Recombinant Pan troglodytes Tripartite motif-containing protein 26 (TRIM26), partial

What is the domain structure of Pan troglodytes TRIM26, and how does it compare to human TRIM26?

Pan troglodytes TRIM26 shares remarkable structural similarity with human TRIM26, exhibiting over 92% homology at the amino acid level. Like human TRIM26, it contains the characteristic RBCC motif: a RING domain, one B-Box domain, and a coiled-coil (CC) domain, followed by a C-terminal PRY/SPRY domain. The PRY/SPRY domain demonstrates higher conservation compared to the RING and B-Box domains across species. Structural analysis using PyMOL visualization reveals that the three-dimensional structures of Pan troglodytes and Homo sapiens TRIM26 are highly comparable, suggesting functional similarities. This conservation pattern indicates evolutionary pressure to maintain TRIM26 function across primate species .

How conserved is TRIM26 across species, and what does this suggest about its evolutionary importance?

TRIM26 demonstrates remarkable conservation across diverse species. Comprehensive comparative analysis using Molecular Evolutionary Genetic Analysis (MEGA) reveals that porcine TRIM26 shares over 90% homology with 45 different species and exceeds 80% homology with 23 additional species. Specifically, Pan troglodytes TRIM26 shows 92.39% homology with human TRIM26. This high degree of conservation, particularly in the PRY/SPRY domain, strongly suggests that TRIM26 serves fundamental biological functions that have been preserved throughout evolution. The conservation pattern also indicates that research findings from one species may have translational relevance to others, making Pan troglodytes TRIM26 a valuable research model for understanding human TRIM26 function .

What are the recommended protocols for producing recombinant Pan troglodytes TRIM26?

Production of recombinant Pan troglodytes TRIM26 typically follows standard molecular cloning and protein expression procedures with key modifications for optimal yield and function. The recommended protocol involves:

• Gene synthesis or PCR amplification of the Pan troglodytes TRIM26 coding sequence (consider codon optimization for expression system)

• Cloning into an expression vector with an appropriate tag (His-tag is commonly used)

• Transformation into a bacterial expression system (E. coli BL21(DE3) strain is preferred)

• Induction with IPTG at 18°C for 16-18 hours to minimize inclusion body formation

• Purification using nickel affinity chromatography followed by size exclusion chromatography

For optimal functionality in ubiquitination assays, the recombinant protein should be tested for E3 ligase activity using in vitro ubiquitination assays with control substrates before experimental use .

What in vitro assays can effectively demonstrate the E3 ubiquitin ligase activity of recombinant TRIM26?

To evaluate the E3 ubiquitin ligase activity of recombinant TRIM26, several complementary in vitro assays are recommended:

• Ubiquitination Assay: Combine purified recombinant His-tagged TRIM26 with E1 enzyme, E2 conjugating enzyme (typically UbcH5a/UBE2D1), ubiquitin, ATP, and potential substrate proteins (such as NEIL1, NTH1, NEIL3, or OGG1). Analyze ubiquitination by western blotting.

• Auto-ubiquitination Assay: Similar to above but without substrate proteins to assess TRIM26 self-ubiquitination capacity.

• Binding Affinity Assessment: Use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding between TRIM26 and potential substrate proteins.

These assays should include appropriate controls such as RING domain mutants that abolish E3 ligase activity to confirm specificity. The successful demonstration of ubiquitination activity confirms proper folding and functional activity of the recombinant protein .

How does TRIM26 function in different viral infections, and why are there seemingly contradictory effects?

TRIM26 demonstrates context-dependent roles in viral infections, functioning either as a protective or detrimental host factor depending on the specific virus. This duality is evident in the following examples:

These contradictory effects likely stem from:

• Different experimental conditions and in vitro models

• Virus-specific interactions with different signaling pathways

• Cell-type specific responses and protein expression levels

To resolve these contradictions, more comprehensive in vivo studies using gene-edited animal models are necessary to understand the physiological role of TRIM26 during natural infection courses .

What structural features of TRIM26 are critical for its interaction with viral proteins?

The interaction between TRIM26 and viral proteins primarily involves domain-specific binding that determines functional outcomes. Key structural features include:

• PRY/SPRY Domain: The most conserved region across species, this domain is often responsible for substrate recognition and binding specificity. For viral interactions, this domain likely mediates direct binding to viral proteins.

• RING Domain: Essential for E3 ubiquitin ligase activity, this domain recruits E2 enzymes and facilitates ubiquitin transfer to target viral proteins, potentially marking them for degradation.

• B-Box Domain: May provide additional substrate recognition surfaces and regulate ligase activity.

