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Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome
Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute.
Rheumatology Unit, Department of Medicine, CMM L8:04 Karolinska Institute.
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2006 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 43, no 6, 588-598 p.Article in journal (Refereed) Published
Abstract [en]

Ro52, one of the major autoantigens in the rheumatic disease Sjögren's syndrome (SS), belongs to the tripartite motif (TRIM) or RING-B-box-coiled-coil (RBCC) protein family, thus comprising an N-terminal RING, followed by a B-box and a coiled-coil region. Several different proteomic functions have been suggested for Ro52, including DNA binding, protein interactions and Zn 2+-binding. To analyze the presence and/or absence of these functions and, in particular, map those to different subregions, the modular composition of the Ro52 protein was experimentally characterized. Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions, respectively. Secondary structure analysis by circular dichroism (CD) spectroscopy indicated that the two subregions are independently structured. The entire RING-B-box region displayed Zn2+-dependent stabilization against proteolysis in the presence of Zn2+, indicating functional Zn2+-binding sites in both the RING and the B-box. However, no stabilization with DNA was detected, irrespective of Zn2+, thus suggesting that the RING-B-box region does not bind DNA. Oligomerization of the coiled-coil was investigated by analytical ultracentrifugation and in a mammalian two-hybrid system. Both methods show weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The C-terminal B30.2 region was rapidly degraded both during cellular expression and refolding, indicating a less stable structure. Immunologic analysis of the stable protein regions with sera from patients with Sjögren's syndrome shows that immunodominant epitopes to a large extent are localized in the structurally stable parts of Ro52. The results form a basis for further Ro52 functional studies on the proteome level. © 2005 Elsevier Ltd. All rights reserved.

Place, publisher, year, edition, pages
2006. Vol. 43, no 6, 588-598 p.
Keyword [en]
Immunologi, Ro52, Biofysik
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-38851DOI: 10.1016/j.molimm.2005.04.013Local ID: 45868OAI: oai:DiVA.org:liu-38851DiVA: diva2:259700
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
In thesis
1. Structure-function studies on TRIM21/Ro52, a protein involved in autoimmune diseases
Open this publication in new window or tab >>Structure-function studies on TRIM21/Ro52, a protein involved in autoimmune diseases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Several members of the tripartite motif (TRIM) protein family are involvedin antiviral activity and immunity and have been linked to severaldiseases. TRIM21, the main object of this thesis, is involved in Sjögrensyndrome (SS) and systemic lupus erythematosus (SLE), where patientsoften have autoantibodies against different epitopes on TRIM21. Duringthe course of this study a role of TRIM21 in regulation of proinflammatorycytokines and autoimmunity emerged. The aim of this thesis is to providea better understanding of the structure-function relationship of TRIM21.A conformational epitope in the coiled-coil domain of TRIM21 has beencharacterized, whose autoantibodies cause congenital heart block. A widerange of biophysical methods were employed to establish a model of theprotein domain arrangement of TRIM21, and functional implications werederived. By sequence comparisons, TRIM proteins were classified into threesubgroups, sharing a conserved amphipathic helix in the region, linkingthe conserved N-terminal Zn2+-binding domains RING and B-box, calledthe RING-B-box linker (RBL). A structural dependence of this region on theRING has been observed and a model of the RING-RBL was derived frombioinformatics and proteolysis data. Anti-RING-RBL antibodies inhibit theE3 ligase activity of TRIM21 in ubiquitination. Interferon regulatory factors(IRFs), the substrate for TRIM21-dependent ubiquitination could thereforeretain their high cellular levels after stress-induced inflammation, increasingthe susceptibility to SS and SLE. According to NMR data, the antibodiesbind to the Zn2+-binding loop regions of the RING, which usually bind tothe E2 conjugating enzyme. Antibodies against the C-terminus of the RBLregion do not inhibit the E3 ligase activity.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 89 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1272
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:liu:diva-52744 (URN)978-91-7393-538-8 (ISBN)
Public defence
2009-10-30, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 13:15 (English)
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Available from: 2010-01-18 Created: 2010-01-12 Last updated: 2010-01-18Bibliographically approved

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Hennig, JanoschSunnerhagen, Maria

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