TRIM69 Antibody

What is TRIM69 and what cellular functions does it perform?

TRIM69 is an E3 ubiquitin-protein ligase that plays important roles in antiviral immunity and cellular processes. It functions primarily by restricting different viral infections including dengue virus, vesicular stomatitis Indiana virus, HIV-1, primate lentiviruses, and SARS-CoV2 . Beyond viral immunity, TRIM69 is involved in cataract formation together with TP53, and mechanistically inhibits UVB-induced cell apoptosis and reactive oxygen species (ROS) production by inducing TP53 ubiquitination . This dual functionality makes TRIM69 an interesting target for both viral immunity and cellular protection studies.

What are the structural and molecular characteristics of TRIM69?

TRIM69 is characterized by its tripartite motif structure typical of the TRIM family of innate immunity regulators. The protein exists in 4 distinct isoforms with molecular weights of 57, 39, 34, and 32 kDa . While the calculated molecular weight based on its 500 amino acid sequence is 57 kDa, the observed molecular weight in experimental settings typically ranges between 60-70 kDa . This discrepancy between calculated and observed weights suggests post-translational modifications that may be relevant to TRIM69's function.

How is TRIM69 expression regulated across different cell types?

TRIM69 expression is highly responsive to interferon stimulation, particularly in cells of myeloid lineage. In THP-1-PMA cells, TRIM69 is expressed at basal levels and shows extreme sensitivity to type I interferon (IFN-I α/β) stimulation, a pattern similar to what is observed in primary macrophages . This cell-type specificity is important methodologically as researchers should select appropriate cell models when studying TRIM69 function. The differential expression pattern helps explain why TRIM69's antiviral effects are more pronounced in myeloid cells compared to other cell types.

What are the validated applications for TRIM69 antibody in research?

The TRIM69 antibody (29907-1-AP) has been validated for multiple research applications including Western Blot (WB), Immunohistochemistry (IHC), and ELISA . For optimal results in Western Blot applications, the recommended dilution range is 1:500-1:2000, while for Immunohistochemistry, the recommended dilution range is 1:50-1:500 . Researchers should note that antibody performance can be sample-dependent, and titration in each testing system is recommended to obtain optimal results.

What experimental conditions yield optimal results with TRIM69 antibody in immunohistochemistry?

For successful immunohistochemistry applications with TRIM69 antibody, antigen retrieval with TE buffer pH 9.0 is strongly recommended . As an alternative methodology, antigen retrieval may be performed with citrate buffer pH 6.0 . The antibody has shown consistent positive detection in mouse cerebellum tissue, making this an appropriate positive control for IHC protocol optimization. Researchers should be aware that protocol optimization may be necessary depending on tissue fixation methods and specific experimental goals.

What cellular models are best suited for TRIM69 antibody studies?

Through what mechanisms does TRIM69 function in cellular antiviral defense?

TRIM69 has been identified as an interferon-stimulated gene (ISG) that serves as a broad-spectrum antiviral factor. Its antiviral activity is uniquely linked to its ability to promote the accumulation of stable microtubules, a specialized subset of the cellular cytoskeleton . This represents a novel mechanism of antiviral defense, as TRIM69 is the first described antiviral innate defense factor that regulates microtubule properties to limit viral spread . Additionally, for certain viruses like dengue virus, TRIM69 employs its E3 ubiquitin ligase activity to specifically ubiquitinate viral nonstructural proteins, thereby interrupting viral replication .

What is the spectrum of viruses inhibited by TRIM69, and how does inhibition vary?

TRIM69 demonstrates remarkable versatility in its antiviral activity, inhibiting viruses from multiple families with different genomic structures:

Research indicates that the magnitude of inhibition depends on the specific combination of virus and cell type . Notably, some studies suggest specificity in TRIM69's antiviral activity against certain viruses, as it reportedly did not interfere with influenza virus H1N1 or herpes virus HSV-1 infection in specific experimental contexts .

At what stages of viral replication does TRIM69 exert its inhibitory effects?

