Recombinant Xenopus laevis F-box-like/WD repeat-containing protein TBL1XR1-B (tbl1xr1-b), partial

What is the molecular structure of Xenopus laevis TBL1XR1-B and how does it compare to mammalian orthologs?

The Xenopus laevis TBL1XR1-B is a 522 amino acid protein with a molecular mass of approximately 56.3 kDa. It contains a distinctive domain architecture featuring:

• An N-terminal LisH (Lis1 homology) domain

• An F-box-like domain adjacent to the LisH domain

• Seven WD40 repeats at the C-terminus that form a β-propeller structure

This structure is evolutionarily conserved across species, with the WD40 repeats being particularly important for mediating protein-protein interactions. The WD40 domain in TBL1XR1 forms a barrel-like structure with aromatic residues exposed on the surface that are critical for its function .

TBL1XR1 belongs to the larger WD repeat EBI family of proteins. The high degree of conservation between Xenopus and human TBL1XR1 suggests fundamental functional importance across vertebrate species .

What are the primary biochemical functions of TBL1XR1-B in Xenopus developmental systems?

TBL1XR1-B functions as an integral component of the N-CoR (Nuclear receptor Corepressor) transcriptional repressor complex in Xenopus laevis. Its primary functions include:

• Regulation of transcriptional repression mediated by unliganded nuclear receptors

• Facilitation of the exchange between corepressors and coactivators during transcriptional regulation

• Recruitment of the 19S proteasome complex that mediates the degradation of the N-CoR complex

• Participation in multiple signaling pathways essential for amphibian development

In Xenopus specifically, TBL1XR1 plays a role in thyroid hormone-regulated processes during metamorphosis, acting as a transcriptional bridge between hormone signals and developmental responses .

How can researchers design functional assays to evaluate the transcriptional regulatory activities of recombinant TBL1XR1-B?

To evaluate the transcriptional regulatory functions of recombinant TBL1XR1-B, researchers should implement these methodological approaches:

• Co-repressor Complex Assembly Assays:

  • Co-immunoprecipitation experiments to assess interactions with N-CoR/SMRT complex components

  • Size exclusion chromatography to verify complex formation

  • FRET or BiFC assays to visualize protein-protein interactions in live cells

• Transcriptional Repression Assays:

  • Luciferase reporter assays using promoters known to be regulated by nuclear receptors

  • ChIP assays to detect TBL1XR1-B recruitment to specific genomic loci

  • RNA-seq to identify genes differentially regulated following TBL1XR1-B overexpression or depletion

• Proteasome Recruitment Analysis:

  • Ubiquitination assays to assess the ability of TBL1XR1-B to promote substrate ubiquitination

  • Proteasome activity assays in the presence/absence of TBL1XR1-B

  • Co-localization studies with proteasome components using fluorescence microscopy

• Nuclear Receptor Interaction Studies:

  • GST pull-down assays with various nuclear receptors

  • Mammalian two-hybrid assays to study TBL1XR1-nuclear receptor interactions

  • Competitive binding assays to assess coregulator exchange dynamics

These assays should be calibrated against known TBL1XR1 activities in other species as benchmarks for functional conservation .

How do mutations in TBL1XR1 affect germinal center development in mammalian systems, and what implications might this have for studying amphibian immune development?

Mutations in mammalian TBL1XR1 have significant effects on germinal center (GC) development with potential implications for comparative amphibian studies:

• Observed Mammalian Phenotypes:

  • TBL1XR1 mutations impair rather than enhance GC reactions

  • Mutations lead to decreased GC B-cell abundance and premature GC resolution

  • Reduced proliferation of GC B-cells without affecting apoptosis rates

  • TBL1XR1 mutations skew humoral immune response toward generating abnormal immature memory B-cells while impairing plasma cell differentiation

• Molecular Mechanisms:

  • TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the memory B-cell transcription factor BACH2

  • This occurs at the expense of interactions with germinal center transcription factor BCL6

  • Results in pre-memory transcriptional reprogramming and cell-fate bias

  • Mutant memory B-cells fail to differentiate into plasma cells upon antigen recall

• Mutation Characteristics:

