Translocation of the Serine Protease Omi/HtrA2 from Mitochondria into the Cytosol Upon Seizure-Induced Hippocampal Injury in the Neonatal Rat Brain

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death upon various brain injuries. However, the role of Omi/HtrA2 in neuronal death induced by status epilepticus (SE) in the immature brain has not been reported. In this study, we analyzed the contribution of serine protease Omi/HtrA2, its substrate X-linked inhibitor of apoptosis protein (XIAP) and the caspase-3 activation to damage of hippocamplal CA1 cells following lithium-pilocarpine SE in P14 rat pups. Status epilepticus in the immature brain significantly induced translocation of Omi/HtrA2 from mitochondria into the cytosol, increased cytosolic accumulation of Omi/HtrA2, induced appearance of XIAP-breakdown products and enhanced caspase-3 activity in the selectively vulnerable hippocampal CA1-subfield. Taken together, these results demonstrate for the first time that SE in the immature brain results in Omi/HtrA2 accumulation in the cytosol, where it probably promotes neuronal death by neutralizing and cleaving XIAP, one of the most potent endogenous inhibitors of apoptosis.     

Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression

A fall in circulating levels of cardiac survival factor insulin-like growth factor 1 (IGF-1) contributes to cardiac aging. To better understand the role of IGF-1 in cardiac aging, we examined the influence of cardiac IGF-1 overexpression on lifespan, cardiomyocyte intracellular Ca²⁺ homeostasis, protein damage, apoptosis and expression of pro- and anti-apoptotic proteins in young and old mice. Mouse survival rate was constructed by the Kaplan–Meier curve. Intracellular Ca²⁺ was evaluated by fura-2 fluorescence. Protein damage was determined by protein carbonyl formation. Apoptosis was assessed by caspase-8 expression, caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay. Pro- and anti-apoptotic proteins including Bax, p53, pp53, Bcl2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) were assessed by Western blot. Aging decreased plasma in IGF-1 levels, elevated myocyte resting intracellular Ca²⁺ levels, reduced electrically stimulated rise in intracellular Ca²⁺ and delayed intracellular Ca²⁺ decay associated with enhanced protein carbonyl formation, caspase-8 expression and caspase-3 activity in FVB mice, all of which with the exception of elevated resting intracellular Ca²⁺ were attenuated by IGF-1. Aging up-regulated expression of Bax, Bcl2 and ARC, down-regulated XIAP expression and did not affect p53, pp53 and Omi/HtrA2. The IGF-1 transgene attenuated or nullified aging-induced changes in Bax, Bcl2 and XIAP. Our data suggest a beneficial role for IGF-1 in aging-induced survival, cardiac intracellular Ca²⁺ homeostasis, protein damage and apoptosis possibly related to pro- and anti-apoptotic proteins.     

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.     

Mitochondrial protease Omi/HtrA2 enhances caspase activation through multiple pathways

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis and promotes cytochrome c (Cyt c)dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs) via its IAP-binding motif. The protease activity of Omi/HtrA2 also contributes to the progression of both apoptosis and caspase-independent cell death. In this study, we found that wild-type Omi/HtrA2 is more effective at caspase activation than a catalytically inactive mutant of Omi/HtrA2 in response to apoptotic stimuli, such as UV irradiation or tumor necrosis factor. Although similar levels of Omi/HtrA2 expression, XIAP-binding activity, and Omi/HtrA2 mitochondrial release were observed among cells transfected with catalytically inactive and wild-type Omi/HtrA2 protein, XIAP protein expression after UV irradiation was significantly reduced in cells transfected with wild-type Omi/HtrA2. Recombinant Omi/HtrA2 was observed to catalytically cleave IAPs and to inactivate XIAP in vitro, suggesting that the protease activity of Omi/HtrA2 might be responsible for its IAP-inhibiting activity. Extramitochondrial expression of Omi/HtrA2 indirectly induced permeabilization of the outer mitochondrial membrane and subsequent Cyt c-dependent caspase activation in HeLa cells. These results indicate that protease activity of Omi/HtrA2 promotes caspase activation through multiple pathways.     

Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.     

The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion

Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.     

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15.     

Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.     

Essential postmitochondrial function of p53 uncovered in DNA damage-induced apoptosis in neurons

In postmitotic sympathetic neurons, unlike most mitotic cells, death by apoptosis requires not only the release of cytochrome c from the mitochondria, but also an additional step to relieve X-linked inhibitor of apoptosis protein (XIAP)'s inhibition of caspases. Here, we examined the mechanism by which XIAP is inactivated following DNA damage and found that it is achieved by a mechanism completely different from that following apoptosis by nerve growth factor (NGF) deprivation. NGF deprivation relieves XIAP by selectively degrading it, whereas DNA damage overcomes XIAP via a p53-mediated induction of Apaf-1. Unlike wild-type neurons, p53-deficient neurons fail to overcome XIAP and remain resistant to cytochrome c after DNA damage. Restoring Apaf-1 induction in p53-deficient neurons is sufficient to overcome XIAP and sensitize cells to cytochrome c. Although a role for p53 in apoptosis upstream of cytochrome c release has been well established, this study uncovers an additional, essential role for p53 in regulating caspase activation downstream of mitochondria following DNA damage in neurons.     

