Recombinant Bovine Tripartite motif-containing protein 2 (TRIM2), partial

Basic Research Questions

• What is the structural composition of bovine TRIM2 and how does it differ from human TRIM2?

Bovine TRIM2, like its human counterpart, belongs to the tripartite motif-containing protein family characterized by an N-terminal tripartite motif (also called RBCC) consisting of:

• A RING finger domain with E3 ubiquitin ligase activity

• One or two B-box zinc-binding domains (typically B1 and B2, though some TRIMs only contain B2)

• A coiled-coil domain (CCD) responsible for protein self-association

Human and bovine TRIM2 share approximately 56% sequence identity, with the SPRY domain being a major determinant of species-specific functions . The RING finger domain is crucial for ubiquitination activity, as demonstrated by studies showing that TRIM2 lacking this domain cannot catalyze autoubiquitination .

• What are the established functions of TRIM2 in bovine cellular physiology?

Bovine TRIM2 functions primarily as a UBE2D1-dependent E3 ubiquitin-protein ligase that mediates the ubiquitination of specific substrates. Key physiological functions include:

• Regulation of neurofilament light chain (NF-L) degradation via the ubiquitin-proteasome pathway

• Neuroprotection against ischemic events through ubiquitination-dependent degradation of pro-apoptotic proteins like Bim

• Potential role in angiogenesis regulation, particularly inflammation-driven pathological angiogenesis

• Antiviral immunity functions, including restricting arenavirus infection independent of its ubiquitin ligase activity

Studies of TRIM2-deficient models show increased NF-L levels in axons and NF-L-filled axonal swellings in various tissues, confirming its crucial role in cytoskeletal protein homeostasis .

• What expression systems are most effective for producing recombinant bovine TRIM2?

For recombinant bovine TRIM2 production, several expression systems have been documented with varying efficacy:

For functional studies of bovine TRIM2, mammalian expression systems are recommended, as they maintain the protein's native E3 ligase activity. When using E. coli systems, refolding protocols using redox couples (e.g., oxidized and reduced glutathione) may be necessary to obtain properly folded protein .

• How can researchers verify the activity of recombinant bovine TRIM2?

Verification of recombinant bovine TRIM2 activity can be performed through multiple complementary approaches:

• Auto-ubiquitination assays: Measuring self-ubiquitination activity using fluorescently labeled ubiquitin (e.g., Ub ATTO). Active TRIM2 constructs show strong auto-ubiquitination bands on SDS-PAGE, with full-length and RBCC constructs demonstrating higher activity than isolated RING domains .

• Lysine-discharge assays: Monitoring the transfer of ubiquitin from pre-loaded E2~Ub conjugates to free lysine. This approach provides a more direct measure of catalytic activity independent of acceptor lysine availability .

• Substrate ubiquitination assays: Measuring ubiquitination of known substrates like NF-L. Knockdown and overexpression studies confirm that TRIM2 regulates NF-L levels through ubiquitination .

• E2 enzyme specificity testing: Assessing activity with different E2 conjugating enzymes. Research shows that bovine TRIM2 preferentially functions with UBE2D1, though it may have activity with other E2s .

Advanced Research Questions

• What experimental approaches can elucidate the role of TRIM2 in neurodegenerative disease progression?

To investigate TRIM2's role in neurodegenerative diseases, several sophisticated experimental approaches have proven effective:

• CRISPR/Cas9-mediated knockout models: Generation of TRIM2-deficient cell lines or mouse models allows for assessment of phenotypes relevant to neurodegeneration. TRIM2 knockout mice show gradual neurodegeneration and axon swelling due to NF-L accumulation .

• siRNA knockdown and lentiviral overexpression in primary neurons: These complementary approaches have demonstrated that TRIM2 knockdown increases NF-L levels and α-synuclein aggregation, while TRIM2 overexpression reduces both, suggesting a neuroprotective role .

• Pre-formed fibril (PFF) models of α-synuclein pathology: Treatment of primary neurons with α-synuclein PFFs induces pathological protein aggregation. TRIM2 manipulation in these models has revealed that TRIM2 knockdown increases pS129+ α-synuclein aggregation, while overexpression reduces it .

• Co-localization studies: Immunostaining for TRIM2, NF-L, and disease-associated proteins like α-synuclein can assess their spatial relationships in disease models. Approximately 20% of pS129+ α-synuclein aggregates also contain NF-L, with TRIM2 knockdown increasing this co-localization .

Research has shown a strong correlation (R²=0.9191, P=0.0007) between NF-L and pS129+ α-synuclein accumulation in PFF-treated neurons, suggesting a mechanistic link that may be therapeutically exploitable .

• How does TRIM2 influence inflammation-driven pathological angiogenesis and how can this be experimentally assessed?