  • TBL1XR1 mutations in lymphoma occur as heterozygous missense alleles (5-10% of DLBCL cases)

  • Mutations primarily affect the WD40 domain, specifically aromatic residues exposed on the barrel structure

  • Evidence suggests mutations function as dominant-negative loss-of-function alleles

• Implications for Amphibian Research:

  • Amphibian TBL1XR1 likely plays conserved roles in immune cell development

  • Xenopus models could be valuable for studying evolutionary conservation of TBL1XR1 functions in lymphocyte development

  • Comparing amphibian vs. mammalian TBL1XR1 could reveal fundamental versus specialized immune regulatory functions

This comparison provides a framework for investigating whether TBL1XR1 functions are conserved in amphibian immune system development and how evolutionary divergence might have shaped specialized roles .

What is known about the role of TBL1XR1 in thyroid hormone signaling during Xenopus metamorphosis compared to its role in mammalian development?

TBL1XR1 plays a crucial role in thyroid hormone signaling during Xenopus metamorphosis, with interesting parallels and differences compared to mammals:

• Xenopus-Specific Regulation:

  • In Xenopus laevis, TBL1XR1 is duplicated due to the tetraploid genome

  • Both copies are regulated by thyroid hormone during metamorphosis

  • A consensus thyroid hormone response element (TRE) lies far upstream of the transcriptional start site of both genes

  • This TRE is nested within a 200-bp region of high sequence conservation between the duplicated genes

  • The TRE acts as a strong response element in transfection assays in both heterologous promoters and native contexts

• Functional Role During Metamorphosis:

  • TBL1XR1 appears to be one of the earliest thyroid hormone-induced genes in tadpoles

  • It functions as a transcription factor predicted to play an important role in downstream gene regulation

  • This regulation leads to growth and remodeling of tissues during metamorphosis

  • The regulation mechanism involves xBTEB1, a Xenopus homolog of basic transcription element-binding protein 1

• Comparative Aspects with Mammals:

  • Both amphibian and mammalian TBL1XR1 are components of nuclear receptor co-repressor complexes

  • In both systems, TBL1XR1 modulates the repression/activation switch of nuclear receptor signaling

  • The evolutionary conservation of TRE elements suggests fundamental importance in hormone responsiveness

  • Mammalian systems lack the metamorphosis-specific developmental transitions seen in amphibians

This comparative analysis highlights the adaptation of a conserved transcriptional regulatory protein to species-specific developmental programs while maintaining core molecular functions .

How do TBL1XR1 mutations contribute to lymphomagenesis, and could Xenopus TBL1XR1-B be utilized as a model system to study these pathological mechanisms?

TBL1XR1 mutations contribute to lymphomagenesis through specific molecular mechanisms that could potentially be modeled using Xenopus TBL1XR1-B:

• Mutation Patterns in Lymphoma:

  • TBL1XR1 mutations occur in 5-10% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma cases

  • Mutations are more frequent in ABC-DLBCL and are highly enriched in the MCD subtype

  • Mutations often co-occur with MYD88 mutations

  • Mutations primarily affect the WD40 domain with a predilection for aromatic residues that mediate protein-protein interactions

• Functional Consequences Leading to Lymphomagenesis:

  • TBL1XR1 mutations drive extranodal lymphoma by disrupting normal B-cell differentiation

  • Mutant TBL1XR1 skews the humoral immune response toward generating abnormal immature memory B-cells

  • Upon antigen recall, TBL1XR1 mutant memory B-cells fail to differentiate into plasma cells

  • Instead, these cells preferentially reenter new germinal center reactions, creating a cyclic reentry lymphomagenesis mechanism

• Molecular Mechanisms:

  • TBL1XR1 mutants redirect SMRT/HDAC3 repressor complexes toward binding the memory B-cell transcription factor BACH2

  • This occurs at the expense of interactions with the germinal center transcription factor BCL6

  • The resulting pre-memory transcriptional reprogramming leads to cell-fate bias and impaired plasma cell differentiation

  • TBL1XR1 mutations appear to function as dominant-negative loss-of-function alleles

• Xenopus Model System Potential:

  • Recombinant Xenopus TBL1XR1-B could be engineered with equivalent mutations found in human lymphomas

  • The evolutionary conservation of TBL1XR1 structure suggests functional mutations would have similar effects

  • Xenopus B-cell development could serve as a simplified model system to study the fundamental aspects of TBL1XR1 function

  • Transgenic Xenopus models expressing mutant TBL1XR1-B could provide insights into early developmental effects of these mutations

A comparative approach using both mammalian and amphibian systems could provide complementary insights into the pathological mechanisms of TBL1XR1 mutations in lymphomagenesis .

