Recombinant Xenopus laevis Denticleless protein homolog B (dtl-b), partial

What is the functional role of DTL-B in Xenopus laevis development?

DTL-B (also known as cdt2-b, dcaf2, or l2dtl) functions as a denticleless E3 ubiquitin protein ligase homolog that plays critical roles in DNA replication and cell cycle control. As a component of the CRL4 (Cullin-RING ligase 4) ubiquitin ligase complex, DTL-B targets specific proteins for ubiquitin-mediated degradation during S phase and after DNA damage. In Xenopus development, this protein helps regulate cell proliferation and differentiation by facilitating the proteolysis of key cell cycle regulators. The temporal regulation of DTL-B expression corresponds with developmental stages that involve rapid cell divisions, making it particularly relevant for studying embryogenesis in this model organism .

How does Xenopus laevis serve as an advantageous model for studying DTL-B?

Xenopus laevis offers several unique advantages for studying DTL-B function. As an amphibian model with extrauterine development, Xenopus embryos are readily accessible for manipulation throughout development. The ability to generate cell-free extracts from Xenopus eggs provides a powerful biochemical system for studying protein interactions and enzymatic functions in a controlled environment . Additionally, Xenopus development occurs rapidly and follows well-characterized stages, allowing researchers to examine DTL-B functions across different developmental contexts. The model also permits the use of semi-intact and isolated preparations for in vitro morphophysiological experimentation, providing insights into developmental and integrative processes . These characteristics make Xenopus particularly valuable for studying ubiquitin ligases like DTL-B that regulate critical developmental processes.

What are the genomic characteristics of DTL-B in Xenopus laevis?

The Xenopus laevis genome contains multiple gene copies for many proteins, a characteristic that extends to the DTL-B gene. Similar to other protein-coding genes in Xenopus that typically have 2-5 copies per haploid genome, DTL-B likely exists in multiple variants . This gene redundancy is partly attributed to the pseudotetraploid nature of the Xenopus laevis genome. Population polymorphism has been observed in genomic regions containing sequences for various proteins in Xenopus, suggesting potential variability in DTL-B expression across different populations . The gene structure of DTL-B includes conserved domains typical of E3 ubiquitin ligase components, particularly the WD40 repeat domains that facilitate protein-protein interactions crucial for substrate recognition.

What are the optimal methods for expressing recombinant DTL-B in Xenopus laevis systems?

For successful expression of recombinant DTL-B in Xenopus systems, researchers should consider several methodological approaches:

• Cell-free extract system: The Nucleoplasmic Extract (NPE) system derived from Xenopus eggs provides an optimal environment for expressing recombinant proteins due to its enrichment in RNA polymerase II and transcription factors . For DTL-B expression, prepare NPE as described in established protocols, using high-speed centrifugation of interphase egg extracts, which yields extracts with robust transcriptional activity.

• Plasmid design: Construct expression vectors containing the DTL-B coding sequence under a strong promoter recognized by Xenopus transcription machinery. The CMV promoter has demonstrated high activity in Xenopus systems, though using endogenous Xenopus promoters like those from the actb gene may provide more context-appropriate expression .

• DNA concentration optimization: Titrate plasmid concentrations between 10-100 ng/μl, as transcription efficiency in Xenopus extracts shows DNA concentration dependence. Higher DNA concentrations may overcome transcriptional suppression mechanisms .

• mRNA approach: Alternatively, synthesize capped mRNA encoding DTL-B using in vitro transcription systems and inject into Xenopus oocytes or early embryos for protein expression. This method circumvents potential limitations of promoter recognition in different extract systems.

What purification strategies yield the highest quality recombinant DTL-B protein?

Purification of recombinant DTL-B from Xenopus systems requires a multi-step approach to ensure high purity and functional integrity:

• Affinity tag selection: Incorporate an appropriate affinity tag (His6, FLAG, or GST) at either the N- or C-terminus of DTL-B. C-terminal tagging is often preferred to avoid interfering with the WD40 domain functionality at the protein's N-terminus.

• Extraction conditions: Since DTL-B functions within protein complexes, use extraction buffers containing 150-300 mM NaCl, 0.1-0.5% NP-40 or Triton X-100, and protease inhibitors to maintain protein integrity while disrupting protein-protein interactions.