The structural basis of these interactions can be investigated through techniques such as co-immunoprecipitation followed by mass spectrometry, yeast two-hybrid screening, and structural studies using X-ray crystallography or cryo-EM. For Pan troglodytes TRIM26, the high homology with human TRIM26 suggests conserved interaction patterns with viral proteins, making it a valuable comparative model .

How does TRIM26 regulate base excision repair through DNA glycosylase targeting?

TRIM26 functions as a critical regulator of base excision repair (BER) by controlling the cellular levels of multiple DNA glycosylases through ubiquitylation-dependent degradation. The regulatory mechanism involves:

• Target Recognition: TRIM26 specifically recognizes DNA glycosylases including NEIL1, NTH1, NEIL3, and OGG1, which are key enzymes in removing oxidative DNA base damage.

• Ubiquitylation: As an E3 ubiquitin ligase, TRIM26 catalyzes the addition of ubiquitin molecules to these glycosylases, marking them for proteasomal degradation.

• Homeostatic Control: This mechanism maintains appropriate levels of glycosylases, as excessive amounts can lead to aberrant DNA processing.

Experimental evidence from in vitro ubiquitylation assays confirms that purified recombinant His-tagged TRIM26 can directly ubiquitinate multiple BER enzymes. Furthermore, knockdown studies in U2OS cells demonstrate that TRIM26 depletion increases resistance to ionizing radiation and oxidative stress due to the accumulation of DNA glycosylases, which accelerates DNA repair as measured by alkaline comet assays .

What experimental approaches are most effective for studying TRIM26-mediated regulation of DNA repair in Pan troglodytes cells?

To effectively study TRIM26-mediated regulation of DNA repair in Pan troglodytes cells, a multi-faceted experimental approach is recommended:

• CRISPR/Cas9 Gene Editing: Generate TRIM26-knockout or domain-specific mutant Pan troglodytes cell lines to evaluate functional consequences.

• Protein Level Analysis: Use western blotting and immunoprecipitation to monitor changes in DNA glycosylase levels (NEIL1, NTH1, NEIL3, OGG1) in response to DNA damage in wild-type versus TRIM26-modified cells.

• DNA Damage and Repair Assays:

  • Alkaline comet assay to measure DNA strand break repair kinetics

  • Immunofluorescence for γH2AX foci formation and resolution

  • BER capacity assays using synthesized DNA substrates containing specific lesions

• Functional Recovery Experiments: Perform rescue experiments by reintroducing wild-type or mutant TRIM26 into knockout cells to identify critical domains.

• Comparative Analysis: Conduct parallel experiments in human cells to identify any species-specific differences in TRIM26 function.

These approaches should be combined with relevant DNA damaging agents, particularly ionizing radiation and hydrogen peroxide, which have been shown to trigger TRIM26-dependent responses in human cells .

What are the key structural and functional differences between human and Pan troglodytes TRIM26?

Despite the high homology (92.39%) between human and Pan troglodytes TRIM26, subtle differences exist that may influence function:

Structural Comparison:
Both proteins share the characteristic RBCC motif and PRY/SPRY domain, with the latter showing higher conservation. Predicted three-dimensional structures using PyMOL visualization demonstrate remarkable similarity, suggesting preservation of general function.

Functional Implications:

• Protein-Protein Interactions: Minor amino acid differences may slightly modify the binding affinity to substrates or interacting partners.

• Enzymatic Activity: The high conservation of the RING domain suggests comparable E3 ligase activity, though subtle differences in substrate specificity may exist.

• Cellular Localization: Both proteins are found in cytoplasmic and nuclear bodies, indicating conserved localization patterns.

Evolutionary Significance:
The high degree of conservation between human and chimpanzee TRIM26 indicates strong evolutionary pressure to maintain function, suggesting shared biological roles in immune response and DNA repair mechanisms. This makes Pan troglodytes an excellent model for investigating TRIM26 functions relevant to human biology and disease .

How do experimental findings from Pan troglodytes TRIM26 studies translate to human applications?

Translating experimental findings from Pan troglodytes TRIM26 studies to human applications is facilitated by several factors:

• High Sequence Homology: The 92.39% amino acid sequence identity between human and chimpanzee TRIM26 suggests strong functional conservation, making findings potentially directly applicable to human contexts.

• Conserved Domain Structure: The preservation of critical domains (RING, B-Box, coiled-coil, and PRY/SPRY) indicates that mechanistic insights regarding domain function are likely transferable.

• Similar Protein Interactions: Due to structural conservation, Pan troglodytes TRIM26 likely interacts with the same partner proteins as human TRIM26, including DNA glycosylases and viral proteins.

