title: Anti-Ro52 autoantibodies from patients with Sjögren's syndrome inhibit the Ro52 E3 ligase activity by blocking the E3/E2 interface.
abstract: Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1-4 and UBE2E1-2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.
authors.affiliation: Rheumatology Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, SE-171 76, Stockholm, Sweden.
authors.forename: Alexander
authors.initials: A
authors.lastname: Espinosa
authors.forename: Janosch
authors.initials: J
authors.lastname: Hennig
authors.forename: Aurélie
authors.initials: A
authors.lastname: Ambrosi
authors.forename: Madhanagopal
authors.initials: M
authors.lastname: Anandapadmanaban
authors.forename: Martina Sandberg
authors.initials: MS
authors.lastname: Abelius
authors.forename: Yi
authors.initials: Y
authors.lastname: Sheng
authors.forename: Filippa
authors.initials: F
authors.lastname: Nyberg
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.forename: Maria
authors.initials: M
authors.lastname: Sunnerhagen
authors.forename: Marie
authors.initials: M
authors.lastname: Wahren-Herlenius
mesh_terms: D001323:Autoantibodies / Q000276:immunology* / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D017434:Protein Structure, Tertiary; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012859:Sjogren's Syndrome / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects / Q000276:immunology*; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D012261:Ribonucleoproteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000099207:SS-A Antigen