TRIM21 Human
Description
Molecular Structure of TRIM21
TRIM21 (Tripartite Motif Containing Protein 21) is a 52–56 kDa intracellular E3 ubiquitin ligase belonging to the TRIM protein family. Its domain architecture includes:
The PRYSPRY domain forms a β-sandwich structure with two anti-parallel β-sheets, enabling high-affinity binding (Kd ~0.6 nM) to the IgG1 Fc fragment at the CH2-CH3 interface . This binding is pH-independent and unaffected by N-glycosylation but sensitive to high salt concentrations .
Functional Mechanism in Intracellular Immunity
TRIM21 acts as a cytosolic antibody receptor, neutralizing pathogens post-cellular entry. Its dual roles include:
Anti-Viral Activity
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Viral Capture: Binds antibody-coated viruses (e.g., adenovirus, rhinovirus) via Fc regions .
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Proteasomal Degradation:
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Immune Signaling:
Experimental Evidence
Pathological Roles
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Autoimmunity: TRIM21 autoantibodies enhance NF-κB signaling in lupus-prone macrophages, exacerbating inflammation .
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Cancer Metabolism:
Therapeutic Applications
Research Gaps and Future Directions
Product Specs
Q&A
What is the primary molecular function of TRIM21 in human cells?
TRIM21 functions as an E3 ubiquitin ligase that targets viral particles and immune signaling proteins for proteasomal degradation. It binds antibody-opsonized pathogens via its PRYSPRY domain, triggering ubiquitination of viral capsids and host factors like IRF3/IRF8 to modulate inflammatory responses . Methodologically, its ligase activity is validated through in vitro ubiquitination assays using purified TRIM21, E1/E2 enzymes, and substrates (e.g., IRF3), followed by Western blotting with anti-ubiquitin antibodies .
How is TRIM21 expression regulated under inflammatory conditions?
TRIM21 is induced by interferons (IFN-α/β/γ) and proinflammatory cytokines (e.g., TNF-α). Researchers quantify this using qRT-PCR for TRIM21 mRNA and Western blotting for protein levels in cytokine-treated cell lines (e.g., THP-1 monocytes) . A 3.5- to 5-fold induction is typical within 6–24 hours post-stimulation .
What techniques confirm TRIM21’s intracellular localization?
Subcellular fractionation combined with immunofluorescence microscopy reveals TRIM21’s dual cytoplasmic/nuclear distribution. Antibodies targeting the N-terminal RING domain (cytoplasmic) versus nuclear epitopes (e.g., phosphorylated residues) are critical . For example, >60% of TRIM21 localizes to the cytoplasm in unstimulated HeLa cells .
How to resolve conflicting data on TRIM21’s role in autoimmunity versus antiviral defense?
Contradiction: TRIM21 deficiency exacerbates viral infections (e.g., mouse adenovirus-1) but reduces autoimmune symptoms in some lupus models .
Resolution:
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Model specificity: Use tissue-specific knockouts (e.g., myeloid vs. lymphoid cells) to dissect compartmentalized roles.
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Pathogen load: Measure viral titers (plaque assays) and autoantibody levels (ELISA) in parallel .
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Kinetic analysis: Track TRIM21 expression phases—early antiviral (ubiquitinating viruses) vs. late immunoregulatory (degrading IRFs) .
What experimental strategies identify TRIM21 interaction partners?
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Immunoprecipitation-MS: Tag TRIM21 with FLAG/HA in HEK293T cells, immunoprecipitate with anti-FLAG beads, and identify co-purified proteins via mass spectrometry .
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Yeast two-hybrid screening: Screen a human spleen cDNA library using TRIM21’s PRYSPRY domain as bait .
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Structural mapping: Mutate key binding residues (e.g., W380A/W382A in the PRYSPRY domain) to disrupt Fc binding, confirmed by surface plasmon resonance (SPR) .
How to address tissue-specific TRIM21 expression discrepancies in cancer studies?
Example: TRIM21 is downregulated in ovarian cancer (OC) but upregulated in certain lymphomas.
Methodological approach:
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Multi-omics validation: Compare RNA-seq (TCGA), proteomics (CPTAC), and IHC data across 10+ cancer types.
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Functional rescue: Overexpress TRIM21 in OC cell lines (OVCAR-3, SKOV-3) and assess proliferation (BrdU assay) versus apoptosis (Annexin V) .
| Cancer Type | TRIM21 Expression | Functional Outcome | Source |
|---|---|---|---|
| Ovarian | ↓ (5.8-fold) | ↑ Proliferation | |
| Diffuse large B-cell lymphoma | ↑ (3.2-fold) | ↓ Apoptosis |
What controls are essential for TRIM21 knockout studies?
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Genotyping: Confirm TRIM21−/− status via PCR using primers flanking exon 2 (deleted in many models) .
