Recombinant Xenopus tropicalis Ubiquitin carboxyl-terminal hydrolase 47 (usp47), partial

What is USP47 and what is its primary function in Xenopus tropicalis?

USP47 (Ubiquitin Specific Peptidase 47) is a deubiquitylase that plays a critical role in regulating protein ubiquitylation states. In Xenopus, USP47 functions as a positive regulator of Wnt signaling by deubiquitylating specific targets. Experimental evidence shows that USP47 is evolutionarily conserved across species, including Drosophila, Xenopus, and humans, where it performs similar functions in the Wnt signaling pathway . The enzyme specifically counteracts the E3 ligase activity of XIAP on Groucho/TLE transcriptional co-repressors, which is essential for proper Wnt-mediated transcriptional activation .

How is USP47 expression regulated during Xenopus development?

USP47 is present throughout Xenopus development, with expression detectable from the unfertilized egg through gastrula, neurula, and late tailbud stages . RT-PCR analysis has confirmed maternal expression of usp47 mRNA in unfertilized eggs, indicating its potential importance in early developmental events prior to zygotic genome activation . In situ hybridization studies demonstrate a dynamic expression pattern similar to that of β-catenin, with localization initially in the animal half of early embryos up to gastrula stage, then at the anterior and posterior ends during neurula stage, and later in the branchial arches, eye, and posterior regions at tailbud stage . By tadpole stage, usp47 expression becomes primarily restricted to the head and spinal cord, suggesting tissue-specific functions during later development .

What protein domains and motifs are important for USP47 function?

The functional activity of USP47 depends on several critical domains and motifs. The catalytic domain contains the cysteine protease activity necessary for deubiquitylation. Based on related deubiquitylases like USP7, USP47 likely contains or interacts with specific binding motifs including P/AxxS motifs and KxxxK motifs . In other systems, these motifs have been shown to mediate protein-protein interactions. For example, in PAF15, P/AxxS motifs (76PSTS79 and 94AGGS97) and a KxxxK motif (101KKPRK105) were found to facilitate binding to USP7 . While the search results don't specify the exact motifs in Xenopus USP47, similar structural elements likely facilitate its interactions with binding partners like XIAP and TLE3.

What are the best approaches for studying USP47 function in Xenopus?

Multiple complementary approaches have proven effective for studying USP47 function in Xenopus:

• Morpholino knockdown: Targeting usp47 mRNA with specific morpholinos injected into dorsal blastomeres can effectively reduce USP47 levels. This approach has been validated through rescue experiments using co-injection of mouse usp47 mRNA, confirming specificity .

• mRNA overexpression: Ventral injection of usp47 mRNA can be used to induce gain-of-function phenotypes, such as partial axis duplication observed in Xenopus embryos .

• In situ hybridization: This technique effectively maps the spatiotemporal expression pattern of usp47 during development .

• RT-PCR analysis: For temporal expression profiling across developmental stages .

• Histological analysis: Techniques such as H&E staining of sectioned embryos can be used to examine detailed phenotypic effects, such as duplicated axes resulting from USP47 overexpression .

How can recombinant Xenopus USP47 be effectively expressed and purified?

For expression and purification of recombinant Xenopus USP47, the following methodology is recommended:

• Expression system selection: E. coli systems may be suitable for partial USP47 constructs, while baculovirus-infected insect cells are generally more effective for full-length deubiquitylases to ensure proper folding and post-translational modifications.

• Fusion tag strategy: GST-fusion constructs have been successfully used with related proteins, facilitating purification via glutathione sepharose affinity chromatography . For USP47, a design incorporating cleavable tags (such as thrombin-cleavable GST) allows tag removal after purification.

• Purification protocol:

  • Affinity chromatography (glutathione sepharose for GST-tagged proteins)

  • Optional tag cleavage with thrombin or similar protease

  • Further purification via ion exchange and size exclusion chromatography

  • Validation of enzymatic activity using fluorogenic ubiquitin substrates

• Storage conditions: Purified USP47 is typically stored in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM DTT, and 10% glycerol at -80°C to maintain stability and activity.