• Column chromatography sequence:

  • Initial capture using affinity chromatography corresponding to the chosen tag

  • Intermediate purification using ion exchange chromatography (typically anion exchange at pH 7.5-8.0)

  • Final polishing step via size exclusion chromatography to isolate monomeric protein

• Quality assessment: Evaluate protein purity using SDS-PAGE (>95% purity threshold), and verify structural integrity using circular dichroism spectroscopy. Functional assessment through in vitro ubiquitination assays is essential to confirm biological activity.

• Storage considerations: Store purified DTL-B in buffer containing 10-20% glycerol at -80°C to maintain long-term stability and prevent freeze-thaw degradation.

How can researchers effectively analyze DTL-B interactions with other proteins in Xenopus systems?

Analysis of DTL-B protein interactions requires complementary approaches to identify both stable and transient binding partners:

• Co-immunoprecipitation (Co-IP): Use anti-DTL-B antibodies or antibodies against the affinity tag to pull down DTL-B complexes from Xenopus extracts. This technique is particularly effective for identifying stable interaction partners within the CRL4 ubiquitin ligase complex.

• Proximity labeling: Employ BioID or APEX2 fusion constructs with DTL-B to biotinylate proteins in close proximity, enabling identification of transient interactors that might be missed by traditional Co-IP approaches.

• Yeast two-hybrid screening: Screen Xenopus laevis cDNA libraries using DTL-B as bait to identify potential interaction partners, followed by validation in Xenopus systems.

• Mass spectrometry-based interactomics: Perform quantitative proteomics on immunoprecipitated DTL-B complexes from different developmental stages or cellular conditions to map dynamic interaction networks.

• In vitro binding assays: Utilize purified recombinant DTL-B for direct binding studies with candidate interactors, determining binding affinities and interaction domains.

According to research on related ubiquitin ligase systems, DTL-B primarily interacts with components of the CRL4 complex including Cullin4, DDB1, and RBX1, as well as various substrate proteins earmarked for ubiquitination .

How does DTL-B function differ between developmental stages in Xenopus laevis?

The function of DTL-B exhibits significant variation across Xenopus developmental stages, reflecting changing cellular requirements during embryogenesis:

• Early embryonic development: During rapid cleavage stages, DTL-B primarily regulates replication licensing factors, preventing re-replication during the abbreviated cell cycles characteristic of early development. Expression begins following the mid-blastula transition when zygotic gene expression is activated.

• Organogenesis: As embryos progress through gastrulation and neurulation, DTL-B function expands to regulate tissue-specific differentiation factors in addition to cell cycle proteins. This developmental shift corresponds to changes in substrate recognition specificity.

• Metamorphosis: During the dramatic tissue remodeling that occurs during metamorphosis, DTL-B likely participates in regulated protein degradation associated with tissue regression and remodeling, particularly in structures undergoing apoptotic removal.

These stage-specific functions can be effectively studied using stage-specific extract preparation protocols, allowing biochemical reconstitution of developmental contexts. Researchers should employ developmental staging according to Nieuwkoop and Faber's standard tables for Xenopus laevis to ensure consistent results when examining stage-dependent DTL-B functions .

What are the recommended approaches for CRISPR/Cas9-mediated modification of the DTL-B gene in Xenopus laevis?

CRISPR/Cas9 editing of DTL-B in Xenopus laevis requires special considerations due to the species' pseudotetraploid genome:

• gRNA design strategy:

  • Target conserved regions present in all DTL-B homeologs to ensure complete knockout

  • Design multiple gRNAs targeting different exons to increase editing efficiency

  • Validate gRNA specificity against the Xenopus laevis genome to minimize off-target effects

• Delivery method optimization:

  • For F0 analysis: Inject Cas9 protein (1-2 ng) complexed with gRNAs (400-500 pg each) into one-cell stage embryos

  • For germline transmission: Target the germ cell lineage through injection into vegetal pole blastomeres at 4-8 cell stages

• Screening protocol:

  • Use T7 endonuclease assays or high-resolution melt analysis for initial mutation detection

  • Confirm edits through deep sequencing to assess allele modification frequencies

  • Validate protein loss through Western blotting using DTL-B specific antibodies

• Phenotypic analysis timeline:

  • Begin morphological assessment at gastrulation (stage 10-12)

  • Conduct detailed analysis during neurulation and organogenesis (stages 14-25)

  • Perform molecular phenotyping through RNA-seq or proteomics at key developmental timepoints

Given DTL-B's likely essential role in development, complete knockout may cause early embryonic lethality. Consider creating conditional knockouts or hypomorphic alleles to circumvent this limitation.