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Heterozygote controls: Include TRIM21+/− mice, as haploinsufficiency impacts viral clearance (e.g., 16-fold higher adenovirus titers in +/− vs. +/+) .
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Rescue experiments: Reintroduce wild-type or mutant TRIM21 (ΔRING) via lentiviral transduction to verify phenotype specificity .
Does TRIM21 enhance or inhibit NF-κB signaling?
Context-dependent mechanism:
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Proinflammatory: TRIM21 ubiquitinates TAB2/TAK1, activating NF-κB in macrophages during lupus .
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Anti-inflammatory: Targets IRF3 for degradation, suppressing IFN-β-driven NF-κB in fibroblasts .
Experimental design: Use cell-type-specific reporters (e.g., RAW264.7 macrophages vs. MEFs) and NF-κB luciferase assays with/without TRIM21 siRNA .
How does TRIM21 achieve species-specific IgG binding despite low sequence conservation?
Structural insight: The PRYSPRY domain binds conserved Fc hot-spot residues (e.g., His433, His435) via a preformed interface. SPR shows a Kd of 37 nM for human IgG1, with <10% affinity loss across primates .
Method: Generate chimeric Fc mutants (e.g., H433A/H435A) and test binding via ELISA with recombinant TRIM21 .
Can TRIM21 be harnessed for targeted protein degradation (e.g., Trim-Away)?
Protocol:
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Electroporate cells with 10 μM TRIM21 plasmid and 50 nM target antibody (e.g., anti-GFP).
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Monitor degradation via live-cell imaging (GFP-tagged proteins) within 30–60 minutes .
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Validate using cycloheximide chase assays to block new protein synthesis .
What are the implications of TRIM21’s dual affinity for IgG/IgA/IgM?
Functional assay: Compare viral neutralization efficiency using isotype-switched antibodies. For example, IgA-opsonized rotavirus shows 40% lower TRIM21-dependent neutralization vs. IgG in HT-29 cells .
Why do TRIM21 autoantibodies interfere with ELISA measurements?
Problem: Serum anti-TRIM21 antibodies (common in SLE) cause false positives/negatives.
Solution:
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Pre-treat sera with protein A/G beads to remove IgGs.
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Use epitope-mapping peptides (e.g., TRIM21 aa 200–300) as competitors in assays .
How to model TRIM21’s role in human autoimmune diseases in vitro?
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Patient-derived cells: Isolate PBMCs from SLE patients (≥50% anti-TRIM21+) and compare TRIM21 mRNA/protein vs. healthy donors .
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CRISPR editing: Introduce SLE-associated SNPs (e.g., rs660) into iPSC-derived macrophages and assess IFN-α secretion .
Does TRIM21 regulate non-immune pathways (e.g., metabolism)?
Preliminary data suggest TRIM21 ubiquitinates ACLY (ATP-citrate lyase), reducing lipid synthesis in hepatocytes. Test via:
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Metabolomics: LC-MS profiling of TRIM21−/− vs. WT HepG2 cells.
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ACLY ubiquitination: Co-IP with anti-ACLY followed by anti-ubiquitin blot .
Can TRIM21’s Fc-binding interface be engineered for antiviral therapeutics?
Approach:
Product Science Overview
Tripartite Motif Containing 21 (TRIM21), also known as RO52, is a protein encoded by the TRIM21 gene in humans. This protein is a member of the tripartite motif (TRIM) family, which is characterized by the presence of three zinc-binding domains: a RING finger domain, a B-box type 1, and a B-box type 2, along with a coiled-coil region . TRIM21 plays a crucial role in the immune system and is involved in various cellular processes, including protein ubiquitination and intracellular antibody-mediated proteolysis .
TRIM21 is expressed in most human tissues and is particularly abundant in immune cells . It functions as an E3 ubiquitin-protein ligase, which means it helps attach ubiquitin molecules to specific protein substrates, marking them for degradation by the proteasome . This activity is essential for maintaining cellular homeostasis and regulating immune responses.
One of the unique functions of TRIM21 is its role in intracellular immunity. It acts as a sensor that intercepts antibody-coated viruses that have evaded extracellular neutralization and breached the cell membrane . By binding to the Fc domain of immunoglobulins, TRIM21 can target these viruses for degradation, providing a last line of defense against viral infections .
TRIM21 is closely associated with autoimmune diseases, particularly systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) . Dysregulation of TRIM21 can lead to aberrant immune responses, contributing to the pathogenesis of these conditions. Autoantibodies against TRIM21 are commonly found in patients with these autoimmune diseases, making it a valuable biomarker for diagnosis and disease monitoring .
Recombinant TRIM21 is a human-derived protein that is expressed in Escherichia coli (E. coli) for research and therapeutic purposes . This recombinant protein retains the functional properties of the native TRIM21 and is used in various studies to understand its role in immune regulation and disease mechanisms. Additionally, recombinant TRIM21 has been shown to interact with the HIV-1 Vpu protein, highlighting its potential in antiviral research .
