mM Tris-HCl (pH 7.6), 20 mM KCl, 1 mM DTT) and incubated with 5 units of human thrombin (Amersham Biosciences) for 1 h at room temperature. The cleaved proteins were eluted from the GST-bound beads by brief centrifugation. Purified proteins were stored at Ϫ70°C in a final glycerol concentration of 20% and 7 mM DTT and were stable for several months.
Proteolytic Cleavage Assays and N-terminal Amino Acid Sequencing-[ 35 S]Methionine-labeled mutant forms of HtrA2 were prepared by in vitro transcription and translation of plasmids encoding various forms of HtrA2 as templates in the TNT T7 Coupled Reticulocyte Lysate system (Promega). Cleavage reactions were initiated by adding proteolytically active or inactive forms of ⌬133 to a final concentration of 0.5 M in 30 l of cleavage buffer (50 mM Tris-HCl (pH 7.5), 1 mM DTT) for 6 h at 37°C. The reaction products were analyzed by 15% SDS-PAGE, and gels were dried and exposed to x-ray film.
Amino acids at the cleavage site were identified by N-terminal se-quencing. The processed HtrA2 proteins were transferred electrophoretically onto a polyvinylidene difluoride membrane, and the transferred proteins were stained with Ponceau S solution. The 36-kDa fragment (ϳ1.2 g) was excised from the membrane, and its N-terminal amino acid residues were identified by using the Procise 491 protein sequencer (Applied Biosystems) (Korea Basic Science Institute). Cell Culture and Transfection-293 cells (American Type Culture Collection), which yield a high transfection efficiency, were maintained at 37°C in a humidified 5% CO 2 incubator in Dulbecco's modified Eagle's minimum essential medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen), 50 units/ml penicillin, and 50 g/ml streptomycin (Invitrogen). For all transfections into 293 cells, the LipofectAMINE reagent (Invitrogen) was used according to the manufacturer's instructions.
Immunoblot Analyses and Immunoprecipitation-Transfected cells were lysed for 2 h at room temperature in radioimmune precipitation assay buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate) containing protease inhibitors, 10 g/ml aprotinin, 10 g/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride, and protein concentrations were determined with the Bio-Rad protein assay kit (Bio-Rad). The protein extracts (30 g) were resolved by 15% SDS-PAGE. HtrA2 expressed from the heterologous system was immunoblotted by IB analysis with anti-HtrA2 or anti-FLAG antibodies, then detected with the enhanced chemiluminescent (ECL) immunoblotting system as described by the manufacturer (Amersham Biosciences).
For immunoprecipitation, the transfected cells were homogenized in radioimmune precipitation assay buffer. Briefly, protein extracts (1 mg) were incubated for 2 h at 4°C with anti-HtrA2 antibody and precipitated with protein G beads (Amersham Biosciences). The resulting immunoprecipitates were resolved by 15% SDS-PAGE, followed by IB analysis with anti-FLAG antibody.
Preparation of S100 Fraction from 293 Cells-S100 extracts were prepared as described previously (41). Briefly, cells (2 ϫ 10 7 ) were harvested by centrifugation at 1,000 ϫ g for 10 min at 4°C and washed with ice-cold phosphate-buffered saline. The cell pellet was resuspended in 1 ml of ice-cold buffer A (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT) containing protease inhibitors, 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 10 g/ml leupeptin. After incubation on ice for 15 min, cells were broken by passing 10 times through a G26 needle. Cell extracts were centrifuged at 1000 ϫ g for 10 min at 4°C. The supernatant was further centrifuged at 50,000 ϫ g for 1 h in a Sorvall Biofuge centrifuge (Kendro Laboratory Products). The resulting supernatant (S-100 fraction) was stored at Ϫ70°C and used for the in vitro caspase activation assay.
