Recombinant Rat Tripartite motif-containing protein 39 (Trim39)

What is Rat Tripartite Motif-Containing Protein 39 (Trim39) and what are its key structural domains?

Rat Tripartite Motif-Containing Protein 39 (Trim39) belongs to the TRIM protein family of E3 ubiquitin ligases. Like other members of this family, Trim39 is characterized by a specific domain architecture consisting of a RING finger domain, a B-box domain, and a coiled-coil region, collectively referred to as the RBCC or tripartite motif. The C-terminal region of Trim39 contains NHL domains, which are involved in protein dimerization and substrate recognition .

The RING domain (located at amino acids 39-42 in mouse Trim39) is essential for its E3 ubiquitin ligase activity, as demonstrated in studies where deletion of this domain (Trim39-ΔRING) abolished its ability to ubiquitinate substrates . The B-box domain (containing SIM2 at position 125-128) and the coiled-coil domain (containing SIM3 at position 211-215) are involved in protein-protein interactions and oligomerization . These structural components work together to facilitate Trim39's diverse cellular functions.

What are the primary cellular functions of recombinant Trim39?

Trim39 functions primarily as an E3 ubiquitin ligase with several important cellular roles:

• Regulation of protein degradation: Trim39 mediates the ubiquitination of specific targets, including the transcription factor NFATc3, marking them for proteasomal degradation .

• SUMO-targeted ubiquitin ligase (STUbL) activity: Trim39 contains SUMO-interacting motifs (SIMs) that allow it to recognize and preferentially ubiquitinate SUMOylated proteins, acting as a genuine STUbL for NFATc3 .

• Cell cycle regulation: Trim39 can directly inhibit the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase that controls cell cycle progression .

• p53 regulation: Trim39 has been identified as capable of ubiquitinating p53, suggesting a role in regulating cellular responses to stress and DNA damage .

• Neuronal apoptosis: Through its regulation of NFATc3 stability and activity, Trim39 modulates neuronal apoptosis, with its silencing leading to increased NFATc3 levels and enhanced apoptotic activity .

How do environmental chemicals affect Trim39 expression in rats?

Several environmental chemicals have been shown to influence Trim39 expression in rats, offering important insights for toxicological research and environmental health studies:

• 2,3,7,8-tetrachlorodibenzodioxine (TCDD): Multiple studies have demonstrated that TCDD exposure increases Trim39 mRNA expression, as confirmed by experimental (EXP) and inferred (ISO) evidence .

• 17α-ethynylestradiol: This synthetic estrogen increases Trim39 expression, and when co-administered with TCDD, produces complex interactive effects on Trim39 expression patterns .

• 1,2-dimethylhydrazine: This chemical decreases Trim39 expression, revealing a different regulatory pattern compared to TCDD and ethynylestradiol .

• 2,4-dinitrotoluene: This compound affects Trim39 expression, though the specific direction of change requires further characterization .

These chemical-gene interactions suggest that Trim39 may play roles in cellular responses to xenobiotic exposure and could serve as a potential biomarker in toxicological studies.

What techniques are optimal for studying Trim39-mediated ubiquitination in vitro?

For researchers investigating Trim39's ubiquitin ligase activity, several methodological approaches have proven effective:

• Cell-based ubiquitination assays:

  • Co-transfection of cells (e.g., Neuro2A) with His-tagged ubiquitin, the substrate of interest (e.g., NFATc3), and Trim39 or its mutant variants (e.g., Trim39-ΔRING)

  • Treatment with proteasome inhibitors (e.g., MG-132) prior to cell lysis to prevent degradation of ubiquitinated proteins

  • Detection of ubiquitinated proteins through western blotting after nickel affinity purification of His-tagged ubiquitin conjugates

• In vitro ubiquitination assays:

  • Using in vitro translated/immunopurified substrates (e.g., NFATc3)

  • Purified recombinant components including E1, E2, ubiquitin, ATP, and GST-Trim39

  • Analysis of ubiquitination products by SDS-PAGE and immunoblotting

  • Inclusion of proper controls, such as reactions lacking ATP or using catalytically inactive Trim39 variants

• Validation through silencing approaches:

  • Implementation of multiple specific shRNAs targeting Trim39 (minimum of three different constructs recommended)

  • Confirmation of knockdown efficiency through western blotting

  • Assessment of changes in substrate ubiquitination levels after Trim39 depletion

These complementary approaches provide robust validation of Trim39's ubiquitination activity toward specific substrates and allow for mechanistic investigations of regulatory factors.

