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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
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Gabriele von Bülow Gymnasium, Berlin.
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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.

Place, publisher, year, edition, pages
2008. Vol. 377, no 2, 431-449 p.
Keyword [en]
Ro52; TRIM21; RING; linker; zinc binding
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-12887DOI: 10.1016/j.jmb.2008.01.005OAI: oai:DiVA.org:liu-12887DiVA: diva2:17296
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Characterization of protein families, sequence patterns, and functional annotations in large data sets
Open this publication in new window or tab >>Characterization of protein families, sequence patterns, and functional annotations in large data sets
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bioinformatics involves storing, analyzing and making predictions on massive amounts of protein and nucleotide sequence data. The thesis consists of six papers and is focused on proteins. It describes the utilization of bioinformatics techniques to characterize protein families and to detect patterns in gene expression and in polypeptide occurrences. Two protein families were bioinformatically characterized - the membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) and the Tripartite motif (TRIM) protein families.

In the study of the MAPEG super-family, application of different bioinformatic methods made it possible to characterize many new members leading to a doubling of the family size. Furthermore, the MAPEG members were subdivided into families. Remarkably, in six families with previously predominantly mammalian members, fish representatives were also now detected, which dated the origin of these families back to the Cambrium ”species explosion”, thus earlier than previously anticipated. Sequence comparisons made it possible to define diagnostic sequence patterns that can be used in genome annotations. Upon publication of several MAPEG structures, these patterns were confirmed to be part of the active sites.

In the TRIM study, the bioinformatic analyses made it possible to subdivide the proteins into three subtypes and to characterize a large number of members. In addition, the analyses showed crucial structural dependencies between the RING and the B-box domains of the TRIM member

Ro52. The linker region between the two domains, denoted RBL, is known

to be disease associated. Now, an amphipathic helix was found to be a

characteristic feature of the RBL region, which also was used to divide the family into three subtypes.

The ontology annotation treebrowser (OAT) tool was developed to detect functional similarities or common concepts in long lists of proteins or genes, typically generated from proteomics or microarray experiments. OAT was the first annotation browser to include both Gene Ontology (GO) and Medical Subject Headings (MeSH) into the same framework. The complementarity of these two ontologies was demonstrated. OAT was used in the TRIM study to detect differences in functional annotations between the subtypes.

In the oligopeptide study, we investigated pentapeptide patterns that were over- or under-represented in the current de facto standard database of protein knowledge and a set of completed genomes, compared to what could be expected from amino acid compositions. We found three predominant categories of patterns: (i) patterns originating from frequently occurring families, e.g. respiratory chain-associated proteins and translation machinery proteins; (ii) proteins with structurally and/or functionally favored patterns; (iii) multicopy species-specific retrotransposons, only found in the genome set. Such patterns may influence amino acid residue based prediction algorithms. These findings in the oligopeptide study were utilized for development of a new method that detects translated introns in unverified protein predictions, which are available in great numbers due to the many completed and ongoing genome projects.

A new comprehensive database of protein sequences from completed genomes was developed, denoted genomeLKPG. This database was of central importance in the MAPEG, TRIM and oligopeptide studies. The new sequence database has also been proven useful in several other studies.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2008. 85 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1159
Keyword
Bioinformatics, sequence analysis, patterns, protein families
National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:liu:diva-10565 (URN)978-91-85523-01-6 (ISBN)
Public defence
2008-02-15, Planck, Fysikhuset, Linköpings Universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-01-28 Created: 2008-01-28 Last updated: 2010-01-13Bibliographically approved
2. 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)
Opponent
Supervisors
Available from: 2010-01-18 Created: 2010-01-12 Last updated: 2010-01-18Bibliographically approved

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Hennig, JanoschBresell, AndersPersson, BengtSunnerhagen, Maria

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