| 1. |
- Anandapadamanaban, Madhanagopal, et al.
(författare)
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E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2
- 2019
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 294:30, s. 11404-11419
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Tidskriftsartikel (refereegranskat)abstract
- The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-angstrom crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called linchpin residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.
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| 2. |
- Anandapadamanaban, Madhanagopal, et al.
(författare)
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Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM211- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.
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| 3. |
- Bossini-Castillo, Lara, et al.
(författare)
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A replication study confirms the association of TNFSF4 (OX40L) polymorphisms with systemic sclerosis in a large European cohort
- 2011
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 1468-2060 .- 0003-4967. ; 70:4, s. 638-641
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Tidskriftsartikel (refereegranskat)abstract
- Objectives The aim of this study was to confirm the influence of TNFSF4 polymorphisms on systemic sclerosis (SSc) susceptibility and phenotypic features. Methods A total of 8 European populations of Caucasian ancestry were included, comprising 3014 patients with SSc and 3125 healthy controls. Four genetic variants of TNFSF4 gene promoter (rs1234314, rs844644, rs844648 and rs12039904) were selected as genetic markers. Results A pooled analysis revealed the association of rs1234314 and rs12039904 polymorphisms with SSc (OR 1.15, 95% CI 1.02 to 1.31; OR 1.18, 95% CI 1.08 to 1.29, respectively). Significant association of the four tested variants with patients with limited cutaneous SSc (lcSSc) was revealed (rs1234314 OR 1.22, 95% CI 1.07 to 1.38; rs844644 OR 0.91, 95% CI 0.83 to 0.99; rs844648 OR 1.10, 95% CI 1.01 to 1.20 and rs12039904 OR 1.20, 95% CI 1.09 to 1.33). Association of rs1234314, rs844648 and rs12039904 minor alleles with patients positive for anti-centromere antibodies (ACA) remained significant (OR 1.23, 95% CI 1.10 to 1.37; OR 1.12, 95% CI 1.01 to 1.25; OR 1.22, 95% CI 1.07 to 1.38, respectively). Haplotype analysis confirmed a protective haplotype associated with SSc, lcSSc and ACA positive subgroups (OR 0.88, 95% CI 0.82 to 0.96; OR 0.88, 95% CI 0.80 to 0.96; OR 0.86, 95% CI 0.77 to 0.97, respectively) and revealed a new risk haplotype associated with the same groups of patients (OR 1.14, 95% CI 1.03 to 1.26; OR 1.20, 95% CI 1.08 to 1.35; OR 1.23, 95% CI 1.07 to 1.42, respectively). Conclusions The data confirm the influence of TNFSF4 polymorphisms in SSc genetic susceptibility, especially in subsets of patients positive for lcSSc and ACA.
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| 4. |
- Bossini-Castillo, Lara, et al.
(författare)
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Confirmation of association of the macrophage migration inhibitory factor gene with systemic sclerosis in a large European population.
- 2011
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Ingår i: Rheumatology (Oxford, England). - : Oxford University Press (OUP). - 1462-0332 .- 1462-0324. ; 50, s. 1976-1981
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Tidskriftsartikel (refereegranskat)abstract
- Objectives. The aim of this study was to confirm the implication of macrophage migration inhibitory factor (MIF) gene in SSc susceptibility or clinical phenotypes in a large European population.Methods. A total of 3800 SSc patients and 4282 healthy controls of white Caucasian ancestry from eight different European countries were included in the study. The MIF -173 single nucleotide polymorphism (SNP) was selected as genetic marker and genotyped using Taqman 5' allelic discrimination assay.Results. The MIF -173 SNP showed association with SSc [P = 0.04, odds ratio (OR) = 1.10, 95% CI 1.00, 1.19]. Analysis of the MIF -173 polymorphism according to SSc clinical phenotype revealed that the frequency of the -173*C allele was significantly higher in the dcSSc group compared with controls (P = 5.30E-03, OR = 1.21, 95% CI 1.07, 1.38). Conversely, the frequency of the MIF -173*C allele was significantly underrepresented in the lcSSc group compared with dcSSc patients, supporting previous findings [(P = 0.04, OR = 0.86, 95% CI 0.75, 0.99); meta-analysis including previous results (P = 0.005, OR = 0.83, 95% CI 0.73, 0.94)].Conclusion. Our results confirm the role of MIF -173 promoter polymorphism in SSc, and provide evidence of a strong association with the dcSSc subgroup of patients. Hence, the MIF -173 variant is confirmed as a promising clinical phenotype genetic marker.