Subcellular Fractionation-The cells were broken by passing 10 times through a G26 needle in buffer D (250 mM sucrose, 20 mM HEPES (pH 7.5), 10 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 10 mM Tris-HCl (pH 7.5) containing protease inhibitors. Cell extracts were centrifuged at 900 ϫ g for 5 min at 4°C to remove supernatants. These supernatants were further centrifuged at 50,000 ϫ g for 1 h at 4°C to recover pellets (the mitochondria-containing fraction) and supernatants (the cytosolic fraction). The subcellular fractions were processed for IB analysis with anti-HtrA2 or anti-cytochrome c antibodies.
Cell Death Assay-293 cells (1 ϫ 10 5 cells/well) in 6-well plates were transfected with 0.3 g of empty vector or plasmids encoding full-length HtrA2 mutants (S306A or R/R/S306A) together with 0.1 g of pEGFP-N3 reporter plasmid. At 24 h post-transfection the cells were treated with increasing concentrations of staurosporine (0 -1 M) for 5 h and were stained with 4Ј,6-diamidino-2-phenylindole. Normal and apoptotic nucleus-containing GFP-positive cells were counted under fluorescence microscopy as described previously (16).
To assess the apoptotic features, DNA strand breaks in apoptotic cells were detected by the terminal deoxynucleotidyltransferase (TdT)mediated dUTP nick-end labeling (TUNEL) assay (In Situ Cell Death kit, Roche Applied Science). Additionally, translocation of phosphatidylserine to the outer membrane of apoptotic cells was detected by the annexin V-fluorescein isothiocyanate apoptosis detection kit (Pharmingen). Images of the stained nuclei were visualized to determine the incidence of apoptotic changes under a fluorescence microscopy.
Phylogenetic Analysis-Homologous protein sequences to human HtrA2 were collected from public databases through BLAST searches. The complete coding sequences or the expressed sequence tags (EST) of HtrA2 were obtained from 12 different animal species. The 12 representing sequences were aligned by using ClustalX (42), and then gaps were further adjusted by using the MacClade program (version 4.0, Sinauer Associates Inc.). The phylogenetic tree of the HtrA2 proteins was reconstructed by both maximum parsimony and neighbor-joining methods using the PAUP program (version 4.0b10, Sinauer). Branchand-bound algorithm with ACCTRAN character transformation option was adopted for maximum parsimony analysis. Branch supports were estimated from 1000 bootstrapping replicates.

HtrA2 Is Autocatalytically Processed in an Intermolecular
Manner-To investigate whether a mature form of HtrA2 observed in mammalian is generated by autocatalytic processing, we synthesized the proteolytically inactive mutant form of HtrA2 in a cell-free transcription and translation system ( Fig.  1). An in vitro translated [ 35 S]Met-labeled ⌬129 (S306A) that contained 4 amino acid residues preceding an IBM (Fig. 1A) was incubated with wild-type or S306A mutant ⌬133 proteins (Fig. 1B). The ⌬133 proteins were expressed as GST fusions in E. coli and purified in a single step from cell lysates by selective binding to glutathione-Sepharose 4B beads. Wild-type ⌬133 exhibited endoproteolytic activity against ␤-casein as an exogenous substrate, whereas the endoproteolytic activity was abolished by mutation at the conserved active site, serine 306 (Fig.  1C). In an in vitro cleavage assay, we observed a 36-kDa band similar in size to the band detected in mammalian cells (15). In contrast, production of the 36-kDa protein fragment was not observed when incubated with ⌬133 (S306A). The result indicates that HtrA2 is directly involved in producing the 36-kDa protein fragment in trans or an intermolecular manner.
