Research on proteasomes is mainly focused on processing within the cytoplasm. About 30% of substrates are derived from newly synthesized, faulty proteins that would never attain native structure and thus are subjected to ubiquitination and proteasome-dependent degradation (Schubert et al., 2000
). A substantial fraction of the resulting peptides is then translocated via the TAP transporter into the endoplasmatic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system (Reits et al., 2000
). However, proteasomes also play an important role in the turnover of flawless proteins that have simply reached the end of their half lives. Thus, the following question arises: how are nuclear proteins processed when they have come to the end of their working lives and/or if they cannot exert their function because of changes in nuclear structure?
A prerequisite for two proteins to interact in a cell is that they are present in the same intracellular region. In the present study we provide evidence that mercury-induced redistribution of fibrillarin leads to colocalization with nucleoplasmic proteasomes and proteasome-dependent processing of fibrillarin within nuclei. These results confirm recent reports describing As2
-induced degradation of nuclear protein PML (Lallemand-Breitenbach et al., 2001
) and intracellular localization of proteasomal proteolysis of a viral antigen (Anton et al., 1999
). Both studies localize proteasome-dependent processing to nucleoplasmic aggregates, thus defining the nucleus as an intracellular site of proteasomal degradation. It has been well established over the last two decades that proteasomes occur in the cytoplasm and the nucleoplasm but not in nucleoli (Hügle et al., 1983
; Stauber et al., 1987
; Amsterdam et al., 1993
; Reits et al., 1997
; Rivett, 1998
). By means of double labeling and confocal analysis, we were able to refine subnuclear localization of 20S as well as 26S proteasomes in nucleoplasmic speckles where they partially colocalize with the splicing factor SC35 (Figure ). Additionally, proteasomes were distributed throughout the nucleoplasm in punctate and speckled aggregates but not within nucleoli, which is in perfect agreement with the literature. Thus it seems highly unlikely that nucleoli represent intracellular sites for proteasomal protein degradation. However, inhibition of nucleolar transcription by mercury induces redistribution of nucleolar protein fibrillarin (Chen and von Mikecz, 2000
), resulting in (1) colocalization with nucleoplasmic proteasomes (Figure ) and (2) proteasome-dependent processing of fibrillarin (Figure ).
The latter observations do not only describe one possible turn over mechanism of a nucleolar protein but may as well provide new insights into the generation of systemic autoimmune responses, because redistribution of fibrillarin and colocalization with proteasomes could be observed in splenic cells from mercury-treated mice (Figure ). Such mice also developed a specific autoimmunity against fibrillarin. The results suggest that fibrillarin, which is normally segregated from proteasomes within the nucleolus is recruited to proteasome-dependent degradation by mercury. This event may represent altered antigen processing, which in turn may lead to presentation of cryptic determinants to the immune system. The following findings corroborate our hypothesis: (1) Susceptibility for mercury-induced autoimmunity seems to be under the control of MHC genes (Hultman et al., 1992
), suggesting that antigen processing and presentation may be involved in the generation of the autoimmune response. (2) Because treatment of IFN-γ knockout mice with subtoxic concentrations of HgCl2
does not provoke any autoimmune response, it was concluded that the prototypic autoimmunity induced by mercury is dependent on IFN-γ (Kono et al., 1998
), the same cytokine that modulates the subunit composition and proteolytic activity of immunoproteasomes (Boes et al., 1994
). (3) Like fibrillarin, major nucleolar proteins B23 and nucleolin redistributed to the nucleoplasm after HgCl2
treatment, but colocalization with proteasomes could not be observed (Figure ). The specificity of subcellular colocalization of fibrillarin with proteasomes might reflect the specificity of mercury-induced autoimmunity, which is exclusively directed against fibrillarin in mice.
Apart from our results on altered degradation of fibrillarin there are recent studies that report similar recruitment of nuclear proteins to proteasomal processing when nuclear structure and function is disturbed. Cells infected by herpes simplex virus type 1 in the G2
phase of the cell cycle become stalled in mitosis. This block correlates with the viral immediate-early protein ICP0-induced, proteasome-dependent degradation of centromere proteins CENP-C (Everett et al., 1999
), and CENP-A (Lomonte et al., 2001
). The antitumor drug camptothecin inhibits the rejoining step of superhelical DNA relaxation, thereby trapping topoisomerase I in covalent linkage with DNA and preventing normal DNA replication. Desai et al. (1997)
showed that the half-life of topoisomerase I dropped from 10–16 h down to 1–2 h and that conjugates of topoisomerase I and ubiquitin emerged upon camptothecin treatment . Because MG-132 and lactacystin prevented camptothecin-induced destruction, it is concluded by the authors that camptothecin stimulates proteasome-dependent processing of topoisomerase I. Interestingly, both examples describe how alteration of nuclear function leads to recruitment of nuclear autoantigens to proteasomal processing, because centromere proteins as well as topoisomerase I constitute frequent targets of the autoimmune response in scleroderma (Tan, 1989
). The exposure to a number of environmental substances has been associated with scleroderma (Galperin and Gershwin, 1998
); thus future studies should focus on further elucidation of proteasome-dependent processing of nuclear antigens and its role in the generation of systemic autoimmune responses.
We consider investigation on proteasomal proteolysis within nuclei an important research topic, because it may provide novel insights into (1) the turn over of nuclear proteins, (2) the regulation of nuclear processes such as transcription and splicing, and (3) the generation of systemic autoimmune responses against nuclear proteins.