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| 5. |
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| 6. |
- Espinosa, Alexander
(författare)
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A study on the E3 ligase TRIM21/Ro52
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Patients with the systemic autoimmune diseases Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE), often have antibodies against the intracellular protein TRIM21/Ro52. Although the presence of anti-TRIM21/Ro52 autoantibodies is used as a diagnostic tool, the biological function of TRIM21/Ro52 is still unknown. The aim of this thesis is to provide a better understanding of the function of TRIM21/Ro52, especially regarding its role in autoimmunity. To achieve this, TRIM21/Ro52 was studied at a molecular and cellular level in vitro and in vivo. By using circular dichroism, limited proteolysis, mass spectrometry, ultracentrifugation, and two-hybrid experiments, it was shown that TRIM21/Ro52 is a Zn2+ binding protein that forms weak dimers. These results also confirmed the presence of the predicted secondary structure domains of TRIM21/Ro52. A RING domain in the N-terminus of TRIM21/Ro52 suggests that TRIM21/Ro52 is a RING dependent E3 ligase. Through ubiquitination assays, it was shown that TRIM21/Ro52 is indeed an E3 ligase and that the E3 ligase activity of TRIM21/Ro52 was dependent on the E2 enzymes UbcH5a-c or UbcH6. Furthermore, anti-RING autoantibodies from patients with SS and SLE blocked the E3 activity of TRIM21/Ro52 in vitro. Since the expression of many TRIM proteins is induced by interferons, the effect of interferon alpha (IFNalpha) and virus on TRIM21/Ro52 expression was investigated. After exposing the cell lines HeLa, Daudi and Raji to IFNalpha, TRIM21/Ro52 expression was increased both at the mRNA and protein level. In addition, TRIM21/Ro52 expression was increased in human peripheral blood mononuclear cells (PBMC) exposed to inactivated herpes simplex virus. TRIM21/Ro52 was also expressed at a higher level in PBMCs from patients with SS and SLE than in PBMCs from healthy individuals, which could be interpreted as an effect of the interferon signature typical of SS and SLE patients. To investigate the effect of overexpressing TRIM21/Ro52 in B cells, stable transfected cell lines were made and characterized. B cells overexpressing TRIM21/Ro52 proliferated slower and were more sensitive to induced cell death; which was in contrast to B cells expressing a TRIM21/Ro52 mutant lacking E3 ligase activity. Thus, TRIM21/Ro52 is involved in regulating proliferation, and activation status, of B lymphocytes. The intracellular localization of TRIM21/Ro52 was determined by transfecting HeLa cells with GFP-Ro52 and GFP-Ro52 mutants. Two stretches of amino acids (aa) were found to be important for the cytoplasmic localization and nuclear import of TRIM21/Ro52. When the aa 203-248 were deleted, TRIM21/Ro52 entered the nucleus; but the deletion of aa 381-470 prevented the nuclear import of TRIM21/Ro52. In addition, exposure to IFNalpha and nitric oxide induced translocation of TRIM21/Ro52 from the cytoplasm to the nucleus. In conclusion, TRIM21/Ro52 is a cytoplasmic E3 ligase that is overexpressed in PBMCs from patients with SS and SLE. TRIM21/Ro52 is induced by IFNalpha and herpes simplex virus, and inhibits cell proliferation.
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| 7. |
- Espinosa, Alexander, et al.
(författare)
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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
- 2011
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 286:42, s. 36478-36491
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Tidskriftsartikel (refereegranskat)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.
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| 8. |
- Espinosa, Alexander, et al.
(författare)
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Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway
- 2009
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 206:8, s. 1661-1671
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Tidskriftsartikel (refereegranskat)abstract
- Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.
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| 9. |
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| 10. |
- Hedlund, Malin, et al.
(författare)
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Type I IFN system activation in newborns exposed to Ro/SSA and La/SSB autoantibodies in utero
- 2020
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Ingår i: RMD Open. - : BMJ. - 2056-5933. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: In utero exposure of the fetus to Ro/La autoantibodies may lead to congenital heart block (CHB). In the mother, these autoantibodies are associated with activation of the type I interferon (IFN)-system. As maternal autoantibodies are transferred to the fetus during pregnancy, we investigated whether the type I IFN-system is activated also in newborns of anti-Ro/La positive mothers, and whether fetal IFN activation is affected by maternal immunomodulatory treatment.METHODS: Blood drawn at birth from anti-Ro/La positive mothers, their newborns and healthy control pairs was separated into plasma and peripheral blood mononuclear cells (PBMC). PBMC were analysed directly or cultured. mRNA expression was analysed by microarrays, cell surface markers by flow cytometry, and IFNα levels by immunoassays.RESULTS: We observed increased expression of IFN-regulated genes and elevated plasma IFNα levels not only in anti-Ro/La positive women, but also in their newborns. CD14+ monocytes of both anti-Ro/La positive mothers and their neonates showed increased expression of Sialic acid-binding Ig-like lectin-1, indicating cellular activation. Notably, the IFN score of neonates born to mothers receiving immunomodulatory treatment was similar to that of controls, despite persistent IFN activation in the mothers. In both maternal and neonatal PBMC, IFNα production was induced when cells were cultured with anti-Ro/La positive plasma.CONCLUSIONS: Ro/La autoantibody-exposed neonates at risk of CHB have signs of an activated immune system with an IFN signature. This study further demonstrates that neonatal cells can produce IFNα when exposed to autoantibody-containing plasma, and that maternal immunomodulatory treatment may diminish the expression of IFN-regulated genes in the fetus.
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