The Autocatalytic Ala 133 -Ala 134 Cleavage of HtrA2 Exposes an IAP Binding Motif-To identify the autocatalytic cleavage site of HtrA2, we constructed in-frame fusions of N-terminaltruncated HtrA2 with GST, and GST fusion proteins expressed in E. coli were purified by glutathione-Sepharose 4B beads (Fig. 2). Full-length HtrA2 was unavailable for biochemical studies because it was highly susceptible to proteolytic degradation during expression in E. coli (37). Fortunately, we found that ⌬129 is a suitable form for biochemical studies of its enzyme activity and proteolytic processing, since the ⌬129 protein expressed in E. coli not only is as soluble as other truncated portions of HtrA2 but also contains additional amino acid residues preceding the putative cleavage site, 130 MVLAAVP-SPP (Fig. 1A) (36).
During expression of GST-HtrA2 in E. coli and purification, several protein fragments of lower molecular mass were detected in the catalytically active wild-type, ⌬133 and ⌬129, whereas these fragments were barely detectable in the catalytically inactive mutants, ⌬133 (S306A) and ⌬129 (S306A) (Fig.  2A). The result suggests that in SDS-PAGE, the faster-migrating proteins were derived from self-proteolytic processing of HtrA2. To verify whether these fragments were autocatalytic processing products of HtrA2, we analyzed them by IB analysis with anti-HtrA2 (Fig. 2B) or anti-FLAG (Fig. 2C) antibodies. Two fragments of relative molecular mass 36 and 43 kDa, which were predicted by comparison with the relative molecular mass of size marker proteins, were specific to anti-HtrA2 or anti-FLAG antibodies.
To identify the amino acid sequence in the N-terminal side of the scissile bond, we selected the 36-kDa fragment, designated p36, with a band size similar to mature HtrA2. The N-terminal amino acid sequence of this proteolytic fragment was identified as AVPSPP by N-terminal amino acid sequencing, representing that the autocatalytic cleavage site starts with alanine 134 (Fig. 2D). Consequently, an IBM of HtrA2 is exposed by the autocatalytic Ala 133 -Ala 134 cleavage of it. Additionally, the presence of a larger, ϳ43-kDa fragment suggests that there are other cleavage sites within GST for HtrA2 endoproteolytic activity, since the 43-kDa fragment was also specific to anti-HtrA2 and anti-FLAG antibodies that recognize the C-terminal region of HtrA2 (37). p36 was not detected from even wild-type ⌬133 that had methionine and alanine residues in the junction between GST and mature HtrA2 (Figs. 2 and 3A). It is probable that the amino acid sequence GSAM in ⌬133 was less susceptible to self-proteolytic processing than the amino acid sequence MVLA surrounding the cleavage site in ⌬129, although both proteins consisted of the same AVPS residues in the P 1 Ј through P 4 Ј subsites (Fig. 3A). The results suggest that the amino acid residues in the P 1 through P 4 sites of the substrate seem to be one of the significant criteria for specificity to HtrA2 processing (5,14,43,44).
p36 Serves as an Inhibitor of XIAP through Interaction with XIAP, Promoting the Activation of Caspases-To determine whether p36 interacts with XIAP, we performed in vitro binding assays with S100 extracts and the recombinant HtrA2 proteins, which were cleaved by thrombin digestion and purified by elution from the GST-bound beads (Fig. 3B). The thrombin-cleaved HtrA2 proteins were unable to interact with XIAP because extra amino acid residues preceding an IBM made them more conformationally restricted by masking a methyl group (-CH 3 ) and the protonated amino group (-NH 3 ϩ ) of the N-terminal alanine of an IBM (Fig. 3, A and B) (26,27,30).
Only wild-type ⌬129 exhibited the interaction with endogenous XIAP, whereas no bands were detectable from ⌬129 (S306A) as well as wild-type ⌬133. The result demonstrates that p36 is essential for the interaction with XIAP (Fig. 3B). Subsequently, we investigated whether p36 promotes cytochrome c-dependent activation of the caspases by eliminating the inhibitory effect of XIAP (Fig. 3C). To analyze this function in vitro, the thrombin-cleaved recombinant HtrA2 proteins were incubated with S100 extracts supplemented cytochrome c and dATP. In the presence of cytochrome c and dATP, procaspase-3 was proteolytically cleaved to the active forms, p20 and p17 (Fig. 3C, lane 2). However, the addition of XIAP completely abolished the activation of procaspase-3 (Fig. 3C, lane 3). p36 was observed only in wild-type ⌬129 but not in ⌬129 (S306A) or wild-type ⌬133 (Fig. 3C, third panel). Likewise, we observed that the inhibitory effect of XIAP on the caspase activation was eliminated by incubating with wild-type ⌬129, resulting in the proteolytic cleavage of procaspase-3 (Fig. 3C, lane 6).