What controls should be included when analyzing USP47 function in developmental assays?

When designing experiments to analyze USP47 function in developmental assays, several controls are essential:

• Morpholino specificity controls:

  • Injection of control/scrambled morpholinos

  • Rescue experiments with co-injection of morpholino-resistant usp47 mRNA (e.g., mouse usp47 for Xenopus knockdown)

  • Use of multiple non-overlapping morpholinos targeting different regions of usp47 mRNA

• Overexpression controls:

  • Injection of control mRNA (e.g., GFP) at equivalent concentrations

  • Dose-response analysis to determine threshold effects

  • Co-injection with pathway components to verify specificity (e.g., β-catenin or XWnt8)

• Phenotypic analysis controls:

  • Comparison to established pathway manipulation phenotypes

  • Molecular marker analysis (e.g., Wnt target genes)

  • Histological validation of gross morphological observations

How does USP47 regulate the Wnt signaling pathway in Xenopus?

USP47 serves as a positive regulator of Wnt signaling in Xenopus through its deubiquitylase activity on specific targets. The experimental evidence for this role includes:

• Loss-of-function effects: Knockdown of USP47 by morpholino injection in Xenopus embryos results in severely ventralized embryos, consistent with inhibition of the Wnt pathway .

• Gain-of-function effects: Overexpression of USP47 by ventral injection of usp47 mRNA induces partial axis duplication, a phenotype characteristic of Wnt pathway activation .

• Interaction with Wnt pathway components: USP47 knockdown suppresses secondary axis formation induced by overexpression of β-catenin or XWnt8 mRNA, demonstrating its requirement for Wnt-mediated developmental processes .

Mechanistically, USP47 likely functions similarly to its role in human cells, where it deubiquitylates Groucho/TLE co-repressors, counteracting the ubiquitylation mediated by XIAP . This regulated cycle of ubiquitylation and deubiquitylation promotes the ability of β-catenin to cycle on and off TCF/LEF transcription factors, ensuring proper transcriptional responses to Wnt signaling .

What are the key differences in USP47 function between Xenopus tropicalis and other model organisms?

The USP47 function shows remarkable evolutionary conservation across species, but with some notable differences:

These comparative insights suggest that while the core function of USP47 in Wnt signaling is conserved, species-specific adaptations may fine-tune its activity in different developmental and cellular contexts.

What are the molecular mechanisms by which USP47 affects TLE/Groucho during Wnt signaling?

The molecular mechanism by which USP47 influences Wnt signaling involves specific interactions with the transcriptional repressor TLE/Groucho and the E3 ubiquitin ligase XIAP:

• Protein-protein interactions: USP47 physically interacts with both XIAP and TLE3, as demonstrated by co-immunoprecipitation studies in human cells . These interactions position USP47 to directly affect the ubiquitylation state of TLE3.

• Deubiquitylation activity: USP47 counteracts XIAP-mediated ubiquitylation of TLE3. In vitro experiments show that USP47 inhibits TLE3 ubiquitylation by XIAP in a dose-dependent manner .

• Dynamic regulation: The interplay between USP47 (deubiquitylation) and XIAP (ubiquitylation) creates a dynamic cycle that regulates TLE3's repressive activity on TCF/LEF transcription factors .

• Competitive binding: Evidence suggests that TLE3 and USP47 may compete for binding to XIAP rather than forming a heterotrimeric complex . This competition adds another layer of regulation to the system.

This mechanism ensures that Wnt target gene expression continues only as long as upstream signaling is present, by enabling β-catenin to cycle on and off TCF transcription factors in a regulated manner .

How can CRISPR-Cas9 genome editing be optimized for studying USP47 function in Xenopus tropicalis?