How can researchers differentiate between DTL-B functions and those of other E3 ligase components in Xenopus systems?

Distinguishing DTL-B-specific functions from those of other E3 ligase components requires sophisticated experimental approaches:

• Substrate specificity profiling:

  • Perform comparative proteomics between wild-type and DTL-B-depleted extracts to identify accumulating substrates

  • Use ubiquitin remnant profiling (K-ε-GG antibody enrichment) to identify proteins with reduced ubiquitination in DTL-B-depleted conditions

  • Compare identified substrates with those of other E3 ligases to establish unique and overlapping targets

• Domain-specific mutations:

  • Generate point mutations in key functional domains of DTL-B to disrupt specific protein interactions while preserving others

  • Create chimeric proteins by swapping domains between DTL-B and related proteins to determine domain-specific contributions to substrate recognition

• Temporal inhibition strategies:

  • Employ auxin-inducible degron (AID) technology for rapid, reversible DTL-B depletion at specific developmental timepoints

  • Use small molecule inhibitors specific to different E3 ligase components for comparative phenotypic analysis

• Interaction network mapping:

  • Create comprehensive interaction maps for DTL-B and related E3 ligase components using affinity purification-mass spectrometry

  • Identify DTL-B-specific interaction partners that may contribute to its unique functions

What strategies can address poor expression of recombinant DTL-B in Xenopus systems?

When encountering poor expression of recombinant DTL-B, researchers should implement the following troubleshooting strategies:

• Optimize promoter selection: Compare expression levels using different promoters. While CMV promoters show strong activity in Xenopus systems, the endogenous actb promoter may provide more consistent expression in certain contexts . Test both to determine optimal conditions for DTL-B expression.

• Assess extract quality: Poor extract preparation can significantly impact transcriptional activity. Ensure extracts contain sufficient levels of transcription machinery by Western blotting for RNA polymerase II, which should be enriched in correctly prepared Nucleoplasmic Extract (NPE) . Consider supplementing extracts with purified transcription factors if necessary.

• Optimize DNA concentration: Titrate plasmid concentrations between 10-100 ng/μl, as transcription efficiency in Xenopus extracts shows DNA concentration dependence. At lower DNA concentrations, transcription from certain promoters may be suppressed .

• Monitor for protein toxicity: If DTL-B overexpression is toxic, consider using inducible expression systems or expressing a catalytically inactive version of the protein that retains structural integrity but lacks enzymatic activity.

• Check for RNA stability issues: If mRNA is rapidly degraded, incorporate stabilizing elements like optimal Kozak sequences and appropriate 5' and 3' UTRs from highly expressed Xenopus genes into your construct design.

How can researchers address solubility issues with recombinant DTL-B protein?

Solubility challenges with recombinant DTL-B can be addressed through multiple approaches:

• Expression temperature optimization: Lower the expression temperature to 16-18°C to reduce aggregation and improve folding kinetics. For Xenopus extract systems, this may require longer incubation times to achieve sufficient protein levels.

• Buffer optimization matrix:

• Co-expression with binding partners: Express DTL-B together with known interaction partners (e.g., DDB1) to promote proper folding and complex formation, thereby enhancing solubility.

• Fusion protein approach: Test multiple solubility-enhancing fusion tags such as:

  • MBP (Maltose Binding Protein)

  • SUMO (Small Ubiquitin-like Modifier)

  • Thioredoxin

  • GST (Glutathione S-transferase)

• Refolding protocol: If inclusion bodies form, develop a refolding protocol using gradual dialysis from denaturing conditions (6-8M urea) to native buffer conditions.

What controls are essential for validating DTL-B function in ubiquitination assays?