What experimental strategies can be employed to investigate the interactome of Xenopus TBL1XR1-B and how it differs from mammalian systems?

Advanced experimental strategies to map the Xenopus TBL1XR1-B interactome include:

• Unbiased Interactome Analysis:

  • Affinity purification coupled with mass spectrometry (AP-MS) using tagged recombinant TBL1XR1-B

  • BioID or APEX proximity labeling in Xenopus cell systems to capture transient interactions

  • Cross-species yeast two-hybrid screening using Xenopus TBL1XR1-B as bait against cDNA libraries

• Domain-Specific Interaction Mapping:

  • Generation of domain deletion/mutation constructs (ΔLisH, ΔF-box, ΔWD40) to identify domain-specific interactors

  • Peptide array analysis to map specific binding interfaces within TBL1XR1-B

  • Competitive binding assays to identify differential binding partners between domains

• Developmental Stage-Specific Analysis:

  • Temporal interactome analysis across key Xenopus developmental stages, particularly during metamorphosis

  • Comparison with equivalent mammalian developmental transitions

  • Correlation of interactome shifts with changes in gene expression

• Comparative Interactome Analysis:

  • Direct comparison between Xenopus TBL1XR1-B and human TBL1XR1 interactomes under equivalent conditions

  • Identification of conserved versus species-specific interactors

  • Functional validation of key interactions using knockdown/rescue experiments

• Data Analysis Framework:

  • Network analysis to identify interaction clusters and functional modules

  • Enrichment analysis for biological processes and signaling pathways

  • Evolutionary conservation scoring of identified interactions

This comprehensive approach would provide insights into both conserved functions of TBL1XR1 and adaptations specific to amphibian biology, particularly in developmental contexts and nuclear receptor signaling pathways .

How can structural studies of TBL1XR1-B WD40 repeats inform the design of targeted therapeutics for TBL1XR1-associated pathologies?

Structural studies of TBL1XR1-B WD40 repeats can significantly advance therapeutic design through:

• High-Resolution Structural Analysis:

  • X-ray crystallography or cryo-EM structures of Xenopus TBL1XR1-B WD40 domain

  • Comparison with mammalian structures to identify conserved binding pockets

  • Molecular dynamics simulations to understand conformational flexibility

  • Identification of "hotspot" residues that mediate critical protein-protein interactions

• Structure-Function Relationship Assessment:

  • Analysis of disease-associated mutations mapped onto the WD40 structure

  • Characterization of how mutations in aromatic residues on the WD40 barrel surface affect protein binding

  • Comparison between wild-type and mutant structures to identify conformational changes

• Therapeutic Target Identification:

  • Mapping of druggable pockets within the WD40 domain

  • Virtual screening against identified pockets to discover potential binding molecules

  • Fragment-based drug discovery approaches focused on the protein-protein interaction surfaces

• Rational Drug Design Strategy:

  • Development of small molecules that can:

    • Disrupt pathological interactions of mutant TBL1XR1

    • Restore normal binding profiles in disease states

    • Stabilize productive conformations of the WD40 domain

  • Peptide-based inhibitors designed to mimic natural binding partners

• Translation to Human Applications:

  • Comparative analysis of binding affinities between amphibian and human proteins

  • Assessment of species-specific differences in drug binding and efficacy

  • Development of broad-spectrum versus selective therapeutic agents

This structure-based approach leverages the evolutionary conservation of TBL1XR1 to develop potential therapeutics for TBL1XR1-associated pathologies including lymphomas, intellectual disability syndromes, and potentially other cancers where TBL1XR1 is dysregulated .

What are the most common technical challenges when working with recombinant Xenopus TBL1XR1-B and how can they be addressed?