Optimizing CRISPR-Cas9 genome editing for USP47 studies in Xenopus tropicalis requires careful consideration of several factors:

• sgRNA design strategy:

  • Target conserved exons encoding catalytic domains

  • Design multiple sgRNAs targeting different regions to compare efficiency

  • Validate sgRNAs using in silico prediction tools to minimize off-target effects

  • Consider the experience from Drosophila studies where multiple sgRNAs targeting different parts of the Usp47 coding region were used

• Delivery method optimization:

  • Injection timing: 1-cell stage for full knockout, later stages for mosaic analysis

  • Injection location: animal pole for optimal distribution

  • Cas9 format: protein (immediate activity) vs. mRNA (delayed expression)

  • Ribonucleoprotein (RNP) complex pre-formation before injection

• Validation strategies:

  • T7E1 or similar mismatch detection assays

  • Direct sequencing of target loci

  • Western blot to confirm protein reduction

  • Phenotypic analysis comparable to morpholino studies

• Potential challenges:

  • Complete knockout may cause embryonic lethality, as suggested by the pupal lethality observed in Drosophila Usp47 null alleles

  • Consider inducible or tissue-specific knockout strategies

What are the best approaches for studying the substrate specificity of Xenopus USP47?

To investigate the substrate specificity of Xenopus USP47, several complementary approaches can be employed:

• In vitro deubiquitylation assays:

  • Purified recombinant USP47 incubated with ubiquitylated candidate substrates

  • Synthetic ubiquitin chains of different linkage types (K48, K63, etc.) to determine chain preference

  • Quantitative measurement of deubiquitylation rates for different substrates

  • Analysis similar to studies showing USP47 inhibits ubiquitylation of TLE3 by XIAP in a dose-dependent manner

• Substrate trapping approaches:

  • Catalytically inactive USP47 mutants to trap substrates

  • Immunoprecipitation followed by mass spectrometry to identify bound proteins

  • Validation of interactions by co-immunoprecipitation experiments

  • Comparison with known substrates like TLE3

• Comparative proteomics:

  • Stable isotope labeling with amino acids in cell culture (SILAC) of control vs. USP47-depleted cells

  • Global ubiquitylome analysis by mass spectrometry

  • Bioinformatic identification of enriched ubiquitylation sites

• Structural studies:

  • Crystallography of USP47 alone and in complex with ubiquitin or substrate peptides

  • Molecular modeling based on related DUBs like USP7, which interacts with specific motifs (P/AxxS and KxxxK)

  • Identification of substrate recognition elements

How does USP47 contribute to cross-talk between Wnt signaling and other developmental pathways in Xenopus?

USP47's role in facilitating cross-talk between Wnt signaling and other developmental pathways can be investigated through several approaches:

• Integrative pathway analysis:

  • Examine USP47's effect on multiple signaling reporters simultaneously

  • Investigate whether USP47 depletion affects not only Wnt targets but also targets of intersecting pathways

  • Analyze temporal coordination of different pathway activities in relation to USP47 function

• Key developmental contexts for investigation:

  • Neural crest formation (Wnt, BMP, FGF cross-talk)

  • Mesoderm induction and patterning (Wnt, Nodal, FGF interactions)

  • Anterior-posterior axis formation (Wnt, retinoic acid pathway)

• Potential mechanisms of cross-talk:

  • Shared transcriptional co-regulators (beyond TLE)

  • Common chromatin remodeling complexes

  • Substrate overlap with other ubiquitin-related enzymes

  • Competition for limiting cofactors

• Experimental approach:

  • Combined pathway perturbation experiments (e.g., USP47 knockdown plus activation/inhibition of other pathways)

  • Epistasis analysis to position USP47 within complex signaling networks

  • Transcriptomic analysis to identify genes co-regulated by multiple pathways dependent on USP47

  • Chromatin immunoprecipitation to identify pathway-specific and shared genomic targets

How can inconsistent phenotypes in USP47 studies be reconciled and interpreted?