TRIM69 primarily targets the early phases of viral life cycles, with mechanistic variations depending on the virus:

• For HIV-1 and other lentiviruses: Inhibition occurs during reverse transcription, after entry of viral complexes into the cell

• For SARS-CoV2: Similar to HIV-1, with no measurable defects in virus entry but an early defect in viral RNA replication

• For VSV: A small but detectable defect is observed at the entry step, followed by a major defect during pioneer transcription

• For Dengue virus: Direct ubiquitination of viral nonstructural proteins, resulting in decreased viral RNA and protein levels

This stage-specific inhibition pattern suggests that researchers investigating TRIM69's antiviral effects should design experiments with appropriate time points to capture these early replication events.

How does TRIM69 specifically inhibit dengue virus replication?

For dengue virus, TRIM69 employs a direct ubiquitination mechanism. Studies have shown that overexpression of TRIM69 significantly decreased viral RNA and proteins in infected cells, as demonstrated through multiple methodological approaches including qRT-PCR, Western Blot, and immunofluorescence assays . This inhibition results in measurably reduced viral release in cell supernatants. The specificity of this mechanism is highlighted by experimental results using a luciferase-based DENV replicon system that confirmed TRIM69's direct impact on viral replication machinery .

How does TRIM69 interact with and modulate the cellular microtubule network?

TRIM69 has been identified as a direct microtubule-binding protein that promotes the accumulation of stable microtubules . This represents a novel finding linking innate immunity factors to cytoskeletal regulation. Methodologically, this interaction can be observed through immunofluorescence techniques using antibodies against acetylated α-tubulin (a marker of stable microtubules) in conjunction with TRIM69 detection . The direct binding of TRIM69 to microtubules indicates a physical interaction rather than an indirect signaling pathway.

What is the relationship between interferon stimulation, TRIM69 expression, and microtubule stabilization?

Interferon stimulation leads to a coordinated program of microtubule stabilization specifically in cells of myeloid lineage (monocytes > macrophages > dendritic cells), but not in activated primary lymphocytes . This program represents a previously unrecognized facet of the innate defense system. TRIM69 plays an instrumental role in this response, as demonstrated by experiments showing that:

• Control cells show increased stable microtubule accumulation following IFN-α stimulation

• TRIM69-overexpressing cells show elevated basal levels of stable microtubules, further enhanced by IFN-α

• TRIM69 knockout cells show severely diminished stable microtubule accumulation, even after IFN-α stimulation

These findings establish TRIM69 as a key mediator of interferon-induced cytoskeletal remodeling in antiviral responses.

Through what experimental approaches can researchers study TRIM69's effects on microtubule dynamics?

To investigate TRIM69's effects on microtubule dynamics, researchers can employ several methodological approaches:

• Immunofluorescence microscopy using antibodies against acetylated α-tubulin to visualize stable microtubules, coupled with TRIM69 detection

• Genetic manipulation approaches (knockout or overexpression) of TRIM69 followed by quantification of stable microtubules on a per-cell basis

• Time-course experiments following interferon stimulation to track the dynamics of microtubule stabilization

• Comparative analysis across different cell types (myeloid vs. lymphoid) to understand cell-type specificity

These approaches allow for both qualitative and quantitative assessment of TRIM69's impact on the microtubule network.

What evolutionary forces have shaped TRIM69 function in primate antiviral defense?

Multiple studies have documented evidence of ongoing genetic conflict in primate TRIM69 genes, as well as polymorphism in the human population . This pattern suggests that TRIM69 has been under strong selective pressure throughout primate evolution. Interestingly, experiments using divergent simian TRIM69 variants suggest that lentiviruses (which have invaded primates relatively recently) may not have been the main drivers of this selective pressure . This raises fascinating questions about which pathogens might have exerted this evolutionary pressure. Researchers investigating this question should consider comparative genomic approaches and functional analyses of TRIM69 variants across primate species.

How might TRIM69's microtubule regulation mechanism be leveraged for broad-spectrum antiviral strategies?

