The Fellowship of the RING: The RING–B-Box Linker Region Interacts with the RING in TRIM21/Ro52, Contains a Native Autoantigenic Epitope in Sjögren Syndrome, and is an Integral and Conserved Region in TRIM Proteins

Hennig, Janosch; Bresell, Anders; Sandberg, Mats; Hennig, Klaus Dm; Wahren‐Herlenius, Marie; Persson, Bengt; Sunnerhagen, Maria

doi:10.1016/j.jmb.2008.01.005

Cited by 24 publications

(23 citation statements)

References 83 publications

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“…These samples produced only few and very broad NMR signals probably due to the formation of large complexes during the purification process. This agrees with our previous study where pH 8 and Tris buffer provided the best achievable conditions for biophysical studies of the RING-RBL (27). Although optimal solubility and stability of a monomeric RING-RBL were achieved at pH 8.0 and 25°C (22), chemical exchange with solvent during these conditions resulted in only 57% of all residues providing observable NMR resonances.…”

Section: Methodssupporting

confidence: 91%

“…A structural model for antibody interference with Ro52-mediated ubiquitination. A, model of the RING domain generated from previous homology modeling and mass spectrometry (27), with residues of the E2 binding interface as determined by NMR spectroscopy highlighted in cobalt blue. Loops L1 and L2 continue N-and C-terminally but are not part of this model.…”

Section: Discussionmentioning

confidence: 99%

“…Two additional regions were involved in E2/E3 interactions as follows: the putative helix region between both loops, as well as residues Lys-77 and Glu-78 in the RBL. This intriguing linker region, conserved throughout TRIM proteins (27), has also been shown to take part in inhibiting HIV infection restriction in TRIM5 (43). Further studies will show if these novel E2 interaction patches are conserved throughout the TRIM protein family.…”

Section: Discussionmentioning

confidence: 99%

“…Mapping of Interaction Surfaces onto the Structure Model of the RING-To obtain a structural understanding of how patient antibodies could block ubiquitination, and in the absence of a complete experimental RING-RBL structure, we evaluated the results on a previously derived homology model of the RING domain (27). The E2 interaction with Ro52 RING involves the L1 and L2 loops, which meet across the ␤-strands sandwiched between the two zinc-binding sites (Fig.…”

Section: Mapping Of the Binding Epitope Of Patient And Monoclonalmentioning

confidence: 99%

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Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface

Espinosa

Hennig

Ambrosi

et al. 2011

Journal of Biological Chemistry

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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. AntiRo52-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 antiRo52 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.Ro52 (TRIM21) is an interferon-inducible protein frequently targeted by autoantibodies in patients with primary Sjögren's syndrome (SS) 4 and systemic lupus erythematosus (SLE) (1, 2). As a member of the tripartite motif (TRIM) protein family (3), Ro52 is characterized by a RING domain (4), a B-box type 2 motif (5), and a coiled-coil region. A B30.2 domain is located in the C-terminal region (6). Several TRIM family members, including Ro52, have been identified as E3 ubiquitin ligases involved in the ubiquitination process (7-10). Ubiquitination is a covalent post-translational modification of proteins by the 76-amino acid residue polypeptide ubiquitin, leading either to proteolysis in the proteasome, internalization from membranes, or functional alteration (11,12). After activation by a ubiquitin-activating enzyme (E1), ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2) followed by a ubiquitin ligase (E3)-mediated transfer of ubiquitin from the E2 to a target protein (13). Ro52 mediates ubiquitination of several members of the interferon regulatory factor (IRF) transcription factor family, thereby regulating cytokine production (14 -17). Gene disruption of Ro52 leads to increased production of proinflammatory cytokines by embryonic fibroblasts and immune cells after Toll-like receptor activation, suggesting that Ro52 negatively regulates IRF activity (16,17).Autoantibodies to Ro52 can appear years before tissue damage is ...

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Section: Methodssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Mapping Of the Binding Epitope Of Patient And Monoclonalmentioning

confidence: 99%

See 2 more Smart Citations

Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface

Espinosa

Hennig

Ambrosi

et al. 2011

Journal of Biological Chemistry

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“…The difference in the spectra for EDTA added and following 6 M guanidine hydrochloride addition indicates that parkin has some residual secondary structure that is independent of Zn 2ϩ coordination. This could arise from portions of the protein that fold in a zinc-independent manner such as the UblD (50) or residual helical structure noted in some RING domains (51,52). Nevertheless, the large differences between spectra in the presence and absence of Zn 2ϩ binding shows that a significant loss of structure occurs in parkin upon the removal of Zn 2ϩ ions.…”

Section: Zinc Binding Is Required For Proper Parkinmentioning

confidence: 99%

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Hristova¹,

Beasley²,

Rylett

et al. 2009

Journal of Biological Chemistry

121

119

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Missense mutations in park2, encoding the parkin protein, account for ϳ50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ϳ160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys 150 -Cys 169 and Cys 196 -His 215 consisting of CX 2 -3 CX 11 CX 2 C and CX 4 -6 CX 10 -16 -CX 2 (H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn 2؉ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease. Autosomal recessive juvenile Parkinson disease (ARJP)2 is a neurodegenerative disorder arising from the loss of dopaminergic neurons in the substantia nigra of the midbrain. ARJP is characterized by the onset of Parkinsonian symptoms such as tremors, rigidity, and bradykinesia. It is distinguished from the idiopathic form of Parkinson disease by the onset of symptoms, prior to the age of forty. The hereditary nature of ARJP implicates a number of mutations in the genes encoding the proteins parkin, PINK1, LRRK2, and DJ-1 as the cause of dopaminergic neurodegeneration (1-4). A variety of deletion, truncation, and point mutations distributed throughout the park2 gene, which encodes the protein parkin, have been reported in ARJP patients (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).Parkin functions as a ubiquitin ligase (E3) and belongs to a family of RBR (RING-between-RING) ubiquitin li...

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A Interacting Model: How TRIM21 Orchestrates with Proteins in Intracellular Immunity

Huang,

Gao,

et al. 2023

Small Methods

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Tripartite motif‐containing protein 21 (TRIM21), identified as both a cytosolic E3 ubiquitin ligase and FcR (Fragment crystallizable receptor), primarily interacts with proteins via its PRY/SPRY domains and promotes their proteasomal degradation to regulate intracellular immunity. But how TRIM21 involves in intracellular immunity still lacks systematical understanding. Herein, it is probed into the TRIM21‐related literature and raises an interacting model about how TRIM21 orchestrates proteins in cytosol. In this novel model, TRIM21 generally interacts with miscellaneous protein in intracellular immunity in two ways: For one, TRIM21 solely plays as an E3, ubiquitylating a glut of proteins that contain specific interferon‐regulatory factor, nuclear transcription factor kappaB, virus sensors and others, and involving inflammatory responses. For another, TRIM21 serves as both E3 and specific FcR that detects antibody‐complexes and facilitates antibody destroying target proteins. Correspondingly delineated as Fc‐independent signaling and Fc‐dependent signaling in this review, how TRIM21's interactions contribute to intracellular immunity, expecting to provide a systematical understanding of this important protein and invest enlightenment for further research on the pathogenesis of related diseases and its prospective application is elaborated.

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The Fellowship of the RING: The RING–B-Box Linker Region Interacts with the RING in TRIM21/Ro52, Contains a Native Autoantigenic Epitope in Sjögren Syndrome, and is an Integral and Conserved Region in TRIM Proteins

Cited by 24 publications

References 83 publications

Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface

Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

A Interacting Model: How TRIM21 Orchestrates with Proteins in Intracellular Immunity

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