How can researchers effectively study Trim39's function as a SUMO-targeted ubiquitin ligase (STUbL)?

Investigating Trim39's STUbL activity requires specialized techniques to analyze the interplay between SUMOylation and ubiquitination:

• Identification and mutation of SUMO-interacting motifs (SIMs):

  • Bioinformatic prediction of SIMs using tools such as GPS-SUMO or JASSA

  • Site-directed mutagenesis of predicted SIMs (e.g., conversion of key hydrophobic residues to alanine)

  • In the case of Trim39, three SIMs have been identified: SIM1 (39-PVII-42), SIM2 (125-VCLI-128), and SIM3 (211-LLSRL-215)

• SUMO-binding assays:

  • GST pull-down experiments using purified recombinant proteins

  • Analysis of binding to different SUMO chains (e.g., di-, tri-, tetra-, and higher-order SUMO-2 chains)

  • Comparison of wild-type Trim39 with SIM mutants (mSIM1: 39-PAAA-42, mSIM2: 125-AAAA-128, mSIM3: 211-AAARA-215)

• Substrate SUMOylation analysis:

  • Identification of SUMOylation sites in substrates (e.g., NFATc3)

  • Generation of SUMOylation-deficient mutants

  • Comparison of ubiquitination efficiency between wild-type and SUMOylation-deficient substrates

• In vitro STUbL activity assays:

  • Preparation of SUMOylated and non-SUMOylated forms of the substrate

  • Parallel ubiquitination assays to compare Trim39's preference for SUMOylated substrates

  • Use of SUMO-specific proteases to confirm the dependence on SUMOylation

Research has shown that mutation of SIM3 in Trim39 strongly reduces its SUMO-binding ability and subsequent interaction with NFATc3, demonstrating the importance of this motif for Trim39's STUbL function .

What experimental approaches can reveal the role of Trim39 in neuronal apoptosis?

To investigate Trim39's involvement in neuronal apoptosis, researchers can employ these methodological strategies:

• Manipulation of Trim39 expression:

  • Silencing using specific shRNAs that have been validated for knockdown efficiency

  • Overexpression of wild-type Trim39 compared to catalytically inactive mutants (e.g., RING domain deletions)

  • Use of SUMOylation pathway modulators to alter Trim39's STUbL activity

• Assessment of NFATc3 stability and activity:

  • Protein stability assays using cycloheximide chase experiments

  • Analysis of NFATc3 transcriptional activity using reporter gene assays

  • Chromatin immunoprecipitation to assess NFATc3 binding to target gene promoters

• Apoptosis detection methods:

  • Morphological analysis of nuclear condensation and fragmentation

  • Measurement of caspase activation

  • TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay for DNA fragmentation

  • Annexin V/PI staining and flow cytometry for quantification of apoptotic cells

• Correlation analysis between SUMOylation, ubiquitination, and apoptotic outcomes:

  • Use of SUMOylation-deficient NFATc3 mutants to assess changes in cellular apoptosis

  • Comparison of apoptotic responses in the presence of wild-type versus SIM-mutant Trim39

Research has demonstrated that silencing Trim39 increases NFATc3 protein levels and transcriptional activity, resulting in enhanced neuronal apoptosis. Similarly, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity, supporting the model that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3 .

How does the interaction between Trim39 and Trim17 regulate NFATc3 stability and function?

The interaction between Trim39 and Trim17 represents a complex regulatory mechanism affecting NFATc3 stability and function:

• Interaction validation methods:

  • Co-immunoprecipitation experiments in cells expressing tagged proteins

  • Verification at endogenous levels in relevant cell types (e.g., C2.7 myoblasts or neuronal cells)

  • Proximity ligation assays (PLA) to detect protein interactions in situ

• Regulatory mechanisms:

  • Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 through two distinct mechanisms:
    a) Reduction of Trim39's E3 ubiquitin-ligase activity
    b) Interference with the NFATc3/Trim39 interaction

• Functional consequences:

  • Increased Trim17 expression leads to stabilization of NFATc3

  • The balance between Trim39 and Trim17 appears to fine-tune NFATc3 levels and activity

  • This regulatory mechanism may be particularly important in contexts where precise control of NFATc3-mediated transcription is required

• Domain mapping approaches:

  • Creation of deletion mutants to identify interaction domains

  • Pull-down assays with purified recombinant proteins

  • Mutational analysis of key residues in interaction interfaces

The Trim39-Trim17 interaction has been confirmed in independent proteome-scale yeast two-hybrid screens and validated through various biochemical approaches, establishing it as a physiologically relevant regulatory mechanism .