ICAD is a caspase-3 substrate that needs to be cleaved to release active caspase-activated DNase (45,46). To investigate whether the active caspase-3 cleaves ICAD, we analyzed IBs with anti-ICAD antibody (Fig. 3C, second panel). Activation of procaspase-3 promoted cleavage of the ICAD precursor to the processed product (ϳ12 kDa) of ICAD. Taken together, the ability that HtrA2 enhances the activation of procaspase-3 and the enzymatic activity of caspase-3 is dependent upon exposing the IBM of HtrA2 via its autocatalytic Ala 133 -Ala 134 cleavage.
The Autocatalytic Ala 133 -Ala 134 Cleavage of HtrA2 Is Required for Enhancement of Apoptotic Cell Death through Antagonizing XIAP-mediated Caspase Inhibition-Before we characterize the role of p36 in mammalian cells, we further examined the autocatalytic processing activity of HtrA2 by the in vitro cleavage assay. Full-length [ 35 S]Met-labeled HtrA2, which was generated with the cell-free expression system from the corresponding pcDNA-HtrA2-FLAG plasmids, was incubated with wild-type ⌬133 (Fig. 4B). Protein fragments with molecular masses of ϳ42 and 36 kDa were detected in HtrA2 (S306A). To verify the autocatalytic cleavage site of HtrA2, leucine and alanine at positions 132 and 133, which are P 1 and P 2 subsites, respectively, were converted to arginine residues (named R/R). The 36-kDa protein fragment, which is designated p36, was almost completely abolished in the HtrA2 (R/ R/S306A), indicating that HtrA2 cleaves by itself on the Nterminal side of alanine 134.
To characterize the proteolytic cleavage of HtrA2 in mammalian cells, we transiently transfected into 293 cells a plasmid encoding full-length HtrA2 with a C-terminal FLAG epitope tag (Fig. 4C). The construct encoding HtrA2 (S306A) directed the expression of one protein that was ϳ50 kDa and two additional proteins with lower molecular masses of ϳ42 and 36 kDa, which were specific to anti-HtrA2 or anti-FLAG antibodies. Consistent with results from the in vitro cleavage assay, the replacement of arginine residues at leucine 132 and alanine 133 in HtrA2 eliminated production of p36, whereas production of the 42-kDa fragment, designated p42, increased in comparison with that of HtrA2 (S306A) (Fig. 4C, lane 3). The results provide evidence that the mature form of HtrA2 observed in mammalian cells is predominantly generated by the autocatalytic Ala 133 -Ala 134 cleavage event of HtrA2.