• Translational Considerations:

  • Findings related to basic mechanisms of E3 ligase activity are highly transferable

  • Insights into DNA repair regulation by TRIM26 likely apply across species

  • Virus-host interactions may show species-specific differences requiring validation

For optimal translation, key findings from Pan troglodytes studies should be validated in human cell lines or tissues. Comparative studies examining both species simultaneously can identify critical differences that might affect therapeutic development targeting TRIM26 .

How can researchers resolve contradictory findings regarding TRIM26's role in antiviral responses?

Resolving contradictory findings regarding TRIM26's role in antiviral responses requires a systematic approach addressing several key aspects:

• Standardized Experimental Systems:

  • Establish consistent cell lines and primary cells for cross-study comparison

  • Develop standardized viral strains and infection protocols

  • Create uniform quantification methods for viral replication and immune responses

• Comprehensive Domain-Function Analysis:

  • Generate domain-specific mutants of TRIM26 to delineate the role of each domain

  • Use chimeric proteins to identify regions responsible for virus-specific effects

• Context-Dependent Investigations:

  • Examine TRIM26 function across different cell types relevant to specific viral infections

  • Analyze temporal dynamics of TRIM26 activity throughout the course of infection

  • Consider concentration-dependent effects of TRIM26 expression

• In Vivo Validation:

  • Develop TRIM26-deficient or domain-specific mutant animal models

  • Study natural infections in these models to understand physiological relevance

• Interaction Networks:

  • Map the complete interactome of TRIM26 under different viral infection conditions

  • Identify common and distinct interaction partners that might explain contradictory effects

These approaches should help reconcile why TRIM26 appears to facilitate replication of certain viruses (HSV-2, PRV) while potentially inhibiting others, and why contradictory findings exist even for the same virus (e.g., PRRSV) .

What are the most promising emerging techniques for studying TRIM26 post-translational modifications and their impact on function?

Several cutting-edge techniques show particular promise for elucidating TRIM26 post-translational modifications and their functional implications:

• Proximity Labeling Proteomics:

  • BioID or TurboID fusion with TRIM26 to identify proximal interacting proteins

  • APEX2-based labeling to map the TRIM26 interaction network in different cellular compartments

• Mass Spectrometry-Based Approaches:

  • Targeted parallel reaction monitoring (PRM) to quantify specific TRIM26 modifications

  • Crosslinking mass spectrometry (XL-MS) to map structural changes induced by modifications

  • Top-down proteomics to analyze intact TRIM26 and its modification patterns

• Advanced Imaging Techniques:

  • STORM/PALM super-resolution microscopy to visualize TRIM26 subcellular localization

  • FRET-based sensors to detect TRIM26 conformational changes upon modification

  • Live-cell imaging with modification-specific biosensors

• CRISPR-Based Screening:

  • CRISPR activation/inhibition screens to identify regulators of TRIM26 modification

  • Base editing to generate modification-resistant TRIM26 variants

• Structural Biology Integration:

  • Cryo-EM analysis of TRIM26 complexes with and without modifications

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to detect structural changes upon modification

These techniques, particularly when applied to both human and Pan troglodytes TRIM26, can provide insights into how post-translational modifications like phosphorylation, SUMOylation, and auto-ubiquitination regulate TRIM26's E3 ligase activity, substrate specificity, and cellular localization .

What are common challenges in measuring TRIM26 E3 ligase activity and how can they be overcome?

Several challenges commonly arise when measuring TRIM26 E3 ligase activity, each requiring specific technical solutions:

• Protein Stability Issues:

  • Challenge: Recombinant TRIM26 may aggregate or lose activity during purification or storage.

  • Solution: Express protein at lower temperatures (16-18°C), include stabilizing agents (10% glycerol, 1mM DTT), and store in small aliquots at -80°C. Consider fusion tags that enhance solubility (MBP, SUMO).

• E2 Enzyme Selection:

  • Challenge: TRIM26 may work preferentially with specific E2 conjugating enzymes.

  • Solution: Screen multiple E2 enzymes (UBE2D1-4, UBE2E1-3) in parallel reactions to identify optimal partners for Pan troglodytes TRIM26.

• Substrate Specificity:

  • Challenge: Identifying true physiological substrates versus non-specific in vitro ubiquitination.

  • Solution: Include negative control proteins, perform competition assays, and validate with mutant TRIM26 lacking E3 ligase activity.

• Detection Sensitivity:

  • Challenge: Low signal-to-noise ratio in ubiquitination assays.

  • Solution: Use fluorescently labeled ubiquitin or anti-ubiquitin antibodies that recognize specific linkages (K48, K63), and employ more sensitive detection methods like LI-COR imaging.

• Reproducibility Issues:

  • Challenge: Variability between experimental replicates.

  • Solution: Standardize protein batches, reaction conditions, and develop positive control reactions that can normalize between experiments .