What techniques can identify novel substrates of Trim39 E3 ubiquitin ligase activity?

Discovering new substrates of Trim39 requires systematic approaches combining biochemical, proteomic, and computational methods:

• Proteome-wide approaches:

  • Quantitative proteomics comparing protein levels in Trim39-deficient versus control cells

  • Ubiquitin remnant profiling using antibodies recognizing the di-glycine remnant left on ubiquitinated lysines after trypsin digestion

  • Stable isotope labeling with amino acids in cell culture (SILAC) combined with immunoprecipitation of ubiquitinated proteins

• Protein interaction screening:

  • Yeast two-hybrid screens using Trim39 as bait

  • Affinity purification mass spectrometry (AP-MS) using tagged Trim39

  • Proximity-dependent biotin identification (BioID) or APEX approaches to identify proteins in the vicinity of Trim39

• Validation methods for candidate substrates:

  • In vivo ubiquitination assays in cells with manipulated Trim39 levels

  • In vitro ubiquitination assays with purified components

  • Protein stability assays in the presence or absence of Trim39

  • Analysis of substrate levels after proteasome inhibition

• Domain-specific interaction mapping:

  • Using deletion mutants of Trim39 (RING/B-box, coiled-coil, NHL repeats) to identify domains responsible for substrate recognition

  • Co-immunoprecipitation experiments with substrate candidates and domain mutants

Known substrates of Trim39 include NFATc3 and p53, suggesting that Trim39 may regulate both transcriptional responses and cellular stress pathways .

How can researchers analyze the effects of Trim39 on cell cycle regulation through APC/C inhibition?

Studying Trim39's role in cell cycle regulation through APC/C inhibition requires specialized cell cycle analysis techniques:

• Cell cycle synchronization and analysis:

  • Synchronization methods appropriate for the cell type under study (e.g., double thymidine block, nocodazole arrest)

  • Flow cytometry with propidium iodide staining to quantify cell cycle distribution

  • Time-course experiments tracking cell cycle progression after manipulation of Trim39 levels

• APC/C activity assays:

  • Monitoring levels of known APC/C substrates (e.g., cyclin B, securin) through western blotting

  • In vitro ubiquitination assays using immunopurified APC/C and recombinant substrates

  • Analysis of APC/C-dependent ubiquitination in the presence of varied concentrations of Trim39

• Interaction studies between Trim39 and APC/C:

  • Co-immunoprecipitation experiments to detect physical association

  • Mapping studies to identify the specific APC/C subunits targeted by Trim39

  • Analysis of APC/C complex integrity in the presence of Trim39

• Functional cell cycle assays:

  • Live-cell imaging with fluorescent reporters of cell cycle phases

  • Analysis of mitotic duration and fidelity

  • Spindle assembly checkpoint activation status

  • Chromosome segregation errors

Research has shown that Trim39 can directly inhibit the APC/C, potentially influencing cell cycle progression and mitotic events, which may have implications for cellular proliferation and genomic stability .

What are the optimal conditions for producing and purifying recombinant Rat Trim39?

Effective production and purification of recombinant Rat Trim39 requires attention to several technical factors:

• Expression systems:

  • Bacterial expression (E. coli): Suitable for producing GST-tagged Trim39 for in vitro assays, though solubility may be challenging

  • Mammalian cell expression: Preferred for obtaining properly folded and post-translationally modified Trim39, especially when studying SUMO-dependent interactions

  • Baculovirus-insect cell systems: Offer a compromise between yield and post-translational modifications

• Purification strategies:

  • Affinity tags: GST fusion proteins have been successfully used for Trim39 purification

  • Buffer optimization: Including reducing agents to maintain RING domain integrity

  • Consideration of detergents when purifying full-length protein due to hydrophobic regions

  • Size exclusion chromatography as a final purification step to ensure homogeneity

• Quality control assessments:

  • SDS-PAGE and western blotting to confirm purity and integrity

  • Mass spectrometry to verify protein identity and detect potential modifications

  • Dynamic light scattering to assess aggregation state

  • In vitro autoubiquitination assays to confirm catalytic activity of purified protein

• Storage considerations:

  • Addition of glycerol (typically 10-20%) to prevent freeze-thaw damage

  • Flash freezing in liquid nitrogen and storage at -80°C

  • Avoidance of repeated freeze-thaw cycles that may compromise activity

Successful examples in the literature include GST-Trim39 purification for in vitro ubiquitination assays and binding studies with SUMO chains .