Furthermore, to investigate the role of the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 in mammalian cells, 293 cells were transiently transfected with a plasmid encoding either full-length HtrA2 (S306A) or (R/R/S306A) (Fig. 5). Several studies show that the protease activity of HtrA2 induces caspase-independent cell death (15,18) or promotes cytochrome c-dependent caspase activation (19). To except these effects of the protease activity of HtrA2, we used the catalytically inactive mutant HtrA2 (S306A). In the HtrA2 (S306A), we observed that p36 and cytochrome c were released from the mitochondria into the cytosolic in response to an apoptotic stimulus, staurosporine (Fig. 5A). The release of p36 was not observed in the autocatalytic mutant, HtrA2 (R/R/S306A). To verify that the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 is necessary for the interaction between HtrA2 and XIAP in mammalian cells, cells transiently overexpressing HtrA2 pro- FIG. 3. The autoproteolytic product of HtrA2 promotes caspase activation through interaction with XIAP. A, amino acid sequence of junctions between GST and HtrA2 fusions. The GST-HtrA2 proteins were cleaved by thrombin proteolytic digestion, and the HtrA2 proteins purified from the GST moiety were used for in vitro interaction and caspase activation assays. The thrombin and autocatalytic cleavage sites are indicated by arrows and an arrowhead, respectively. B, interaction of XIAP with p36. The indicated HtrA2 recombinant proteins were immunoprecipitated (IP) with anti-HtrA2 antibody. The HtrA protein-bound protein G beads were incubated with 293-S100 extracts for 2 h at 4°C and washed with phosphate-buffered saline. The proteins bound to the beads were resolved by 15% SDS-PAGE followed by IB analysis with the anti-XIAP (top panel) or anti-HtrA2 (bottom panel) antibodies. The asterisks in B and C indicate p36. C, effect of p36 on cytochrome c (Cyt c)-mediated caspase-3 activation. Cytochrome c (10 M) and dATP (1 M) were supplemented to S100 extracts. The indicated recombinant proteins were added to S100 extracts with or without XIAP (20 nM). After incubation at 30°C for 2 h, the reaction mixtures were resolved by 15% SDS-PAGE followed by IB analysis with anticaspase-3, anti-ICAD, anti-HtrA2, or anti-XIAP antibodies. Lane 2, procaspase-3 (procas-3) was converted to active caspase-3 by cytochrome c and dATP; lane 3, XIAP inhibited the procaspase-3 activation; lane 6, autocatalytic processing of HtrA2 promoted activation of caspase-3 by antagonizing XIAP inhibition. Filled and open arrowheads indicate the ICAD precursor and the cleaved product (ϳ12 kDa), respectively. teins were analyzed by coimmunoprecipitation assay (Fig. 5B). The interaction between XIAP and HtrA2 was observed in cells expressing HtrA2 (S306A) but not in HtrA2 (R/R/S306A), indicating that p36 preferentially binds to XIAP. Because endogenous mature HtrA2 was also coimmunoprecipitated with XIAP, a tiny amount of XIAP was also detected in cells that were transiently transfected with empty vector or HtrA2 (R/R/ S306A). Full-length HtrA2, p50, was localized in the cytosol as well as in the mitochondria; nonetheless, the cytosolic p50 did not coimmunoprecipitated with XIAP. Thus, the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 is significant for binding of HtrA2 to XIAP with potent caspase inhibitory activity.
To assess the role of the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 in the induction of apoptosis, we measured the extent of apoptotic cell death by directly counting the numbers of cells with apoptotic nuclei under fluorescence microscopy (Fig. 5C). After 1 M staurosporine treatment, HtrA2 (S306A)-transfected cells revealed significantly higher cell death than empty vector-or HtrA2 (R/R/S306A)-transfected cells (Fig. 5D). To verify the apoptotic features, we monitored the key apoptotic events in the cells. The fragmentation of the genomic DNA, a biochemical hallmark of apoptosis, was visualized by the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nickend labeling (TUNEL) assay (Fig. 5E, left panel). Cell surface exposure of phosphatidylserine during apoptosis was detected by annexin V binding to phosphatidylserine as an additional marker for apoptotic cells (Fig. 5E, right panel). At 12 h of exposure to staurosporine, HtrA2 (S306A)-transfected cells revealed more effective activation of caspases-3 than HtrA2 (R/ R/S306A)-transfected cells, resulting in a decrease of levels of procaspase-3 along with an increase of the levels of active caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP) (Fig. 5F). Taken together, these results support evidence that the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 plays an indispensable role in enhancement of apoptosis.