Researchers frequently encounter variability in phenotypes when studying USP47. These inconsistencies can be addressed through:

• Sources of variability and their management:

  • Maternal contribution: Control by using defined depletion methods targeting both maternal and zygotic transcripts

  • Genetic background differences: Maintain consistent strain backgrounds

  • Dosage effects: Establish clear dose-response relationships for all interventions

  • Timing differences: Precisely control the developmental stage of intervention

• Integrative analysis approach:

  • Combine multiple phenotypic readouts (morphological, molecular, cellular)

  • Quantify phenotypic severity using standardized scoring systems

  • Use statistical methods appropriate for developmental biology (e.g., ordinal regression for phenotypic categories)

• Reconciliation strategies for contradictory findings:

  • Determine if differences reflect partial vs. complete loss of function

  • Consider potential compensation mechanisms in different experimental contexts

  • Evaluate contribution of maternal vs. zygotic transcripts

  • Compare acute (morpholino) vs. chronic (genetic) loss of function

• Validation across experimental systems:

  • Compare findings across species (e.g., Xenopus tropicalis vs. laevis)

  • Validate key findings in complementary systems (e.g., explant cultures)

  • Use multiple independent methods to manipulate USP47 levels or activity

What are effective solutions for common technical challenges in recombinant USP47 production?

Production of active recombinant USP47 presents several technical challenges:

How can researchers differentiate between direct and indirect effects of USP47 manipulation?

Distinguishing direct from indirect effects of USP47 manipulation requires rigorous experimental design:

• Temporal analysis approaches:

  • Utilize rapid induction systems (e.g., hormone-inducible constructs)

  • Perform time-course experiments following USP47 manipulation

  • Identify immediate vs. delayed responses through transcriptomic/proteomic profiling

  • Apply mathematical modeling to distinguish direct from feedback effects

• Direct target validation strategies:

  • Demonstrate physical interaction (co-immunoprecipitation, proximity ligation assay)

  • Show enzymatic activity on purified substrates in vitro

  • Identify ubiquitylation sites on targets that change with USP47 manipulation

  • Use ubiquitylation-resistant substrate mutants for rescue experiments

• Specificity controls:

  • Compare effects of USP47 manipulation with manipulation of other DUBs

  • Use catalytically inactive USP47 mutants as controls

  • Perform substrate competition experiments

  • Test whether effects can be rescued by manipulating proposed direct targets

• Integrated approach example: To determine if effects on Wnt signaling are direct, researchers could:

  • Demonstrate direct deubiquitylation of TLE by USP47 in vitro

  • Show that USP47 and TLE co-localize at Wnt target gene promoters

  • Demonstrate that TLE ubiquitylation status changes rapidly upon USP47 inhibition

  • Test whether ubiquitylation-resistant TLE mutants bypass the requirement for USP47

What are the most promising therapeutic applications targeting USP47 for developmental disorders?

Based on USP47's crucial role in development and signaling, several therapeutic applications can be envisioned:

• Developmental disorders with Wnt signaling dysregulation:

  • Neural tube defects where Wnt signaling is aberrant

  • Craniofacial abnormalities involving neural crest development

  • Limb development disorders with disrupted Wnt gradient formation

• Therapeutic strategies:

  • Small molecule inhibitors of USP47 for conditions with excessive Wnt activation

  • USP47 mimetics or stabilizers for conditions with insufficient Wnt signaling

  • Targeted delivery systems for tissue-specific modulation of USP47 activity

• Challenges and considerations:

  • Specificity among DUB family members

  • Developmental stage-specific requirements

  • Potential compensatory mechanisms

  • Tissue-specific delivery methods

• Preliminary evidence: While direct therapeutic applications remain to be developed, the essential role of USP47 in development across species suggests its potential as a target . The severe developmental defects observed in Xenopus and Drosophila upon USP47 manipulation highlight both the importance and challenges of targeting this pathway .

What novel techniques could advance our understanding of USP47 function in development?

