Recombinant Xenopus laevis Ubiquitin carboxyl-terminal hydrolase 10-B (usp10-b), partial

What is Xenopus laevis USP10-B and what is its primary function?

USP10-B is a deubiquitylating enzyme (DUB) belonging to the peptidase C19 family, USP10 subfamily, expressed in Xenopus laevis (African clawed frog). Its primary function is to remove conjugated ubiquitin from target proteins through a catalytic process involving binding to the ubiquitin C-terminus via its USP domain, followed by conformational changes in the catalytic domain. The conserved residues form a catalytic triad that facilitates nucleophilic attack on the carbonyl carbon, ultimately removing ubiquitin from target proteins . This hydrolase activity plays critical roles in regulating protein stability and various cellular processes including DNA damage response and autophagy regulation .

What are the known substrates of USP10-B in Xenopus systems?

While the complete substrate profile specifically for Xenopus USP10-B hasn't been fully characterized in the provided research, studies on homologous USP10 proteins suggest several important substrates. Based on research in other systems, USP10 targets include:

In Xenopus extract systems specifically, USP10-B likely participates in similar regulatory pathways, though research continues to fully map these interactions in the amphibian model system .

How can researchers effectively express and purify recombinant Xenopus USP10-B for in vitro studies?

For effective expression and purification of recombinant Xenopus USP10-B, researchers should consider:

• Expression system selection: E. coli BL21(DE3) or insect cell-based systems (Sf9/Sf21) are recommended for USP10-B expression, with the latter often providing better folding for complex eukaryotic proteins.

• Construct design: Include appropriate affinity tags (His6, GST, or MBP) at either N or C-terminus with TEV or PreScission protease cleavage sites for tag removal. Avoid tag placement that might interfere with the catalytic domain.

• Purification protocol:

  • Initial capture using affinity chromatography

  • Ion exchange chromatography to remove contaminants

  • Size exclusion chromatography for final polishing

  • Maintain buffers with reducing agents (1-5 mM DTT or TCEP) to protect catalytic cysteine

  • Include 5-10% glycerol to enhance stability

• Quality control: Verify purity by SDS-PAGE and activity using fluorogenic ubiquitin substrates (Ub-AMC) before experimental use.

For functional studies, protein should be stored in small aliquots at -80°C with minimal freeze-thaw cycles to maintain enzymatic activity .

What are recommended assays to measure USP10-B deubiquitinating activity in Xenopus extracts?

Several complementary approaches can be employed to measure USP10-B deubiquitylating activity in Xenopus extracts:

• UbVS labeling assay: Using HA-tagged ubiquitin vinyl sulfone (UbVS) to covalently label active DUBs including USP10-B, followed by immunoblotting to detect labeled enzymes. This approach allows visualization of multiple active DUBs simultaneously .

• Substrate degradation assays: Monitoring the degradation of known USP10 substrates in Xenopus extract with and without USP10-B inhibition or depletion. This approach often employs 35S-labeled substrates to track degradation rates over time .

• Ubiquitin chain cleavage assays: Using purified di- or poly-ubiquitin chains of specific linkage types (K48, K63, etc.) to assess the chain-type specificity of USP10-B when incubated with Xenopus extract or purified enzyme.

• Quantitative proteomics approach: Employing Tandem Mass Tag (TMT)-based proteomics to identify proteins whose stability changes upon USP10-B inhibition or depletion in Xenopus extract, as demonstrated in research with other DUBs .

When performing these assays, it's critical to include appropriate controls such as catalytically inactive USP10-B mutants and specific USP10 inhibitors to distinguish USP10-B activity from other DUBs present in the extract .

How can researchers distinguish between the activities of USP10-B and redundant DUBs in experimental systems?

Distinguishing between USP10-B and redundant DUBs requires a multi-faceted approach:

• Specific inhibitors: Utilize small molecule inhibitors with selectivity for USP10 (such as compounds analogous to the USP7 inhibitor XL-188 mentioned in search results) to specifically inhibit USP10-B activity while leaving other DUBs functional .