Researchers frequently encounter several technical challenges when working with recombinant Xenopus TBL1XR1-B. Here are the most common issues and evidence-based solutions:

• Protein Solubility Issues:

  • Challenge: TBL1XR1-B with its multiple WD40 repeats often forms inclusion bodies in bacterial expression systems.

  • Solution:

    • Use lower induction temperatures (16-18°C) and reduced IPTG concentrations (0.1-0.5 mM)

    • Express as fusion proteins with solubility tags (MBP, SUMO, or TrxA)

    • Consider insect cell or mammalian expression systems for improved folding

    • Include low concentrations (1-5%) of non-ionic detergents during purification

• Protein Stability Concerns:

  • Challenge: The multi-domain structure of TBL1XR1-B can lead to degradation during purification.

  • Solution:

    • Include protease inhibitor cocktails throughout purification

    • Maintain samples at 4°C and minimize freeze-thaw cycles

    • Consider addition of stabilizing agents (5-10% glycerol, 1-5 mM DTT)

    • Screen buffer conditions using thermal shift assays to identify optimal stability conditions

• Complex Formation Difficulties:

  • Challenge: Reconstituting functional complexes with N-CoR/SMRT components.

  • Solution:

    • Co-expression of interaction partners in the same system

    • Stepwise assembly of complexes with controlled stoichiometry

    • Validation of complex formation using analytical size exclusion chromatography

    • Implementation of gradient purification techniques to isolate intact complexes

• Activity Assessment Problems:

  • Challenge: Verifying that recombinant TBL1XR1-B retains native functionality.

  • Solution:

    • Compare activity to known mammalian orthologs as benchmarks

    • Develop robust in vitro assays for specific functions (co-repressor binding, proteasome recruitment)

    • Use rescue experiments in TBL1XR1-depleted systems to verify functionality

    • Check for proper post-translational modifications that might affect function

These methodological solutions are based on general principles for working with complex multi-domain proteins containing WD40 repeats and should be optimized for specific experimental conditions and research objectives .

How can researchers distinguish between the effects of wild-type and mutant TBL1XR1-B in experimental systems?

Distinguishing between wild-type and mutant TBL1XR1-B effects requires rigorous experimental design:

• Molecular and Biochemical Differentiation:

  • Binding Affinity Analysis:

    • Quantitative interaction studies (SPR, ITC, MST) to measure differences in binding affinities to key partners

    • Competition assays between wild-type and mutant proteins for limiting interaction partners

    • Detailed kinetic analyses to identify differences in association/dissociation rates

  • Structural Analysis:

    • Comparative circular dichroism to detect secondary structure differences

    • Thermal shift assays to identify stability differences

    • Limited proteolysis to detect conformational changes between wild-type and mutant forms

• Cellular and Functional Differentiation:

  • Transcriptional Readouts:

    • RNA-seq analysis comparing global transcriptional effects of wild-type vs. mutant TBL1XR1-B

    • ChIP-seq to identify differential genomic binding sites

    • Reporter gene assays using known TBL1XR1-responsive promoters

  • Protein Complex Analysis:

    • IP-MS to identify differential interactomes between wild-type and mutant proteins

    • Size exclusion chromatography to detect differences in complex formation

    • Live-cell imaging with fluorescently tagged proteins to observe differences in localization or dynamics

• Experimental Controls and Validation:

  • Rescue Experiments:

    • Depletion of endogenous TBL1XR1 followed by complementation with either wild-type or mutant forms

    • Quantitative assessment of rescue efficiency across multiple phenotypic readouts

    • Dose-response studies to detect dominant-negative effects of mutants

  • Domain Swap/Chimeric Proteins:

    • Creation of chimeric proteins to map functionally important regions

    • Point-mutation series to identify critical residues

    • Structure-guided mutagenesis to confirm mechanism-based hypotheses

Based on lymphoma studies, researchers should particularly focus on:

• Germinal center development phenotypes

• B-cell differentiation markers

• Interactions with SMRT/HDAC3 complexes

• Binding to transcription factors BCL6 and BACH2

• Cell proliferation without effects on apoptosis

These approaches provide a framework for distinguishing the subtle but important differences between wild-type and mutant TBL1XR1-B in experimental systems .

