TRIM23 Antibody

What is TRIM23 and why is it a significant research target?

TRIM23 (Tripartite Motif Containing 23) is a multifunctional protein that acts as an E3 ubiquitin-protein ligase and plays essential roles in selective autophagy, particularly during viral infections. It contains both a RING E3 ligase domain and an ADP-ribosylation factor (ARF) GTPase domain, making it unique among TRIM family proteins. TRIM23 is particularly significant because it:

• Activates TANK-binding kinase 1/TBK1 by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor SQSTM1

• Mediates 'Lys-27'-linked auto-ubiquitination of its ARF domain to induce its GTPase activity and recruitment to autophagosomes

• Functions as a core component of the selective autophagic machinery

• Plays a critical role in antiviral defense mechanisms through autophagy-mediated restriction of multiple viruses

Research on TRIM23 contributes to our understanding of fundamental cellular processes and potential therapeutic targets for viral infections.

How do I select the appropriate TRIM23 antibody for my research?

Selecting the optimal TRIM23 antibody requires consideration of several experimental factors:

For most comprehensive studies, consider using antibodies targeting different epitopes of TRIM23, such as the N-terminal region (AA 1-110), middle region (AA 147-175), or full-length protein (AA 1-574) . When studying TRIM23's role in autophagy, antibodies recognizing the ARF domain may be particularly relevant due to its critical role in TRIM23's autophagic function .

What are the validated applications for TRIM23 antibodies in viral research?

TRIM23 antibodies are instrumental in studying virus-induced autophagy mechanisms. Key applications include:

• Western Blotting: To detect TRIM23 expression levels and post-translational modifications in virus-infected cells. Recommended dilution: 1:500-1:2000 .

• Immunofluorescence: To visualize TRIM23 recruitment to autophagosomes during viral infection. Recommended dilution: 1:50-1:500 .

• Co-immunoprecipitation: To investigate interactions between TRIM23 and autophagy proteins (TBK1, SQSTM1/p62) during viral infection.

• Immunohistochemistry: To examine tissue expression patterns of TRIM23. Recommended dilution: 1:35-1:200 .

When studying virus-triggered autophagy, TRIM23 antibodies can be used with LC3B antibodies to monitor autophagosome formation. Research has validated TRIM23 antibodies for studying various viruses including HSV-1, encephalomyocarditis virus (EMCV), influenza A virus (IAV), adenovirus, and Sindbis virus .

How can I design experiments to investigate TRIM23's role in virus-induced autophagy?

To effectively study TRIM23's role in virus-induced autophagy, consider the following experimental design:

• Baseline characterization:

  • Assess endogenous TRIM23 expression levels in your cell type using Western blot

  • Visualize subcellular localization by immunofluorescence staining

  • Compare with autophagy markers like LC3B and p62/SQSTM1

• Loss-of-function studies:

  • Use TRIM23 siRNA or shRNA to knockdown expression

  • Consider TRIM23 knockout models (TRIM23^-/-^ MEFs)

  • Measure effects on:

    • LC3B puncta formation (by immunofluorescence)

    • LC3B-I to LC3B-II conversion (by Western blot)

    • p62/SQSTM1 degradation

    • Viral replication efficiency

• Structure-function analysis:

  • Complement knockdown/knockout models with:

    • Wild-type TRIM23

    • RING domain mutant (to disrupt E3 ligase activity)

    • ARF domain mutant (to disrupt GTPase activity)

  • Assess restoration of autophagy function with each construct

• Viral infection models:

  • Compare autophagy induction with different viruses (HSV-1, EMCV, IAV, adenovirus)

  • Use wild-type viruses and autophagy-deficient viral mutants (e.g., mutHSV-1)

  • Monitor viral replication kinetics in presence/absence of TRIM23

Evidence indicates that TRIM23 is specifically important for virus-triggered autophagy but has minimal effects on basal or starvation-induced autophagy , making experimental controls particularly important.

What are common troubleshooting issues with TRIM23 antibodies in Western blotting?

When using TRIM23 antibodies for Western blotting, researchers may encounter several challenges. Here are solutions to common issues:

For optimal detection of TRIM23 (predicted molecular weight: 64 kDa), ensure proper sample preparation. TRIM23 can undergo auto-ubiquitination, particularly K27-linked polyubiquitination of its ARF domain , which may result in higher molecular weight bands. To visualize these modifications, consider using denaturing conditions that preserve ubiquitin chains.

How should I optimize immunofluorescence protocols for detecting TRIM23 recruitment to autophagosomes?

