Mitochondrial Serine Protease Research Articles

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.

N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: rapid purification of a proteolytically active HtrA2/Omi

HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death; however, the underlying mechanism of HtrA2/Omi-mediated apoptosis remains to be elucidated. Using the pGEX bacterial expression system, we investigated the expression patterns of various forms of HtrA2/Omi. Full-length mouse HtrA2/Omi (mHtrA2/Omi) was successfully expressed in E. coli and purified as a proteolytically active protein. In contrast, the expression of full-length human HtrA2/Omi (hHtrA2/Omi) in E. coli was barely detected. On the basis of this result, we characterized further the expression patterns of N- or C-terminally truncated hHtrA2/Omi proteins. We found that three copies of the PRA XXT XXTP motif, which exist only in hHtrA2/Omi, might serve as a primary site that is highly susceptible to proteolytic degradation by host proteases. Removal of the N-terminal region containing the PRA XXT XXTP motifs produced a form resistant to proteolytic degradation during expression in E. coli and purification, consequently improving the production of a catalytically active, mature hHtrA2/Omi. Our study provides a method for generating useful reagents to investigate molecular mechanism by which HtrA2/Omi contributes to regulating apoptotic cell death and to identify natural substrates of HtrA2/Omi.

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.

Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice.

The mouse mutant mnd2 (motor neuron degeneration 2) exhibits muscle wasting, neurodegeneration, involution of the spleen and thymus, and death by 40 days of age. Degeneration of striatal neurons, with astrogliosis and microglia activation, begins at around 3 weeks of age, and other neurons are affected at later stages. Here we have identified the mnd2 mutation as the missense mutation Ser276Cys in the protease domain of the nuclear-encoded mitochondrial serine protease Omi (also known as HtrA2 or Prss25). Protease activity of Omi is greatly reduced in tissues of mnd2 mice but is restored in mice rescued by a bacterial artificial chromosome transgene containing the wild-type Omi gene. Deletion of the PDZ domain partially restores protease activity to the inactive recombinant Omi protein carrying the Ser276Cys mutation, suggesting that the mutation impairs substrate access or binding to the active site pocket. Loss of Omi protease activity increases the susceptibility of mitochondria to induction of the permeability transition, and increases the sensitivity of mouse embryonic fibroblasts to stress-induced cell death. The neurodegeneration and juvenile lethality in mnd2 mice result from this defect in mitochondrial Omi protease.

Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis.

Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility.

HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi. Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated 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.

OMInously, a mitochondrial serine protease is now unveiled as the third ‘MAC’

OMInously, a mitochondrial serine protease is now unveiled as the third ‘MAC’

The Serine Protease from Rat Liver and Hepatoma 8999

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.