Subcellular Recruitment of Fibrillarin to Nucleoplasmic Proteasomes: Implications for Processing of a Nucleolar Autoantigen

Abstract

A prerequisite for proteins to interact in a cell is that they are present in the same intracellular compartment. Although it is generally accepted that proteasomes occur in both, the cytoplasm and the nucleus, research has been focusing on cytoplasmic protein breakdown and antigen processing, respectively. Thus, little is known on the functional organization of the proteasome in the nucleus. Here we report that within the nucleus 20S and 26S proteasomes occur throughout the nucleoplasm and partially colocalize with splicing factor-containing speckles. Because proteasomes are absent from the nucleolus, a recruitment system was used to analyze the molecular fate of nucleolar protein fibrillarin: Subtoxic concentrations of mercuric chloride (HgCl 2 ) induce subcellular redistribution of fibrillarin and substantial colocalization (33%) with nucleoplasmic proteasomes in different cell lines and in primary cells isolated from mercury-treated mice. Accumulation of fibrillarin and fibrillarin-ubiquitin conjugates in lactacystin-treated cells suggests that proteasome-dependent processing of this autoantigen occurs upon mercury induction. The latter observation might constitute the cell biological basis of autoimmune responses that specifically target fibrillarin in mercury-mouse models and scleroderma. INTRODUCTIONThe bulk of nonlysosomal proteolysis is carried out by the ATP-powered 26S proteasome, which is involved in the regulation of major cellular processes such as progression of the cell cycle, transcription, flux of substrates through metabolic pathways, elimination of abnormal proteins, and antigen processing (Hershko and Ciechanover, 1998;Kloetzel, 2001). In most cultured mammalian cells 80 -90% of the protein breakdown occurs by the proteasome pathway (Lee and Goldberg, 1998). The 26S proteasome is composed of two distinct subcomplexes: the central 20S proteasome, in which proteins are degraded, and two flanking 19S complexes, which provide substrate specificity and regulation. The 20S proteasome forms the core subunit harboring multiple catalytic centers located within the hollow cavity of a cylinder (Finley, 2002). This topology sequesters the catalytic sites from potential substrates (Voges et al., 1999). Most of the substrates of the eukaryotic 26S proteasome must be marked by ubiquitination in order to be destroyed. This involves the covalent attachment of multiple ubiquitin molecules to the target protein (Ciechanover, 1998). However, exceptions to the rule such as ornithine decarboxylase (ODC), which is degraded by the 26S proteasome without ubiquitinylation are known, and more are currently under investigation (Murakami et al., 1992;Verma and Deshaies, 2000).Proteasomes generate oligopeptides, most of which are further degraded by distinct endopeptidases and aminopeptidases into amino acids. However, a fraction of these peptides escape complete destruction and are subjected to antigen presentation. The peptides are transported to the endoplasmic reticulum via the transporter associated with antigen pre...

Recruitment of topoisomerase I (Scl‐70) to nucleoplasmic proteasomes in response to xenobiotics suggests a role for altered antigen processing in scleroderma

“…Alterations in the molecular context of cryptic nuclear proteins by environmental factors might lead to efficient processing and presentation of the proteins (16,17). Results from other studies and our previous investigations in cell culture and animal models have shown that xenobiotics, such as heavy metals, drugs, and viruses, recruit SSc autoantigens for antigen processing via proteasomes (18,19). Based on these results, we propose the following hypothesis for the generation of systemic autoimmune responses.…”

“…Recently we showed that xenobiotics such as heavy metals induce redistribution and colocalization of fibrillarin in cell culture and in animal models (18). Although ANAs to fibrillarin are specific for SSc, their frequency is rather low (3-8%) (24).…”

“…Indirect immunofluorescence was performed on human DCs on slides or HEp-2 cells on coverslips, as previously described (18). Rabbit polyclonal antibodies to 20S proteasomes, human serum against topo I, and mouse monoclonal antibody to CD11c (BD PharMingen, San Diego, CA) were used as primary antibodies for triple labeling.…”

“…For dual staining, human serum against topo I and goat anti-actin antibody were incubated with HEp-2 cells on coverslips, and FITC-labeled anti-human and rhodamine-conjugated anti-goat IgG antibodies were used as secondary antibodies. Images were obtained by laser confocal microscopy, as previously described (18). Signals were defined as colocalizing in the following ranges: hue 31-54, intensity 0-255, and saturation 106-251 (HIS color model, MetaMorph Imaging System; Universal Imaging, Downingtown, PA).…”

“…Quantitative analysis of fluorescence intensities was performed using the MetaMorph Imaging System, as previously described (18). To measure fluorescence intensity within nuclei, regions of interest (ROIs) were positioned manually based on corresponding differential interference contrast images.…”