What controls are critical for validating Trim39-mediated ubiquitination experiments?

Robust validation of Trim39-mediated ubiquitination requires comprehensive controls:

• Negative controls:

  • Catalytically inactive Trim39 (e.g., Trim39-ΔRING mutant)

  • Omission of key components (E1, E2, ATP, or ubiquitin) in in vitro reactions

  • Non-substrate proteins to demonstrate specificity

  • Reactions with unrelated E3 ligases to exclude non-specific effects

• Positive controls:

  • Known Trim39 substrates (e.g., NFATc3, p53) when establishing new experimental systems

  • Autoubiquitination of wild-type Trim39 to confirm catalytic activity

  • Inclusion of proteasome inhibitors (e.g., MG-132) to stabilize ubiquitinated species

• Specificity controls:

  • Multiple independent shRNAs targeting Trim39 to rule out off-target effects

  • Rescue experiments with shRNA-resistant Trim39 constructs

  • Ubiquitin mutants (e.g., K48R, K63R) to determine ubiquitin chain topology

• Additional validation approaches:

  • Protein stability assays (cycloheximide chase)

  • Proteasomal degradation confirmations (with/without proteasome inhibitors)

  • Mass spectrometry to identify ubiquitination sites

Research has shown that silencing Trim39 using three different specific shRNAs strongly decreased NFATc3 ubiquitination, providing strong evidence for the specificity of this E3 ligase-substrate relationship .

What challenges exist in studying Trim39 SUMOylation and SUMO-dependent interactions?

Investigating SUMO-dependent interactions of Trim39 presents several technical challenges:

Research has demonstrated that mutation of SIM3 (211-LLSRL-215) in Trim39 strongly reduces its SUMO-binding ability and subsequent interaction with NFATc3, highlighting the importance of validating SIM functionality in cellular contexts .

How might Trim39 function differ across neuronal subtypes and developmental stages?

Understanding Trim39's role across different neuronal contexts represents an important frontier:

• Cell type-specific expression analysis:

  • Single-cell RNA sequencing to profile Trim39 expression across neuronal subtypes

  • Immunohistochemistry with cell type-specific markers to localize Trim39 protein

  • Analysis of regulatory elements controlling Trim39 expression in different neuronal populations

• Developmental profiling:

  • Temporal expression patterns during neurodevelopment

  • Investigation of Trim39's role in neuronal differentiation versus mature neuron function

  • Conditional knockout models to distinguish developmental versus maintenance roles

• Context-dependent regulation of apoptosis:

  • Comparison of Trim39 effects in:
    a) Developing neurons undergoing programmed cell death
    b) Mature neurons responding to stress or injury
    c) Neurodegenerative disease models

• Differential substrate targeting:

  • Investigation of whether Trim39 targets different substrates in different neuronal subtypes

  • Analysis of NFATc3-dependent and -independent functions

  • Co-expression patterns of Trim39 with potential interactors like Trim17

Research has established that Trim39 modulates neuronal apoptosis through regulation of NFATc3, but its function may vary across neuronal populations and developmental contexts in ways that require further investigation .

What role might Trim39 play in neurological disorders and potential therapeutic approaches?

Exploring Trim39's involvement in neurological conditions offers promising research directions:

• Expression and function in disease models:

  • Analysis of Trim39 levels in neurodegenerative disease tissues/models

  • Investigation of SNPs or mutations in Trim39 associated with neurological disorders

  • Manipulation of Trim39 levels in disease models to assess potential protective or detrimental effects

• Pathway-focused approaches:

  • Examination of the NFATc3-Trim39 axis in contexts of neuronal stress or degeneration

  • Investigation of APC/C dysregulation in neurodegenerative conditions

  • Analysis of p53 regulation by Trim39 in the context of neuronal DNA damage responses

• Small molecule modulator development:

  • High-throughput screening for inhibitors or enhancers of Trim39's E3 ligase activity

  • Development of compounds that specifically disrupt Trim39-substrate interactions

  • Design of molecules targeting the SUMO-dependency of Trim39's activity

• Gene therapy approaches:

  • Viral vector-mediated delivery of Trim39 or dominant-negative variants

  • CRISPR-based strategies to modify Trim39 expression or function

  • Cell-specific targeting to modulate Trim39 activity in relevant neuronal populations