TRIM69's ability to inhibit diverse viruses through microtubule stabilization represents a potential target for developing novel broad-spectrum antiviral approaches . Unlike traditional antivirals that target specific viral components, therapies based on enhancing TRIM69's microtubule stabilization function could potentially be effective against multiple viral families. Research approaches might include:

• High-throughput screening for small molecules that mimic TRIM69's microtubule-stabilizing effects

• Development of cell-penetrating peptides derived from TRIM69's microtubule-binding domains

• Targeted activation of the interferon-TRIM69-microtubule pathway in specific tissues

• Identification of the minimal TRIM69 domains necessary for antiviral activity

These approaches could open new avenues for antiviral therapeutics that leverage cytoskeletal regulation rather than direct targeting of viral components.

How does the cell-type specificity of TRIM69 function contribute to viral tropism and pathogenesis?

The antiviral effects of TRIM69 show pronounced cell-type specificity, with more robust effects in cells of myeloid lineage compared to other cell types like lymphocytes . This pattern raises important questions about how TRIM69 might contribute to viral tropism and differential susceptibility of tissues to viral infection. This cell-type specificity is not unprecedented, as other cellular factors like SAMHD1 and APOBEC3A also inhibit HIV-1 in a cell type-specific manner . Researchers investigating this aspect should consider:

• Comprehensive profiling of TRIM69 expression across human tissues and cell types

• Investigation of viral replication in matched cell types with varying TRIM69 expression levels

• Analysis of TRIM69 polymorphisms in relation to susceptibility to specific viral infections

• In vivo studies of viral pathogenesis in TRIM69-modulated animal models

These approaches could yield important insights into the role of TRIM69 in determining patterns of viral pathogenesis and tissue tropism.

What are the optimal storage and handling conditions for TRIM69 antibody reagents?

For maximum stability and performance, TRIM69 antibody should be stored at -20°C, where it remains stable for approximately one year after shipment . The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for -20°C storage, simplifying laboratory workflows. For small volume formats (20μl), the preparation contains 0.1% BSA . Researchers should avoid repeated freeze-thaw cycles to maintain antibody performance.

What control experiments should be included when studying TRIM69 function?

When investigating TRIM69 function, several important controls should be included:

• Cell viability assessments for both TRIM69 knockout and overexpression conditions to rule out non-specific effects

• Envelope-independent controls when studying effects on viruses (e.g., testing both VSVg-pseudotyped and native envelope viruses)

• Time-course experiments to capture the dynamics of TRIM69-mediated effects

• Cell-type matched controls given the cell-type specificity of TRIM69 function

• IFN-stimulated and unstimulated conditions to distinguish TRIM69-specific from broader IFN effects

These methodological controls help ensure that observed effects are specifically attributable to TRIM69 function rather than experimental artifacts.

What model systems are most appropriate for studying TRIM69's diverse functions?

Based on the current literature, several model systems have proven valuable for studying different aspects of TRIM69 function:

Researchers should select model systems appropriate to their specific research questions, with particular attention to cell-type specific effects that may influence experimental outcomes.

How does TRIM69 function relate to other members of the TRIM protein family?

TRIM69 is part of the larger TRIM family of innate immunity regulators, many of which have established roles in antiviral defense . While several TRIM proteins function as E3 ubiquitin ligases targeting viral components, TRIM69's microtubule regulatory function represents a distinct mechanism within this family . This raises interesting questions about functional diversification within the TRIM family. Researchers investigating these relationships should consider comparative functional studies across multiple TRIM family members, focusing on both shared and unique mechanisms of antiviral activity.

What is the relationship between TRIM69 and other interferon-stimulated genes in coordinated antiviral responses?

TRIM69 functions within the broader context of interferon-stimulated gene (ISG) responses . Understanding how TRIM69's microtubule-stabilizing activity complements other ISG functions could provide insights into the orchestration of innate immune responses. Methodological approaches to this question might include:

• Transcriptomic and proteomic profiling of cells with TRIM69 modulation (knockout or overexpression)

• Network analysis of ISG interactions during viral infection

• Combinatorial manipulation of TRIM69 and other key ISGs to identify synergistic effects

• Temporal analysis of different ISG functions during the course of viral infection

These approaches could help position TRIM69 within the broader landscape of interferon-mediated antiviral immunity.