Expression, Purification, and Functional Analysis of the Human Serine Protease HtrA2

Savopoulos, John; Carter, Paul S.; Turconi, Sandra; Pettman, Gary; Karran, Eric; Gray, Charles A.; Ward, Robin V.; Jenkins, Owen; Creasy, Caretha L.

doi:10.1006/prep.2000.1240

Cited by 54 publications

(43 citation statements)

References 14 publications

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“…19 During mitochondrial import the first 133 N-terminal amino acids are removed generating mature Omi protein. 24 This processing leads to the N-terminal exposition of a conserved AVPA sequence, which can allow interaction with the BIR3-domain in XIAP. A similar interaction was previously described for DIABLO/Smac, caspase-9 and the Drosophila proteins Hid, Grim and Reaper.…”

Section: Discussionmentioning

confidence: 99%

“…24 Sequence comparison of the N-terminus of the mature mouse and human Omi protein revealed the presence of the conserved XIAP BIR3-interaction motif AVPA and AVPS, respectively. This conserved IAP-binding motif is also seen at the N-terminus of the small subunit of mature caspase-9 and mature DIABLO/Smac ( Figure 3A) and it was also described for the Drosophila proteins Reaper, Hid and Grim.…”

Section: Omi Binds To Xiap and Promotes Caspase Activitymentioning

confidence: 99%

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The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity

et al. 2002

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Proteome analysis of supernatant of isolated mitochondria exposed to recombinant tBid, a proapoptotic Bcl-2 member, revealed the presence of the serine protease Omi, also called HtrA2. This release was prevented in mitochondria derived from Bcl-2-transgenic mice. Release of Omi under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Omi release also occurs in apoptotic dying but not in necrotic dying fibrosarcoma L929 cells, treated with anti-Fas antibodies and TNF, respectively. The amino acid sequence reveals the presence of an XIAP interaction motif at the N-terminus of mature Omi. We demonstrate an interaction between endogeneous Omi and recombinant XIAP. Furthermore we show that endogenous Omi is involved in enhanced activation of caspases in cytosolic extracts.

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Section: Discussionmentioning

confidence: 99%

Section: Omi Binds To Xiap and Promotes Caspase Activitymentioning

confidence: 99%

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity

et al. 2002

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“…A proapoptotic serine protease Omi/HtrA2 has recently been found to be released from mitochondria as a result of a number of apoptotic stimuli. [64][65][66] One target of Omi/ HtrA2 is XIAP, an antiapoptotic protein that is sequestered by Smac, a mitochondrial proapoptotic protein. [67][68][69][70][71][72] In LNCaP cells, microinjection of either cytochrome c or Smac alone are insufficient to induce substantial apoptosis, but combined, the two are proapoptotic.…”

Section: Discussionmentioning

confidence: 99%

Role of ceramide in mediating apoptosis of irradiated LNCaP prostate cancer cells

et al. 2003

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The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by c-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to c-irradiation-induced cell death. LNCaP cells can be sensitized to c-irradiation by tumor necrosis factor a (TNF-a) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-a activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-a, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysinechloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH 2 F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-a, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-a with or without cirradiation. TNF-a and c-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.

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“…This does seem to be the case since catalytically active Omi/HtrA2 is able to cleave itself generating a IAP interacting reaper motif. 11 Three of the recent publications seem to argue that the serine protease activity of Omi/HtrA2 is also important in the process of apoptosis independently of the presence of the reaper motif. Among these Hegde et al 4 and Suzuki et al 1 claim Figure 1 In healthy cells (left) a plethora of pro-apoptotic proteins including the RPs Omi/HtrA2, Smac/DIABLO are sequestered in the mitochondria.…”

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confidence: 99%

The serine protease Omi/HtrA2: a second mammalian protein with a Reaper-like function

Martins

2002

Cell Death Differ

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Expression, Purification, and Functional Analysis of the Human Serine Protease HtrA2

Cited by 54 publications

References 14 publications

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity

Role of ceramide in mediating apoptosis of irradiated LNCaP prostate cancer cells

The serine protease Omi/HtrA2: a second mammalian protein with a Reaper-like function

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