Emerging technologies offer new opportunities to elucidate USP47 function:

• Single-cell approaches:

  • Single-cell RNA-seq to map USP47-dependent transcriptional changes with cellular resolution

  • Single-cell proteomics to identify cell type-specific USP47 substrates

  • Spatial transcriptomics to correlate USP47 activity with developmental patterning

• Live imaging innovations:

  • FRET-based sensors for monitoring USP47 activity in real-time

  • Optogenetic tools for spatiotemporal control of USP47 function

  • Light-sheet microscopy for whole-embryo imaging of USP47-dependent processes

• Genomic engineering advancements:

  • Base editing for precise modification of USP47 catalytic sites

  • Prime editing for introducing specific mutations without double-strand breaks

  • Tissue-specific CRISPR systems for spatial manipulation of USP47

• Structural biology approaches:

  • Cryo-EM studies of USP47 in complex with substrates and regulators

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic protein interactions

  • AlphaFold2 and related computational methods for predicting interaction interfaces

How might environmental factors influence USP47 function during Xenopus development?

Environmental influences on USP47 function represent an important frontier for research:

• Temperature effects:

  • Impact on USP47 enzymatic activity and substrate specificity

  • Interaction with temperature-sensitive developmental timing in poikilothermic organisms

  • Potential for temperature to affect USP47-dependent developmental processes differently from other pathways

• Chemical exposures:

  • Effects of environmental toxicants on USP47 expression or activity

  • Interaction between USP47 and xenobiotic-responsive pathways

  • Potential for USP47 inhibition to sensitize embryos to environmental stressors

• Experimental approaches:

  • Controlled environmental exposure studies with USP47 as readout

  • Comparison of wild-type and USP47-manipulated embryos under environmental stress

  • Multi-omics profiling to identify environment-sensitive USP47 targets

• Potential significance: Understanding environmental influences on USP47 function could provide insights into gene-environment interactions in developmental disorders, particularly those involving Wnt signaling dysregulation. The evolutionary conservation of USP47 function suggests that findings in Xenopus may have relevance to environmental impacts on development across species.

How does USP47 function compare between Xenopus tropicalis and Xenopus laevis?

A comparative analysis of USP47 between these closely related species reveals:

What insights can be gained from comparing USP47 structure and function across diverse vertebrate species?

Evolutionary analysis of USP47 across vertebrates provides valuable insights:

• Structural conservation patterns:

  • Catalytic domains are highly conserved across vertebrates

  • Substrate recognition regions show more divergence

  • Comparison with USP7 suggests conservation of key functional motifs like P/AxxS and KxxxK recognition

• Functional adaptations:

  • Core role in Wnt signaling is conserved from Drosophila to mammals

  • Species-specific adaptations in regulation and substrate specificity

  • Different requirements during development correlating with species-specific patterning mechanisms

• Evolutionary significance:

  • Conservation suggests fundamental importance in development

  • Co-evolution with Wnt pathway components

  • Potential role in the evolution of body plan complexity

• Comparative methodological approaches:

  • Cross-species rescue experiments (e.g., mouse USP47 mRNA rescuing Xenopus phenotypes)

  • Domain swapping between species to identify critical regions

  • Comparative functional genomics to map conserved and divergent targets

How has the role of USP47 in Wnt signaling evolved from amphibians to mammals?

The evolutionary trajectory of USP47's role in Wnt signaling from amphibians to mammals reveals both conservation and adaptation:

• Core mechanism conservation:

  • USP47 functions as a positive regulator of Wnt signaling across species

  • The deubiquitylation of TLE/Groucho appears to be a conserved mechanism

  • Nuclear localization and action downstream of β-catenin stabilization is maintained

• Species-specific adaptations:

  • Differential expression patterns adapted to species-specific developmental programs

  • Potential differences in regulation and post-translational modifications

  • Possible expansion of substrate repertoire in more complex organisms

• Contextual differences:

  • In Xenopus, USP47 plays a crucial role in early axis formation

  • In mammals, USP47 affects Wnt-dependent processes in multiple tissues and contexts

  • The relative importance of maternal vs. zygotic contributions varies across species

• Evolutionary implications:

  • The conservation of USP47 function in Wnt signaling across diverse species (from flies to frogs to humans) suggests it represents an ancient and fundamental regulatory mechanism that evolved before the divergence of these lineages

  • The ability of mouse USP47 to rescue Xenopus phenotypes demonstrates functional conservation across considerable evolutionary distance