What emerging technologies could enhance our understanding of TBL1XR1-B function in development and disease?

Several cutting-edge technologies show promise for advancing TBL1XR1-B research:

• CRISPR-Based Technologies:

  • Base and Prime Editing: For precise introduction of disease-relevant mutations in Xenopus models

  • CRISPRi/CRISPRa: For temporal and spatial control of TBL1XR1-B expression

  • CRISPR Screens: To identify synthetic lethal interactions with TBL1XR1-B mutations

• Single-Cell Omics Approaches:

  • scRNA-seq: To resolve cell-type specific effects of TBL1XR1-B modulation during development

  • scATAC-seq: To map chromatin accessibility changes governed by TBL1XR1-B activity

  • Spatial Transcriptomics: To map TBL1XR1-B activity in tissue contexts during amphibian development

  • Multi-omics Integration: To correlate TBL1XR1-B activity with epigenetic and transcriptional outcomes

• Advanced Structural Biology Methods:

  • Cryo-EM: For visualization of complete TBL1XR1-containing complexes

  • Hydrogen-Deuterium Exchange MS: To map dynamic interactions and conformational changes

  • Integrative Structural Biology: Combining multiple techniques to build comprehensive structural models

• Live Imaging Technologies:

  • Optogenetic Control: For temporal manipulation of TBL1XR1-B activity

  • Biosensors: To visualize TBL1XR1-B interactions and activity in living cells and tissues

  • Super-Resolution Microscopy: To visualize TBL1XR1-B-containing complexes at near-molecular resolution

• Computational and AI-Based Approaches:

  • Deep Learning: For prediction of TBL1XR1-B interaction networks and functional outcomes

  • Molecular Dynamics Simulations: To model the effects of mutations on protein behavior

  • Network Analysis: To position TBL1XR1-B within developmental and disease-relevant pathways

These technologies, particularly when applied to comparative studies between amphibian and mammalian systems, could provide unprecedented insights into the fundamental biology of TBL1XR1 and its role in disease processes like lymphomagenesis and developmental disorders .

How might comparative studies between amphibian and mammalian TBL1XR1 inform evolutionary aspects of nuclear receptor signaling?

Comparative studies between amphibian and mammalian TBL1XR1 offer unique insights into the evolution of nuclear receptor signaling:

• Evolutionary Conservation Analysis:

  • Sequence Conservation Patterns:

    • Identification of invariant residues across species suggesting fundamental functional importance

    • Analysis of lineage-specific adaptations in TBL1XR1 structure

    • Mapping of evolutionary rates across different protein domains to identify selection pressures

  • Functional Domain Evolution:

    • Comparative analysis of WD40 repeat structures between species

    • Assessment of LisH and F-box domain conservation and specialization

    • Identification of species-specific insertions/deletions that might confer specialized functions

• Nuclear Receptor Co-regulator Network Evolution:

  • Interactome Comparison:

    • Systematic comparison of TBL1XR1 binding partners across species

    • Identification of conserved versus species-specific interactions

    • Analysis of co-evolution between TBL1XR1 and its binding partners

  • Regulatory Mechanism Conservation:

    • Comparison of thyroid hormone response elements across species

    • Analysis of the evolution of SMRT/NCoR complexes and their regulation

    • Assessment of proteasome recruitment mechanisms across vertebrates

• Developmental Context Analysis:

  • Metamorphosis vs. Mammalian Development:

    • Comparison of TBL1XR1 functions in amphibian metamorphosis versus mammalian development

    • Analysis of how TBL1XR1 regulation has adapted to different developmental programs

    • Investigation of tissue-specific roles across species

  • Immune System Evolution:

    • Comparative analysis of TBL1XR1 functions in amphibian versus mammalian immune development

    • Study of how TBL1XR1 mechanisms in germinal center regulation evolved

    • Investigation of lineage-specific adaptations in immune function

• Hormone Response Evolution:

  • Response Element Architecture:

    • Analysis of the 200-bp conserved region containing the thyroid hormone response element

    • Comparison of response element positioning and strength across species

    • Investigation of the evolution of hormone responsiveness in TBL1XR1 regulation