Involvement of OMI in regulation of nuclear envelope permeability

One of the serine proteases known to be involved in apoptosis is the mitochondrial serine protease Htra2/OMI. OMI is released from the mitochondrial intermembrane space during apoptosis and in this way gains access to the cytoplasm and the nuclear envelope [55].

Thus, the question arose whether OMI might represent the permeabilising activity measured with the NPA in S-20 extracts from apoptotic Jurkat cells.

To see whether OMI is present in the S-20 extracts used for NPA, a Western Blot was performed. OMI could be detected in S-20 extracts from control and apoptotic Jurkat cells (Figure 23). Compared to lysates from 293T cells overexpressing OMI at a known concentration the OMI-specific band in the Jurkat S-20 extracts migrated at an apparent lower molecular weight. As the same shift was observed for actin this is most likely due to different buffer compositions of control lysates and S-20 extracts.

The presence of OMI in extracts from untreated non-apoptotic control Jurkat cells might be due to the freeze-thaw lysis procedure by which the extracts were prepared. It is possible that organelles such as the mitochondria are damaged by this method leading to the release of OMI.

kDa

45 αOMI 45

αActin 31

Co Apo

50µg 100µg 50µg 100µg

OMI in 293T lysate

5µg 10µg 15µg

Jurkat S20 extracts

Figure 23 OMI is present in S-20 extract from control or apoptotic Jurkat cells

50 or 100 µg of S-20 extract from control (Co) or apoptotic (Apo) Jurkat cells as well as different amounts of OMI in lysates from 293T cells were separated on a 12% Lämmli PAGE and transferred onto a nitrocellulose membrane. The membrane was probed first with an αOMI antibody and then with an αActin antibody.

In a next step it was investigated whether OMI could be inhibited by Pefablock, the serine protease inhibitor which is able to inhibit the permeabilising activity in S-20 extracts from apoptotic Jurkat cells.

To directly test the influence of OMI on nuclear permeability, an NPA using recombinant OMI was the method of choice. Thus, in addition to testing for the inhibitory effect of Pefablock, it was investigated if recombinant OMI is active in the reaction buffer needed for performing the NPA (TRB).

To this end, an in-vitro activity assay with recombinant OMI and its substrate β-casein was performed. It was found that OMI was active in TRB as assessed by cleavage of β-casein but it was not inhibited by Pefablock even at high concentrations (1 mM). In contrast, OMI was efficiently inhibited by Ucf-101, a specific inhibitor of OMI [145]

(Figure 24). The resistance of OMI activity to Pefablock is in accordance with observations of P. Vandenabeele (up to 1 M Pefablock was tested, personal communication).

Omi β-casein

β-casein cleavage product

1 2 3 4 5 6 7

24 kDa 40 kDa 33 kDa

Figure 24 OMI is active in TRB and its activity cannot be inhibited by Pefablock

300 ng recombinant OMI were incubated with or without 2 µg of the substrate β-casein for 45 min at 37°C.

Where inhibitors where added, OMI was pre-incubated with inhibitors for 10 min at room temperature before addition of β-casein. Samples were separated on a 14 % Lämmli gel and stained with Coomassie.

1: positive control (20 mM Tris pH 7.4), 2: negative control without OMI, 3: NPA-buffer (TRB), 4: 400 µM Pefablock in TRB, 5: 80 µM Ucf-101 in specific phosphate buffer, 6: 80 µM Ucf-101 in TRB, 7: 1 mM Pefablock in TRB. Ucf-101: specific inhibitor for OMI.

To control if OMI directly influences nuclear envelope permeability, an NPA was performed with different amounts of recombinant OMI. As the nuclei of digitonin-permeabilised cells were not stable in TRB alone (after the necessary incubation at room temperature), OMI was added to S-20 extracts prepared from untreated control Jurkat cells. Addition of OMI to these cells resulted in a significant increase in nuclear envelope permeability at a concentration of 600 nM (Figure 25). Lower concentrations were not sufficient to increase nuclear envelope permeability significantly.

Co

Figure 25 OMI influences nuclear envelope permeability

NPA was performed with HeLa cells. Digitonin-permeabilised cells were incubated with S-20 extracts from control Jurkat cells alone (Co) or supplemented with the indicated amount of recombinant OMI. n.s.: not significant. **: p<0.01

Because OMI influenced the nucleocytoplasmic barrier in the NPA the question arose whether OMI is directly able to cleave nuclear pore complex proteins. To answer this question another in-vitro assay was performed. Nuclei of Jurkat and HeLa cells were isolated and incubated with different amounts of recombinant OMI. As a positive control the nuclei were incubated with recombinant caspase-3-GFP (kindly provided by Dr. T.

Meergans, University of Konstanz). A Western Blot was performed to screen for nucleoporin degradation products. mAb414, recognizing a subset of FG-repeat nucleoporins, namely Nup358 (RanBP2), Nup214, Nup153 and p62 was employed as a nucleoporin specific antibody. Incubation of OMI with purified nuclei resulted in formation of an immunoreactive fragment of ~ 90 kDa. This fragment was not present in untreated control nuclei and different from a fragment resulting from caspase-3

cleavage, which was migrating slightly faster (Figure 26 A). Activity of OMI in the respective buffer used for the in-vitro reaction was confirmed by an activity assay (Figure 26 B).

Figure 26 OMI induces formation of an mAb414 immunoreactive fragment

A: Isolated Jurkat cell nuclei were incubated with recombinant OMI or recombinant caspase-3-GFP (C3G) for 20 min at 37°C. Samples were separated by a 10% Thomas and Kornberg-PAGE and blotted onto a nitrocellulose membrane. mAb414 was used to detect immunoreactive nucleoporins. B: OMI activity assay. 300 ng recombinant Omi were incubated with or without 2 µg of the substrate β-casein for 45 min at 37°C. Samples were separated on a 14 % Lämmli gel and stained with Coomassie. 1: negative control without OMI, 2: OMI in respective buffer.