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Structure-function studies on TRIM21/Ro52, a protein involved in autoimmune diseases
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-52744ISBN: 978-91-7393-538-8 (print)OAI: oai:DiVA.org:liu-52744DiVA: diva2:285460
Public defence
2009-10-30, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 13:15 (English)
Opponent
Supervisors
Available from: 2010-01-18 Created: 2010-01-12 Last updated: 2010-01-18Bibliographically approved
List of papers
1. Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein
Open this publication in new window or tab >>Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein
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2005 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 61, no 2, 109-118 p.Article in journal (Refereed) Published
Abstract [en]

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly a-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45521 (URN)10.1111/j.0300-9475.2005.01542.x (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
2. Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome
Open this publication in new window or tab >>Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome
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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.

Keyword
Immunologi, Ro52, Biofysik
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-38851 (URN)10.1016/j.molimm.2005.04.013 (DOI)45868 (Local ID)45868 (Archive number)45868 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
3. Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52
Open this publication in new window or tab >>Structural organization and Zn2+ -dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 39, 33250-33261 p.Article in journal (Refereed) Published
Abstract [en]

Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-50421 (URN)10.1074/jbc.M503066200 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12
4. The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins
Open this publication in new window or tab >>The fellowship of the RING: The RING-B-box linker region (RBL) interacts with the RING in TRIM21/Ro52, contributes to an autoantigenic epitope in Sjögren's syndrome, and is an integral and conserved region in TRIM proteins
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 2, 431-449 p.Article in journal (Refereed) Published
Abstract [en]

Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn2+-binding region, which comprises an N-terminal RING followed by a B-box, separated by a ∼40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn2+ increases its stability. Regions stabilized by Zn2+ binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.

Keyword
Ro52; TRIM21; RING; linker; zinc binding
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-12887 (URN)10.1016/j.jmb.2008.01.005 (DOI)
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2017-12-14Bibliographically approved
5. Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
Open this publication in new window or tab >>Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, 36478-36491 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-53170 (URN)10.1074/jbc.M111.241786 (DOI)000296538300033 ()
Note

Funding agencies|Swedish Research Council||Swedish Foundation for Strategic Research||VINNOVA||CeNano||Swedish Cancer Society||Karolinska Institutet||Linkoping University||King Gustaf Vs 80-Year Foundation||Heart-Lung Foundation||Stockholm County Council||Gustafsson Foundation||Soderberg Foundation||National Cancer Institute of Canada||Swedish Rheumatism Association||Wallenberg Foundation||

Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2017-12-12Bibliographically approved
6. MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins
Open this publication in new window or tab >>MTMDAT: Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins
2008 (English)In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 10, 1310-1312 p.Article in journal (Refereed) Published
Abstract [en]

In structural biology and -genomics, nuclear magnetic resonance (NMR) spectroscopy and crystallography are the methods of choice, but sample requirements can be hard to fulfil. Valuable structural information can also be obtained by using a combination of limited proteolysis and mass spectrometry, providing not only knowledge of how to improve sample conditions for crystallization trials or NMR spectrosopy by gaining insight into subdomain identities but also probing tertiary and quaternary structure, folding and stability, ligand binding, protein interactions and the location of post-translational modifications. For high-throughput studies and larger proteins, however, this experimentally fast and easy approach produces considerable amounts of data, which until now has made the evaluation exceedingly laborious if at all manually possible. MTMDAT, equipped with a browser-like graphical user interface, accelerates this evaluation manifold by automated peak picking, assignment, data processing and visualization. © 2008 The Author(s).

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-42790 (URN)10.1093/bioinformatics/btn116 (DOI)68779 (Local ID)68779 (Archive number)68779 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
7. MTMDAT-HADDOCK: high-throughput, data-driven protein complex structure modeling
Open this publication in new window or tab >>MTMDAT-HADDOCK: high-throughput, data-driven protein complex structure modeling
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: MTMDAT is a recently developed program facilitating the analysis of mass spectrometry data of proteins and biomolecular complexes (Hennig et al, 2008), which were structurally probed by limited proteolysis. This can provide information about stable fragments of multidomain proteins, yields tertiary and quaternary structure data, and residue-wise origins of stability changes can be determined.

Results: A new feature allows for the direct identification of residues that are involved in complex formation bycomparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unboundcomponents are available, this data can be used to restrain data-driven docking to calculate the structure ofthe complex. Here, we provide a new implementation of MTMDAT, with a pipeline to the data-driven dockingprogram HADDOCK (Dominguez et al., 2003; De Vries et al., 2007), streamlining the entire procedure. This,together with usability improvements in MTMDAT, enables direct high-throughput modelling of protein complexesfrom mass spectrometry data.

Conclusions: MTMDAT can be downloaded from http://cms.ifm.liu.se/chemistry/molbiotech/maria¯sunnerhagens¯group/mtmdat/ (windows- or unix-based, with or without HADDOCK pipeline) together withthe manual and example files. The program is free for academic/non-commercial purposes.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-53169 (URN)
Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2010-01-18

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