To effectively visualize TRIM23 recruitment to autophagosomes during viral infection:

• Fixation and permeabilization:

  • Compare paraformaldehyde (4%) with methanol fixation

  • Optimize permeabilization (0.1-0.5% Triton X-100) to preserve autophagosomal structures

• Antibody incubation:

  • Use recommended dilutions (1:50-1:500)

  • Consider overnight incubation at 4°C for maximum sensitivity

  • Perform sequential double-staining with LC3B to verify autophagosomal localization

• Controls and markers:

  • Include uninfected controls

  • Use rapamycin-treated cells as positive controls for autophagy induction

  • Co-stain with SQSTM1/p62 to visualize selective autophagy

• Imaging considerations:

  • Use confocal microscopy to accurately assess colocalization

  • Perform z-stack analysis to capture complete autophagosomal structures

  • Quantify TRIM23-LC3B colocalization using appropriate software

• Specific virus considerations:

  • For HSV-1: Use mutHSV-1 (γ34.5 mutant) which efficiently induces autophagy

  • For RNA viruses: Fix cells at optimal time points post-infection (typically 6-24 hours)

Research shows that TRIM23 recruitment to autophagosomes is dependent on its ARF GTPase activity, which is activated by K27-linked auto-ubiquitination . This specific localization pattern is critical for its function in antiviral autophagy.

How does TRIM23 regulate selective autophagy during viral infection through its dual E3 ligase and GTPase activities?

TRIM23 represents a unique case of functional integration between ubiquitination and GTPase activities in regulating selective autophagy during viral infection:

• Dual-domain coordination mechanism:

  • The RING E3 ligase domain of TRIM23 mediates K27-linked auto-ubiquitination of its own ARF domain

  • This auto-ubiquitination activates the GTPase activity of the ARF domain

  • Activated GTPase function enables TRIM23 recruitment to autophagosomal membranes

• TBK1 activation pathway:

  • Once localized to autophagosomes, TRIM23 facilitates TBK1 dimerization

  • Dimerized TBK1 phosphorylates the selective autophagy receptor SQSTM1/p62

  • Phosphorylated p62 efficiently targets viral components for autophagic degradation

• Virus-specific considerations:

  • Different viruses may engage TRIM23-dependent autophagy through distinct mechanisms

  • HSV-1 (DNA virus) and Sindbis virus (RNA virus) are both restricted by TRIM23, suggesting a broad antiviral mechanism

  • TRIM23 knockout enhances replication of mutHSV-1, adenovirus, and Sindbis virus by approximately 2-log

To study this dual functionality, researchers should employ mutational analysis targeting specific domains:

• RING domain mutations to disrupt E3 ligase activity

• ARF domain mutations to impair GTPase activity

• Lysine-to-arginine mutations to prevent auto-ubiquitination

Both domains are required for TRIM23's function in autophagy, as evidenced by experiments showing that neither domain alone can rescue autophagy defects in TRIM23-deficient cells .

How do different TRIM family proteins coordinate virus-specific autophagy responses, and what are the methodological approaches to study their interplay?

TRIM proteins exhibit remarkable specificity in regulating autophagy induced by different viral pathogens. To investigate this coordinated response:

• Comparative analysis of virus-specific TRIM regulators:

  • TRIM13: Specifically required for EMCV-induced autophagy

  • TRIM25: Essential for IAV-induced autophagy

  • TRIM56: Critical for mutHSV-1-induced autophagy

  • TRIM21, TRIM23, TRIM41: Required for autophagy induced by multiple viruses

• Hierarchical screening methodology:

  • Primary screen: Test 61 TRIM proteins for induction of GFP-LC3B puncta

  • Secondary screen: Validate hits using siRNA knockdown in virus-infected cells

  • Tertiary validation: Confirm with genetic knockout models

• Protein interaction network analysis:

  • Identify TRIM protein interactomes during virus infection using:

    • Proximity-dependent biotin labeling (BioID)

    • Co-immunoprecipitation followed by mass spectrometry

  • Map interactions with core autophagy machinery (ATGs)

  • Create protein-protein interaction networks for each virus

• Biochemical dissection of signaling pathways:

  • Study ubiquitination patterns and specificity using:

    • Ubiquitin linkage-specific antibodies (K27, K48, K63)

    • Mass spectrometry to identify ubiquitinated residues

    • Reconstituted in vitro ubiquitination assays

• Evolutionary comparison approach:

  • TRIM23 is highly conserved in vertebrates

  • Compare TRIM protein function across species to identify conserved vs. evolved mechanisms

This comprehensive approach reveals that while some TRIM proteins function in autophagy pathways specific to certain viruses, others like TRIM23 serve as core components of the selective autophagy machinery against a broad spectrum of viral pathogens .