How can researchers effectively differentiate between direct and indirect effects of TRIM26 on DNA repair pathways?

Differentiating between direct and indirect effects of TRIM26 on DNA repair pathways requires a combination of complementary approaches:

• In Vitro Reconstitution:

  • Develop fully reconstituted in vitro systems using purified components (TRIM26, E1/E2 enzymes, ubiquitin, and DNA repair proteins)

  • Demonstrate direct ubiquitination of DNA repair proteins by TRIM26

  • Use recombinant domains to map interaction surfaces

• Rapid Temporal Analysis:

  • Employ rapid protein depletion systems (e.g., auxin-inducible degron) to observe immediate effects of TRIM26 loss

  • Use real-time assays to track DNA repair protein stability upon TRIM26 manipulation

  • Distinguish acute versus chronic adaptations to TRIM26 depletion

• Proximity-Based Methods:

  • Apply proximity ligation assays (PLA) to detect direct TRIM26-substrate interactions in situ

  • Use FRET/BRET approaches to monitor real-time interaction dynamics

• Substrate Mutational Analysis:

  • Identify and mutate TRIM26 ubiquitination sites on DNA glycosylases

  • Generate ubiquitination-resistant mutants of DNA repair proteins

  • Test if these mutants are refractory to TRIM26-mediated regulation

• Comparative Pathway Analysis:

  • Perform phospho-proteomics and interaction proteomics after TRIM26 depletion

  • Use systematic correlation analysis to distinguish primary from secondary effects

  • Employ network analysis to identify direct TRIM26 interactors versus downstream pathway components

This multifaceted approach has successfully demonstrated that TRIM26 directly ubiquitinates DNA glycosylases like NEIL1, NTH1, NEIL3, and OGG1, leading to their degradation and subsequent effects on DNA repair efficiency .

What are the most critical unanswered questions regarding Pan troglodytes TRIM26 function?

Several critical questions about Pan troglodytes TRIM26 remain unanswered, representing important areas for future research:

• Species-Specific Functional Differences:

  • Despite high sequence homology, do subtle functional differences exist between human and Pan troglodytes TRIM26?

  • Are there differences in substrate specificity or regulatory mechanisms?

• Structural Determinants of Function:

  • What is the complete three-dimensional structure of Pan troglodytes TRIM26?

  • How do conformational changes regulate its E3 ligase activity?

• Tissue-Specific Roles:

  • Does TRIM26 function differently across various chimpanzee tissues?

  • Are there tissue-specific interaction partners that modify its function?

• Physiological Regulation:

  • What are the upstream regulators of TRIM26 expression and activity?

  • How is TRIM26 itself regulated by post-translational modifications?

• Evolutionary Adaptations:

  • What selective pressures have maintained TRIM26 conservation?

  • Are there specific viral challenges that have shaped TRIM26 evolution in Pan troglodytes?

• Comprehensive Substrate Landscape:

  • What is the complete set of proteins targeted by TRIM26 for ubiquitination?

  • How does substrate specificity change under different cellular conditions?

Addressing these questions will require integrated approaches combining structural biology, comparative genomics, and functional studies in relevant cellular and animal models .

How might TRIM26 research in Pan troglodytes inform our understanding of human disease mechanisms?

Research on TRIM26 in Pan troglodytes has significant translational potential for understanding human disease mechanisms:

• Viral Pathogenesis:

  • Comparative studies can reveal how TRIM26 modulates susceptibility to viruses that affect both species

  • May explain differential susceptibility to certain viral infections between humans and chimpanzees

  • Could identify critical virus-host interactions as therapeutic targets

• DNA Damage and Cancer Biology:

  • Given TRIM26's role in regulating DNA repair enzymes, comparative studies may reveal mechanisms underlying species differences in cancer susceptibility

  • May identify novel targets for enhancing DNA repair in human cells

  • Could explain variations in response to DNA-damaging agents between species

• Inflammatory Disorders:

  • TRIM26's involvement in immune signaling may contribute to understanding inflammatory disease mechanisms

  • Comparative analysis could identify species-specific regulatory networks affecting inflammatory responses

• Neurological Conditions:

  • If TRIM26 functions in neuronal maintenance through DNA repair regulation, comparative studies could inform understanding of neurodegenerative diseases

  • May explain species differences in susceptibility to age-related neurodegeneration

• Therapeutic Development:

  • Identification of conserved functional domains across species can guide development of targeted therapeutics

  • Understanding species-specific differences can help predict limitations in translating therapies from animal models

The high homology between human and Pan troglodytes TRIM26 (92.39%) provides a strong foundation for translational research, while careful attention to species-specific differences can reveal unique aspects of human disease mechanisms .