TRIM2 selectively regulates inflammation-driven pathological angiogenesis without affecting physiological hypoxia-mediated angiogenesis, as demonstrated by several experimental approaches:

• Periarterial collar model: In CRISPR/Cas9-generated Trim2-/- mice, this model revealed significantly reduced adventitial macrophage infiltration (33.9 ± 10.2% vs. WT: 100.0 ± 27.4%, p < 0.05) and fewer Ki67+CD31+ proliferating neovessels (40.5 ± 10.5% vs. WT: 100.0 ± 16.4%, p < 0.01) .

• Hindlimb ischemia model: Surprisingly, Trim2-/- mice showed no differences in blood flow reperfusion to ischemic hindlimbs compared to wild-type littermates, suggesting a selective role in inflammation-driven rather than hypoxia-driven angiogenesis .

• In vitro angiogenic signaling analysis: TRIM2 knockdown in human coronary artery endothelial cells attenuated TNFα-induced nuclear HIF-1α protein levels (32.9 ± 8.3% vs. control: 100.0 ± 25.4%, p < 0.05) and reduced eNOS phosphorylation (63.5 ± 9.3% vs. control: 100.0 ± 8.5%, p < 0.05) .

This selective influence suggests TRIM2 could be a therapeutic target for diseases driven by pathological angiogenesis without affecting beneficial physiological angiogenesis .

• What genetic approaches can be used to study TRIM2 variants associated with disease progression?

Several genetic approaches have proven valuable for studying TRIM2 variants and their association with disease:

• GWAS and eQTL analysis: Studies have identified TRIM2 SNPs associated with Parkinson's disease progression. eQTL analysis across 13 brain regions can help determine how these variants affect TRIM2 expression in disease-relevant tissues .

• Colocalization analyses: Techniques that identify variants affecting both disease progression and gene expression. This approach has been used with PPMI genotype and baseline gene expression data to study TRIM2 variants in Parkinson's disease .

• CRISPR/Cas9-mediated gene editing: For introducing specific TRIM2 mutations in cell or animal models. This approach has been used to generate TRIM2-null mice and to study the D667A mutation associated with Charcot-Marie-Tooth disease .

• Chimeric protein construction: Creation of chimeric molecules between bovine TRIM2 and human TRIM5α has helped identify the SPRY domain as the major determinant of species-specific activity .

• What are the optimal experimental conditions for studying TRIM2's E3 ubiquitin ligase activity in vitro?

For robust assessment of TRIM2's E3 ubiquitin ligase activity, researchers should consider the following optimized conditions:

Research has demonstrated that TRIM2 RING domains bound to UBE2D1~Ub conjugates exhibit strong catalytic activity, with all TRIM2 constructs able to discharge ubiquitin within minutes, though activity is enhanced for longer RBCC and full-length constructs .

• How can researchers differentiate between TRIM2 and its paralog TRIM3, and what are their functional distinctions?

Despite high sequence identity, TRIM2 and TRIM3 exhibit distinct self-association properties and catalytic activities that can be differentiated through specific experimental approaches:

• Biochemical activity assays: In auto-ubiquitination assays, TRIM2 RB2, RBCC, and full-length constructs demonstrate strong activity, while only full-length TRIM3 shows minimal activity. In lysine-discharge assays, all TRIM2 constructs discharge ubiquitin within minutes, while TRIM3 RING and RB2 constructs are inactive .

• Structural analysis: Crystal structure studies of the dimeric RING domain of TRIM2 bound to UBE2D1~Ub conjugate reveal molecular details of interaction. Despite high sequence similarity, TRIM2 and TRIM3 RING domains exhibit different self-association properties that impact catalytic activity .

• Activity restoration experiments: TRIM3 catalytic activity can be partially restored in full-length proteins and completely recovered upon enforced homodimerization of its RING domain or heterodimerization with TRIM2, highlighting structural differences affecting function .

• Substrate specificity: While both proteins bind Myosin V via their NHL domains, they exhibit different substrate preferences for ubiquitination, offering another means of differentiation .

• What are the methodological considerations for using recombinant bovine TRIM2 in studies of protein-protein interactions?

When investigating protein-protein interactions involving recombinant bovine TRIM2, researchers should consider:

• Domain-specific interactions: Different domains of TRIM2 mediate distinct interactions. The NHL domain interacts with Myosin V, while the RING domain interacts with E2 enzymes like UBE2D1 .

• Expression system selection: For interaction studies, mammalian expression systems provide proteins with native conformation and post-translational modifications. E. coli-expressed proteins may require refolding to achieve proper structure .

• Dimerization status: TRIM2 self-associates through its coiled-coil region, forming larger protein complexes that affect interaction dynamics. Ensure experimental conditions maintain appropriate oligomerization state .

• Co-immunoprecipitation controls: When studying TRIM interactions, be aware that IFN-β treatment strongly upregulates several TRIMs that may co-immunoprecipitate. TRIM21 can function as an intracellular antibody receptor that binds antibodies used for immunoprecipitation .

• Control proteins for specificity: Using paralogous proteins like TRIM3 as controls can help establish interaction specificity, as demonstrated in studies comparing TRIM2 and TRIM3 interactions with UBE2D1 .