The Autocatalytic Ala 133 -Ala 134 Cleavage Site of HtrA2 Is Evolutionally Conserved in Mammals-To investigate the evolutionary relationships of the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2, we performed the phylogenetic analysis (Fig. 6). Twelve aligned HtrA2 protein sequences are 515 amino acids in length, and completed sequences of HtrA2 range from 403 to 458 amino acids in length. The protease and PDZ domains of HtrA2 are well conserved among animals; however, the HtrA2 N-terminal regions that consist of 134 amino acids are conserved only in mammals. The mammalian HtrA2 protein sequences form a strongly supported monophyletic group by 100% bootstrapping supports (Fig. 6A). The same results were also obtained from the neighbor-joining analysis. The phylogenetic tree based on multiple sequence alignment also suggests that the Ala 133 -Ala 134 sequences are conserved in the mammalian HtrA2, with the exception of a serine substitution at the 134-amino acid residue of the bovine HtrA2 (Fig. 6B). These six mammalian HtrA2 orthologs contain identical amino acid residues at the P 1 and P 2 sites, which are critical for the specificity to the autocatalytic processing of the human HtrA2. Therefore, the amino acid substitutions at the alanine 134 did not abolish the autocatalytic cleavage of HtrA2 between residues 133 and 134 (Fig. 6C). DISCUSSION We have described here that the Ala 133 -Ala 134 cleavage of HtrA2 into the mature form, p36, is catalyzed by its own trypsin-like serine protease activity. In the in vitro cytochrome c-dependent caspase activation assay, we have shown that wild-type ⌬129, but not wild-type ⌬133, ⌬129 (S306A), and ⌬133 (S306A), neutralizes XIAP-mediated inhibition of caspases through interaction with XIAP, promoting activation of caspase. In the presence of wild-type HtrA2, ⌬129 and ⌬133, the amounts of XIAP were reduced by ϳ50%, indicating that XIAP may serve as a substrate for HtrA2 (Fig. 3C) (47, 48).
Regardless of the amounts of XIAP that were irreversibly degraded, the activation of caspase-3 was observed only in wildtype ⌬129 but not in ⌬133. The difference between wild-type ⌬129 and ⌬133 is that ⌬129 contains additional amino acid residues in the N-terminal side of the IBM (Fig. 3A). This flanking sequence permits ⌬129 to be autocatalytically processed to generate p36, exposing an IBM to allow interaction with XIAP. The autocatalytic Ala 133 -Ala 134 cleavage of HtrA2, therefore, is an essential event for regulating caspase-dependent apoptotic cell death through the interaction between XIAP and HtrA2.
The notion that the mature form of HtrA2 is generated by other endogenous proteases or its autoproteolytic activity still remains controversial because endogenous HtrA2 protein exists in the most of mammalian cells (8,10). To overcome the technical problem that was accompanied by pre-existing mature HtrA2 in mammalian cells, we characterized the proteolytic processing event of HtrA2 by incubating an in vitro translated [ 35 S]Metlabeled HtrA2 as a substrate and purified recombinant HtrA2 as an enzyme. Moreover, we have developed a method to identify the cleavage site of HtrA2 by using a pGEX expression and purification system, followed by an in vitro cleavage assay and the N-terminal amino acid sequencing.
When a plasmid encoding full-length HtrA2 was transfected into 293 cells, we observed three differently migrating proteins that were immunoreactive to anti-HtrA2 or anti-FLAG antibodies: the fully processed mature form, p36, the precursor form, p50, and the other proteolytic doublet between p50 and p36. The molecular mass of 42 kDa of the doublet raises the possibility that the cleavage occurs at amino acid residues close or within the TM domain of HtrA2. If so, the doublet might be generated by mitochondrial intramembrane cleaving protease (I-Clip), which cleaves within a TM domain of the substrate and liberates its biologically active form (49 -52). It is likely that HtrA2 could be not only a substrate of a mitochondrial I-Clip but also serve as a protease, cleaving other membraneanchored proteases. We presently cannot identify the N-terminal amino acid residues of the doublet by using the pGEX expression system and an in vitro cleavage assay because fulllength HtrA2 was extremely susceptible to proteolysis during expression in E. coli (37).
