Characterization of a Novel and Specific Inhibitor for the Pro-apoptotic Protease Omi/HtrA2

Abstract

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134 -458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (؊/؊) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.

Regulation of HAX-1 Anti-apoptotic Protein by Omi/HtrA2 Protease during Cell Death

“…Bacterially made HisOmi 134 -458 is an active serine protease and has been described previously (13). ucf-101 Inhibitor Prevents HAX-1 Degradation-We investigated whether the Omi protease is specifically responsible for the degradation of HAX-1 observed in HK-2 cells during apoptosis.…”

“…ucf-101 is a specific inhibitor of the proteolytic activity of Omi and has been described previously (13). When HK-2 cells were treated with cisplatin in the presence of ucf-101, the percentage of apoptotic cells decreased and the inhibitor significantly blocked HAX-1 degradation.…”

“…For this the full-length cDNA for HAX-1 protein was cloned in the pET-28 vector (Novagen) containing the T7 promoter and used in an in vitro transcription-translation system (Promega) in the presence of [ 35 S]methionine and [ 35 S]cysteine (Amersham Biosciences). Bacterially expressed recombinant His-Omi 134 -458 was purified on nickel-nitrilotriacetic acid affinity resin as described (13). HisOmi 134 -458 (0.5 g) was incubated with 35 S-labeled HAX-1 in 20 l of reaction volume in assay buffer (20 mM Na 2 HPO 4 (pH 8), 10% glycerol, 200 mM NaCl).…”

Variations in the protein level of Omi/HtrA2 in the heart of aged rats may contribute to the increased susceptibility of cardiomyocytes to ischemia/reperfusion injury and cell death

“…Ten minutes before reperfusion, animals were randomized to receive vehicle (DMSO) or ucf-101 (1.5 mol/kg; estimated plasma concentrations, 20 mol/L) via intraperitoneal injection. Ucf-101 has been shown to inhibit 50 to 90 % of Omi/HtrA2 activity but has no effect on other proteases at these concentrations (Cilenti et al 2003).…”

Intramitochondrial serine protease activity of Omi/HtrA2 is required for caspase-independent cell death of human neutrophils

“…These data indicate that under certain conditions Omi/HtrA2 may mediate cell death through its serine protease properties from within the mitochondria. Indeed, when we studied the subcellular localization of ucf-101 in neutrophils by utilizing the autofluorescent properties of this compound, 7 it was found that the ucf-101 red autofluorescence and the signal from the mitochondrial marker MitoTracker GreenFM colocalized (Figure 1e; a shift in fluorescence to yellow depicts colocalization). Colocalization was confirmed by semiquantitative analysis with the LSM510 Image software (Figure 1f), which showed the peaks of ucf-101 fluorescence (red) in close proximity with the peaks of MitoTracker fluorescence (green).…”