High mobility group proteins B1 and B2 (HMGB1/B2)

The proteome analysis of apoptotic nuclei revealed changes in protein levels for both, HMGB1 and HMGB2 proteins. The level of HMGB1 was found to be decreased in all proteomic experiments, being detected in only one single spot, whereas the level of HMGB2 was always increased and this was determined for three independent spots. Both proteins were running at the expected positions corresponding to their molecular weight in the 2DE-gels.

High mobility group proteins are DNA binding proteins predominantly localised within the nucleus (Bonaldi et al., 2003), although they have also been described to occur in the cytosol (Mosevitsky et al., 1989). In the nucleus, HMGB proteins bind in the minor groove of DNA and induce a sharp bend within one superhelical turn. This action is known to promote the interaction of transcription factors (for review see (Langst and Becker, 2004)). Although early reports proposed a possible nucleocytoplasmic shuttling of HMGB1 and B2 (Isackson et al., 1980), the cytosolic function of these proteins still remains unclear. Recent findings show that in addition to its role in the nucleus, HMGB1 mediates inflammation when secreted, as a consequence of cellular damage and necrosis (Scaffidi et al., 2002).

the components of the cell-free apoptosis reaction prior and after their coincubation was performed. Subsequently, to verify the behaviour of HMGB1/B2 during apoptosis in intact cells, HeLa cells were induced to undergo apoptosis by treatment with TRAIL. TRAIL binds to and triggers apoptosis via the death receptors TRAIL-R1 and TRAIL-R2 similarly to CD95-L to which HeLa cells, however, are insensitive. To study whether apoptosis induction is associated with HMGB1 and B2 translocation, immunocytochemical studies of TRAIL-stimulated HeLa cells with antibodies specific for HMGB1 and B2 were performed.

Western blot analysis of cytosolic extracts and nuclei before and after the cell-free apoptotic reaction were performed with an antibody specific for HMGB1 (Fig.15A). This revealed in all samples examined a single specific band. The intensity of the HMGB1-specific signal did not change in the course of apoptosis. This result shows that no proteolysis of HMGB1 occurs during apoptosis and indicates additionally that no translocation of HMGB1 in or out of the nucleus occurs in the cell-free apoptosis reaction.

To study the distribution of HMGB1 at the cellular level, immunostainings of control cells and of cells stimulated to undergo apoptosis were performed (Fig. 15B). The nuclear distribution can be clearly seen in control cells. In the course of apoptosis, this distribution did not change and the protein remained strictly confined to the nucleus.

Since neither proteolysis nor protein translocation of HMGB1 could be detected by two independent experimental methods, Western blot analysis (Fig. 15A) and immunostainings (Fig. 15B), post-translational modifications would be a possible explanation for the results of the proteome analysis of apoptotic nuclei are. Further experiments, which were beyond the scope of this work, are needed to explore this possibility.

Figure 15: Analysis of HMGB1 in cell-free reactions and intact cells.

(A) Western blot analysis of isolated mouse liver nuclei and cytosolic extracts, before and after the cell-free apoptosis reaction. 1 x 10⁶ nuclei and cytosol extracted from 2 x 10⁶ cells were loaded on a 12 % Laemmli gel and blotted onto nitrocellulose membranes. The blot was probed with antibodies specific for HMGB1. As a loading control for cytosolic proteins, the blot was reprobed with an actin-specific antibody.

(B) Immunocytochemical staining of HeLa cells with HMGB1-specific antibodies. HeLa cells were treated with TRAIL [300 ng/ml]. At the indicated time points, cells were fixed with 4 % paraformaldehyde and immunocytochemical staining was performed. Chromatin was stained with H-33342. The figure shows

Similarly to HMGB1, Western blot analysis of cytosolic extracts and nuclei before and after the cell-free apoptosis reaction was performed with an antibody specific for HMGB2 (Fig. 16A). Two variants of HMGB2 were detected: the first one with a molecular weight of 19 kDa is present predominantly in nuclear samples; the second one with a molecular weight of 28 kDa is mainly detected in the cytosolic samples. No additional cleavage band was detected, thus implying that HMGB2 is not proteolytically processed in the course of apoptosis. The cytosolic 28 kDa form was present also in the nuclei recovered after the in vitro reaction; nuclei from late stage apoptotic reactions showed slightly higher signal intensity compared to the other time points. In contrast, the nuclear signal only appeared in cytosols recovered from late stage apoptotic reactions, indicating that there might be an exchange of HMGB2 between the two components of the cell-free reaction.

The results from the proteome analysis, in which the level of HMGB2 was constantly increased in three independent spots running at molecular weights ranging from approximately 24 to 28 kDa, might thus be explained by a translocation of HMGB2 from the late apoptotic extracts to the nucleus. However, possible post-translational modifications leading to the separation into distinct spots on 2DE-gels cannot be excluded and need further analysis.

In order to examine the protein localisation in living cells, an immunocytochemical analysis of HMGB2 was performed, as described above. Confocal images of untreated HeLa cells show that HMGB2 is predominantly located in the nucleus, but to a minor extent is also present in the cytosol (Fig. 16B). In cells treated to undergo apoptosis, this HMGB2 specific staining was not changed.

From these experiments it is possible to draw the following conclusion: First, the HMGB2 protein is not proteolytically processed in the course of apoptosis. Second, translocation of the protein might occur in late stage cell-free apoptotic reactions, as demonstrated by Western blot analysis (Fig. 16A). Whether this occurs also in cells undergoing apoptosis is still unclear. The third possible explanation, post-translational modifications occurring in the course of apoptosis, needs further experimental investigation.

Figure 16: Analysis of HMGB2 in cell-free reactions and intact cells.

(A) Western blot analysis of isolated mouse liver nuclei and cytosolic extracts, before and after the cell-free apoptosis reaction. 1 x 10⁶ nuclei and cytosol extracted from 2 x 10⁶ cells were loaded on a 10 % Laemmli gel and blotted onto nitrocellulose membranes. The blot was probed with antibodies specific for HMGB2. As a loading control, the Ponceau staining of the same blot was used (not shown). (B) Immunocytochemical staining of HeLa cells with HMGB2-specific antibodies. HeLa cells were treated with TRAIL [300 ng/ml]. At the indicated time points, cells were fixed with 4 % paraformaldehyde and immunocytochemical staining was performed. Chromatin was stained with H-33342. The figure shows representative confocal images. The scale