A study on the E3 ligase TRIM21/Ro52

Abstract: Patients with the systemic autoimmune diseases Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE), often have antibodies against the intracellular protein TRIM21/Ro52. Although the presence of anti- TRIM21/Ro52 autoantibodies is used as a diagnostic tool, the biological function of TRIM21/Ro52 is still unknown. The aim of this thesis is to provide a better understanding of the function of TRIM21/Ro52, especially regarding its role in autoimmunity. To achieve this, TRIM21/Ro52 was studied at a molecular and cellular level in vitro and in vivo. By using circular dichroism, limited proteolysis, mass spectrometry, ultracentrifugation, and two-hybrid experiments, it was shown that TRIM21/Ro52 is a Zn2+ binding protein that forms weak dimers. These results also confirmed the presence of the predicted secondary structure domains of TRIM21/Ro52. A RING domain in the N-terminus of TRIM21/Ro52 suggests that TRIM21/Ro52 is a RING dependent E3 ligase. Through ubiquitination assays, it was shown that TRIM21/Ro52 is indeed an E3 ligase and that the E3 ligase activity of TRIM21/Ro52 was dependent on the E2 enzymes UbcH5a-c or UbcH6. Furthermore, anti-RING autoantibodies from patients with SS and SLE blocked the E3 activity of TRIM21/Ro52 in vitro. Since the expression of many TRIM proteins is induced by interferons, the effect of interferon alpha (IFNalpha) and virus on TRIM21/Ro52 expression was investigated. After exposing the cell lines HeLa, Daudi and Raji to IFNalpha, TRIM21/Ro52 expression was increased both at the mRNA and protein level. In addition, TRIM21/Ro52 expression was increased in human peripheral blood mononuclear cells (PBMC) exposed to inactivated herpes simplex virus. TRIM21/Ro52 was also expressed at a higher level in PBMCs from patients with SS and SLE than in PBMCs from healthy individuals, which could be interpreted as an effect of the interferon signature typical of SS and SLE patients. To investigate the effect of overexpressing TRIM21/Ro52 in B cells, stable transfected cell lines were made and characterized. B cells overexpressing TRIM21/Ro52 proliferated slower and were more sensitive to induced cell death; which was in contrast to B cells expressing a TRIM21/Ro52 mutant lacking E3 ligase activity. Thus, TRIM21/Ro52 is involved in regulating proliferation, and activation status, of B lymphocytes. The intracellular localization of TRIM21/Ro52 was determined by transfecting HeLa cells with GFP-Ro52 and GFP-Ro52 mutants. Two stretches of amino acids (aa) were found to be important for the cytoplasmic localization and nuclear import of TRIM21/Ro52. When the aa 203-248 were deleted, TRIM21/Ro52 entered the nucleus; but the deletion of aa 381-470 prevented the nuclear import of TRIM21/Ro52. In addition, exposure to IFNalpha and nitric oxide induced translocation of TRIM21/Ro52 from the cytoplasm to the nucleus. In conclusion, TRIM21/Ro52 is a cytoplasmic E3 ligase that is overexpressed in PBMCs from patients with SS and SLE. TRIM21/Ro52 is induced by IFNalpha and herpes simplex virus, and inhibits cell proliferation.