What are the methodological considerations for studying the relationship between TRIM23-mediated autophagy and interferon responses during viral infection?

TRIM23 operates at the intersection of autophagy and interferon (IFN) responses, requiring careful experimental design to dissect these interrelated but distinct functions:

• Differential experimental systems:

  • Use HSV-1 variants with distinct autophagy/IFN antagonism profiles:

    • Wild-type HSV-1: Antagonizes both autophagy and IFN responses

    • mutHSV-1 (γ34.5 mutant): Lacks autophagy antagonism but retains IFN antagonism

  • Compare outcomes in:

    • Wild-type cells

    • TRIM23^-/-^ cells

    • ATG5^-/-^ or ATG7^-/-^ cells (autophagy-deficient)

    • IFNAR^-/-^ cells (IFN signaling-deficient)

• Pathway-specific readouts:

  • Autophagy markers: LC3B-I/II conversion, p62 degradation, autophagosome ultrastructure

  • IFN pathway markers: ISG protein expression, STAT1 phosphorylation, IRF3 activation

  • Viral replication: Plaque assays, viral protein expression, viral genome quantification

• Temporal dynamics analysis:

  • Monitor both autophagy and IFN responses over time (0-48h) post-infection

  • Determine if one pathway precedes and potentially influences the other

  • Use time-course experiments with pathway-specific inhibitors

• Domain-specific functional analysis:

  • Assess TRIM23 RING domain mutants for effects on:

    • Ubiquitination activity

    • IFN pathway activation

    • Autophagy induction

  • Evaluate ARF domain mutants for similar parameters

• Confounding variables control:

  • Account for cell-type specific differences in autophagy/IFN responses

  • Consider virus dose-dependent effects

  • Monitor cell viability to exclude cell death-related effects

Research indicates that while TRIM23 has been implicated in both autophagy and type I IFN responses , its autophagy function appears critical for restricting viruses like mutHSV-1, adenovirus, and Sindbis virus, even in contexts where IFN antagonism remains intact .

What quality control measures should be implemented when using TRIM23 antibodies in research?

Implementing rigorous quality control is essential for generating reliable data with TRIM23 antibodies:

For immunohistochemistry applications, antibodies should be validated on tissue microarrays containing both positive and negative tissues. Some commercial TRIM23 antibodies have been validated on arrays containing 44 normal human tissues and 20 common cancer types , providing extensive characterization data.

When using TRIM23 antibodies for autophagy research, additional quality control measures include:

• Confirming that autophagy induction (e.g., by rapamycin) produces the expected pattern of TRIM23 localization

• Verifying that TRIM23 colocalizes with established autophagy markers

• Ensuring detection of expected molecular weight species, including potential ubiquitinated forms

How do post-translational modifications of TRIM23 impact antibody selection and experimental design?

TRIM23 undergoes several critical post-translational modifications that significantly impact experimental approaches:

• K27-linked auto-ubiquitination:

  • This specific ubiquitination is essential for activating TRIM23's GTPase activity

  • Some antibodies may have differential affinity for ubiquitinated vs. non-ubiquitinated forms

  • Considerations:

    • Use denaturing conditions that preserve ubiquitin chains in sample preparation

    • Select antibodies targeting epitopes distant from major ubiquitination sites

    • Consider using deubiquitinating enzymes as controls to confirm band identity

• GTP/GDP-bound states:

  • The ARF domain of TRIM23 cycles between GTP and GDP-bound conformations

  • These conformational changes may affect epitope accessibility

  • Approaches:

    • Compare antibody performance under conditions that promote GTP binding (e.g., GTPγS treatment)

    • Use ARF domain mutants locked in specific nucleotide-bound states as controls

• Phosphorylation state:

  • TRIM23 may be phosphorylated during signaling events

  • Phosphorylation can alter protein migration and epitope recognition

  • Strategies:

    • Treat samples with phosphatases as controls

    • Use phosphorylation site prediction to avoid antibodies targeting potential phospho-sites

• Experimental design implications:

  • Include appropriate controls for each experiment (e.g., TRIM23 RING domain mutants that cannot undergo auto-ubiquitination)

  • Consider the timing of sample collection relative to stimulation or viral infection

  • Be aware that virus-induced modifications may differ from basal conditions