Relatively high amino acid sequence similarities among animal HtrA2 proteins on the serine protease and PDZ domains suggest that the HtrA2 orthologs are likely to have the same evolutionary origin in the serine protease activity. However, the N-terminal regions are conserved among the mammalian HtrA2 proteins, thus implying that the functions related to this region might be conserved in the mammalian HtrA2 orthologs. The phylogenetic analysis, therefore, suggests that the autocatalytic cleavage of HtrA2 between the positions 133 and 134 is derived before the mammalian diversification and conserved in mammals.
A recent study has reported that other mitochondrial proteases may be involved in maturation of HtrA2, as demonstrated by comparing the extent of processing of the wild-type and the active site mutant of HtrA2 in mnd2 (motor neuron degeneration 2) mouse embryonic fibroblasts (53). However, further detailed investigation of the HtrA2 processing, specially by using the HtrA2 knock-out system, can provide conclusive evidence for the HtrA2 processing mechanism in mammals because mnd2 cells express transiently inactive HtrA2, which contains a cysteine substitution at serine 276.
HtrA2 might be a multifunctional protein with many different aspects, providing an endoproteolytic function for removing aberrantly unfolded or damaged proteins in the mitochondria (5, 7, 53), proapoptotic activity through an antagonizing inhib- FIG. 6. Evolutionary relationships of the HtrA2 proteins from various animal species. A, the phylogenetic tree of the HtrA2 proteins. The solid triangle on the tree indicates the Ala 133 -Ala 134 preservation among the mammalian HtrA2 proteins along with the conserved N-terminal regions. The position, however, changed slightly in sequence to Ala 133 -Ser 134 in bovine HtrA2 protein (open triangle) by a single nucleotide point mutation. The tree is one of two most parsimonious topologies. The other tree differs only on the placement of Tetraodon, which positions between zebrafish and Xenopus. As a result, the strict consensus tree collapsed on the dashed line. The tree was oriented by treating the two invertebrates, Drosophila and mosquito, as a basal group. Branch lengths are proportional to numbers of hypothesized amino acid substitutions under the accelerated transformation character optimization. The scale bar indicates 50 amino acid changes. Numbers at nodes are maximum parsimony-bootstrapping support values in % scale from 1000 replications. B, sequence alignment of the cleavage site of the vertebrate HtrA2 orthologs. The mammalian HtrA2 orthologs are represented by bold type. IBM is shaded, and the autocatalytic cleavage site is indicated by an arrowhead. C, the autocatalytic cleavage of HtrA2 with a serine substitution at Ala 134 . The cells transfected with the indicated plasmids were analyzed by anti-HtrA2 antibody. Ser 134 indicates a full-length HtrA2 construct with a serine substitution at Ala 134 . wt, wild type. itory effect of XIAP on caspases in the cytosol, and an autocatalytic processing activity for producing active, mature p36 in addition to other, yet uncharacterized functions. The autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 is responsible for exposing an IAP binding motif that resembles the motif of IAP antagonists, such as Smac/DIABLO, Drosophila Grim, Reaper, and HID (14,(23)(24)(25)(26)(27)(28)(29)(30). A potent apoptosis-inducing activity of HtrA2 might be tightly regulated by localization at the different compartments as well as by its autocatalytic processing event. Our study supports the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 as a possibly pivotal scheme for a biologically significant mode of regulation of its functions in vivo. Moreover, sequence information for the cleavage site that is targeted by the autocatalytic Ala 133 -Ala 134 cleavage of HtrA2 offers insights into the processing of proteins containing an IBM. Eventually, identifying physiologically relevant substrates for HtrA2 will be important for elucidating the molecular mechanisms in regulating essential biological processes, including protein quality control and the apoptotic cell death pathway.