Tyrosine Phosphorylation of the E3 Ubiquitin Ligase TRIM21 Positively Regulates Interaction with IRF3 and Hence TRIM21 Activity

Department Molecular and Cellular Therapeutics, Royal College Surgeons in Ireland Research Institute, Royal College of Surgeons in Ireland, Dublin, Ireland.
PLoS ONE (Impact Factor: 3.23). 03/2012; 7(3):e34041. DOI: 10.1371/journal.pone.0034041
Source: PubMed

ABSTRACT Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F). We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

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Available from: Caroline A Jefferies, Sep 29, 2015
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    • "As already observed for other IRF family members, our results demonstrate that the interaction between TRIM21 and IRF5 requires the PRY/SPRY domain, thus indicating a common mechanism for TRIM21 to target this family of transcription factors. Importantly, TLR-mediated phosphorylation of tyrosine residues in this domain was shown to be necessary to enhance affinity of TRIM21 for IRF3 [27]. The recombinant PRY/SPRY TRIM21 we used in this study, although an invaluable tool to examine the interaction properties of various overexpressed IRF5 isoforms or deletion mutants, likely does not mimic TLR-activated TRIM21. "
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    ABSTRACT: IRF5 is a member of the Interferon Regulatory Factor (IRF) family of transcription factors activated downstream of the Toll-Like receptors (TLRs). Polymorphisms in IRF5 have been shown to be associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) and other autoimmune conditions, suggesting a central role for IRF5 in the regulation of the immune response. Four different IRF5 isoforms originate due to alternative splicing and to the presence or absence of a 30 nucleotide insertion in IRF5 exon 6. Since the polymorphic region disturbs a PEST domain, a region associated with protein degradation, we hypothesized that the isoforms bearing the insertion might have increased stability, thus explaining the association of individual IRF5 isoforms with SLE. As the E3 ubiquitin ligase TRIpartite Motif 21 (TRIM21) has been shown to regulate the stability and hence activity of members of the IRF family, we investigated whether IRF5 is subjected to regulation by TRIM21 and whether dysregulation of this mechanism could explain the association of IRF5 with SLE. Our results show that IRF5 is degraded following TLR7 activation and that TRIM21 is involved in this process. Comparison of the individual IRF5 variants demonstrates that isoforms generated by alternative splicing are resistant to TRIM21-mediated degradation following TLR7 stimulation, thus providing a functional link between isoforms expression and stability/activity which contributes to explain the association of IRF5 with SLE.
    PLoS ONE 08/2014; 9(8):e103609. DOI:10.1371/journal.pone.0103609 · 3.23 Impact Factor
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    • "Such environmental stresses as viral or bacterial infection may increase the opportunity for the exposure of TRIM21 to immunocytes , causing anti-TRIM21 autoantibody production. TRIM21 negatively regulates the production of type I IFN, such as INF-α, via ubiquitination of IFN regulatory factors: TRIM21 represses type I IFN production, resulting in protecting cells from prolonged IFN overproduction (Stacey et al., 2012). TRIM21 also functions as a cytosolic IgG receptor, and contributes to ubiquitination-mediated degradation of IgG-bound viral particles in virus-infected cells (Mallery et al., 2010). "
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    ABSTRACT: Synovial tissue in rheumatoid arthritis (RA) shows dense infiltration of plasmacytes. The purpose of the present study is to identify and localize autoantibodies produced in these immunocytes in RA synovitis. We developed a novel screening system for detecting specific autoantigens. Protein antigens recognized by antibodies in the serum and synovial tissue extract from five RA patients were screened with the AlphaScreen method. For screening, a biotinylated human autoantigen library was constructed by the wheat germ cell-free protein synthesis system. The AlphaScreen analysis of 2183 proteins detected a limited number of antigens reactive with the serum and synovial tissue extract. Eighteen biotinylated proteins, containing top five showing high signals in each synovitis tissue extract, were utilized as probes for the enzyme-labeled antigen method, in order to visualize the site of specific antibody production in synovial lesions. Specific antibodies against two proteins, tripartite motif-containing 21 (TRIM21, also known as SSA/Ro52) and F-box only protein 2 (FBXO2), were visualized in the cytoplasm of plasmacytes in two RA synovitis lesions, respectively. Absorption experiments using unlabeled proteins confirmed the specificity of staining. No positive signals against these two proteins were identified in the additionally evaluated RA and osteoarthritis synovial lesions. The present study indicated 1) the usefulness of screening the human autoantigen library with the AlphaScreen assay for detecting autoantibodies in RA synovitis, and 2) the applicability of biotinylated proteins to the enzyme-labeled antigen method for visualizing the site of autoantibody production within the lesion.
    Journal of immunological methods 10/2012; 387(1-2). DOI:10.1016/j.jim.2012.09.011 · 1.82 Impact Factor
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    ABSTRACT: The SPla/Ryanodine receptor (SPRY)/B30.2 domain is one of the most common folds in higher eukaryotes. The human genome encodes 103 SPRY/B30.2 domains, several of which are involved in the immune response. Approximately 45% of human SPRY/B30.2-containing proteins are E3 ligases. The role and function of the majority of SPRY/B30.2 domains are still poorly understood, however, in several cases mutations in this domain have been linked to congenital disorders. The recent characterization of SPRY/B30.2-mediated protein interactions has provided evidence for a role of this domain as an adaptor module to assemble macromolecular complexes, analogous to Src homology (SH)2, SH3, and WW domains. However, functional and structural evidence suggests that SPRY/B30.2 is a more versatile fold, allowing a wide range of binding modes.
    Trends in Biochemical Sciences 11/2012; 38(1). DOI:10.1016/j.tibs.2012.10.001 · 11.23 Impact Factor
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