Rigorous validation of DTL-B function in ubiquitination assays requires comprehensive controls:

• Essential negative controls:

  • Omission of ATP to demonstrate energy dependence of ubiquitination

  • Exclusion of E1 or E2 enzymes to confirm the complete ubiquitination cascade requirement

  • Use of catalytically inactive DTL-B mutant (typically affecting the substrate-binding WD40 domain)

  • Substitution of wild-type ubiquitin with methylated ubiquitin to confirm polyubiquitination specificity

• Substrate specificity controls:

  • Include known DTL-B substrates (e.g., CDT1, p21) as positive controls

  • Test proteins not targeted by DTL-B as negative controls

  • Use substrate mutants lacking the PIP-box degron that mediates interaction with DTL-B

• System validation markers:

  • Monitor levels of free and conjugated ubiquitin to ensure the ubiquitination system is functional

  • Assess the integrity of the CRL4 complex through Western blotting for component proteins

  • Include positive control E3 ligase reactions running in parallel to validate assay conditions

• Kinetic measurements:

  • Perform time-course experiments to establish ubiquitination rates

  • Generate substrate concentration curves to determine kinetic parameters

  • Compare wild-type DTL-B activity with partially active mutants to establish dose-response relationships

How might DTL-B function in non-canonical developmental processes in Xenopus laevis?

Recent research suggests DTL-B may have unexplored roles beyond canonical cell cycle regulation, particularly in specialized developmental contexts:

• Neural development regulation: DTL-B likely participates in neurogenesis by controlling the degradation of proneural factors and neural stem cell maintenance proteins. Xenopus laevis offers an excellent model for studying these processes due to its well-characterized neural development pathway and the amenability to electrophysiological studies of neural network assembly .

• Metamorphosis-associated remodeling: During the dramatic transition from tadpole to frog, extensive tissue remodeling occurs. DTL-B may regulate the proteolytic degradation of larval-specific proteins during this transition. The accessibility of both pre- and post-metamorphic stages in Xenopus makes it uniquely suited for studying these processes .

• Stress response pathways: DTL-B likely participates in protein quality control during cellular stress responses. Xenopus egg extracts provide a biochemically tractable system for reconstituting stress response pathways in vitro and analyzing how DTL-B targeting is modified under different stress conditions.

• Organ-specific development: Emerging evidence suggests tissue-specific roles for ubiquitin ligases in organ development. For instance, studies of lung-specific marker expression in Xenopus demonstrate how developmental timing affects organ-specific protein expression, potentially regulated by ubiquitin ligases like DTL-B .

What analytical techniques are emerging for studying the structural dynamics of DTL-B complexes?

Cutting-edge structural biology approaches offer new insights into DTL-B function:

• Cryo-electron microscopy (Cryo-EM): High-resolution structural analysis of DTL-B within the larger CRL4 complex can reveal conformational changes associated with substrate binding and ubiquitin transfer. Sample preparation from Xenopus extracts allows visualization of physiologically relevant complexes.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique identifies regions of DTL-B that undergo conformational changes upon substrate binding or complex formation, providing dynamic structural information complementary to static structural techniques.

• Integrative structural biology approaches: Combining multiple techniques including small-angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), and computational modeling to generate comprehensive structural models of DTL-B complexes.

• Single-molecule FRET: Monitoring conformational changes in real-time by labeling specific domains of DTL-B and its binding partners with fluorescent probes, enabling analysis of the dynamics of complex assembly and substrate processing.

• In-cell NMR: Emerging methods for performing NMR studies in Xenopus oocytes allow investigation of DTL-B structural dynamics in a native cellular environment rather than in vitro.

How can multi-omics approaches enhance understanding of DTL-B regulation networks?

Integrated multi-omics strategies provide comprehensive insights into DTL-B function:

• Temporal proteogenomics: Combine RNA-seq and quantitative proteomics across Xenopus developmental timepoints to correlate DTL-B expression with substrate levels, revealing regulatory networks and developmental stage-specific functions.

• Ubiquitinome analysis: Apply ubiquitin remnant profiling (K-ε-GG antibody enrichment) in DTL-B knockdown versus control conditions to globally identify DTL-B-dependent ubiquitination events across the proteome.

• Phospho-ubiquitin crosstalk mapping: Investigate how phosphorylation of substrates or DTL-B itself modulates ubiquitination activity through combined phosphoproteomics and ubiquitinomics.

• Spatial transcriptomics integration: Correlate tissue-specific expression patterns of DTL-B with substrate distributions using in situ techniques combined with laser capture microdissection and RNA-seq.

• Dynamic interactome profiling: Apply BioID or APEX2 proximity labeling at defined developmental timepoints to map changing DTL-B interaction networks throughout development.