Caspase-7 is directly activated by the approximately 700-kDa apoptosome complex and is released as a stable XIAP-caspase-7 approximately 200-kDa complex

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce approximately 1.4-MDa and approximately 700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This approximately 700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/HtrA2, active caspase-7 is released from the apoptosome and forms a stable approximately 200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable approximately 200-kDa complex with active caspase-7.     

HIV-1gp120 induces neuronal apoptosis through enhancement of 4-aminopyridine-senstive outward K+ currents

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) usually occurs late in the course of HIV-1 infection and the mechanisms underlying HAD pathogenesis are not well understood. Accumulating evidence indicates that neuronal voltage-gated potassium (Kv) channels play an important role in memory processes and acquired neuronal channelopathies in HAD. To examine whether Kv channels are involved in HIV-1-associated neuronal injury, we studied the effects of HIV-1 glycoprotein 120 (gp120) on outward K+ currents in rat cortical neuronal cultures using whole-cell patch techniques. Exposure of cortical neurons to gp120 produced a dose-dependent enhancement of A-type transient outward K+ currents (IA). The gp120-induced increase of IA was attenuated by T140, a specific antagonist for chemokine receptor CXCR4, suggesting gp120 enhancement of neuronal IA via CXCR4. Pretreatment of neuronal cultures with a protein kinase C (PKC) inhibitor, GF109203X, inhibited the gp120-induced increase of IA. Biological significance of gp120 enhancement of IA was demonstrated by experimental results showing that gp120-induced neuronal apoptosis, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase-3 staining, was attenuated by either an IA blocker 4-aminopyridine or a specific CXCR4 antagonist T140. Taken together, these results suggest that gp120 may induce caspase-3 dependent neuronal apoptosis by enhancing IA via CXCR4-PKC signaling.     

A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death

X chromosome-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspase-3, -7, and -9. Smac/DIABLO, an inhibitor of XIAP, is released from mitochondria upon receiving apoptotic stimuli and binds to the BIR2 and BIR3 domains of XIAP, thereby inhibiting its caspase-inhibitory activity. Here we report that a serine protease called HtrA2/Omi is released from mitochondria and inhibits the function of XIAP by direct binding in a similar way to Smac. Moreover, when overexpressed extramitochondrially, HtrA2 induces atypical cell death, which is neither accompanied by a significant increase in caspase activity nor inhibited by caspase inhibitors, including XIAP. A catalytically inactive mutant of HtrA2, however, does not induce cell death. In short, HtrA2 is a Smac-like inhibitor of IAP activity with a serine protease-dependent cell death-inducing activity.     

Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide, i.e., caspase-3 activation, nuclear translocation of apoptosis-inducing factor (AIF), and induction of p53, and prevented staurosporine-induced apoptosis by blocking caspase-8 activation upstream of caspase-3 activation and AIF nuclear translocation. Intracerebral APC infusion dose dependently reduced NMDA excitotoxicity in mice. By using different anti-PARs antibodies and mice with single PAR1, PAR3, or PAR4 deletion, we demonstrated that direct neuronal protective effects of APC in vitro and in vivo require PAR1 and PAR3. Thus, PAR1 and PAR3 mediate anti-apoptotic signaling by APC in neurons, which may suggest novel treatments for neurodegenerative disorders.     

Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.     

The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif

The inhibitor-of-apoptosis proteins (IAPs) play a critical role in the regulation of apoptosis by binding and inhibiting caspases. Reaper family proteins and Smac/DIABLO use a conserved amino-terminal sequence to bind to IAPs in flies and mammals, respectively, blocking their ability to inhibit caspases and thus promoting apoptosis. Here we have identified the serine protease Omi/HtrA2 as a second mammalian XIAP-binding protein with a Reaper-like motif. This protease autoprocesses to form a protein with amino-terminal homology to Smac/DIABLO and Reaper family proteins. Full-length Omi/HtrA2 is localized to mitochondria but fails to interact with XIAP. Mitochondria also contain processed Omi/HtrA2, which, following apoptotic insult, translocates to the cytosol, where it interacts with XIAP. Overexpression of Omi/HtrA2 sensitizes cells to apoptosis, and its removal by RNA interference reduces cell death. Omi/HtrA2 thus extends the set of mammalian proteins with Reaper-like function that are released from the mitochondria during apoptosis.     

Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction

To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.     

Binding specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi

Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. We have used peptide libraries to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, we show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins.     

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.