• Immunodepletion approach: Selectively remove USP10-B from Xenopus extracts using specific antibodies against USP10-B, then assess the remaining deubiquitinating activity.

• Rescue experiments: Deplete endogenous USP10-B and add back recombinant wild-type or catalytically inactive USP10-B to determine if observed effects can be specifically attributed to USP10-B enzymatic activity .

• Combinatorial DUB inhibition: Compare the effects of broad DUB inhibition (using UbVS) with specific inhibition of individual DUBs to identify redundancies. For example, research has shown that USP7 can rescue stability of multiple proteins when other DUBs are inhibited, suggesting functional redundancy .

• Substrate profiling: Analyze substrate specificity patterns using proteomics approaches like TMT-based quantitative proteomics to identify unique vs. shared substrates .

The redundant nature of DUBs was highlighted in research showing that specific inhibition of USP7 alone was insufficient to promote degradation of most substrates that were degraded when multiple DUBs were inhibited simultaneously, emphasizing the importance of considering functional redundancy in DUB research .

How does USP10-B contribute to DNA damage response pathways in Xenopus systems?

USP10-B plays critical roles in DNA damage response pathways in Xenopus systems, primarily through regulation of key proteins involved in these processes:

• p53 regulation: USP10-B appears to regulate p53 stability in a manner similar to mammalian USP10. Following DNA damage, USP10 can translocate to the nucleus and deubiquitinate p53, thereby stabilizing it and promoting the DNA damage response pathway .

• Cell cycle checkpoint regulation: Through its deubiquitinating activity, USP10-B likely contributes to cell cycle checkpoint control following DNA damage, though specific mechanisms in Xenopus systems require further characterization.

• DNA repair protein stabilization: USP10-B may regulate the stability and activity of proteins involved in DNA repair pathways, similar to how mammalian USP10 has been shown to regulate PCNA and DNA polymerase eta .

• Evolutionary conservation: Research indicates that many of the DUB-substrate interactions involved in DNA damage response are evolutionarily conserved between Xenopus and mammalian systems, suggesting that insights from Xenopus studies may have translational relevance .

To fully elucidate USP10-B's role in DNA damage response, researchers should employ techniques such as immunofluorescence microscopy to track USP10-B localization following DNA damage, coupled with proteomic approaches to identify changes in the ubiquitination status of potential substrates under various DNA damage conditions .

What is the relationship between USP10-B and cancer-relevant pathways based on comparative studies with human USP10?

While the provided research doesn't directly address Xenopus USP10-B in cancer contexts, comparative studies with human USP10 reveal several important cancer-relevant pathways that may be conserved:

• Prognostic significance: Human USP10 expression has significant prognostic value across multiple cancer types, with high expression correlating with poor prognosis in pancreatic adenocarcinoma (PAAD), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and breast cancer (BRCA) .

• Diagnostic potential: ROC curve analysis shows USP10 has significant diagnostic value across multiple cancers:

Given the evolutionary conservation of many USP10 functions between species, these findings suggest potential areas for investigating Xenopus USP10-B in developmental and cellular contexts relevant to cancer biology .

How do redundancy mechanisms among DUBs in Xenopus affect experimental design when studying USP10-B?

DUB functional redundancy significantly impacts experimental design when studying USP10-B in Xenopus systems, requiring careful methodological approaches:

• Limitations of single-DUB inhibition: Research has demonstrated that specific inhibition of individual DUBs (including USP7) was insufficient to promote degradation of most substrates that were degraded when multiple DUBs were inhibited simultaneously . This suggests:

  • Single USP10-B knockout/inhibition studies may underestimate its physiological roles

  • Negative results should be interpreted cautiously, as redundant DUBs may compensate

• Combinatorial approaches: Researchers should design experiments incorporating:

  • Simultaneous inhibition of multiple DUBs (using broad inhibitors like UbVS together with ubiquitin supplementation)

  • Combinatorial inhibition of USP10-B with other DUBs (particularly USP7 and USP14, which have shown functional overlap)

  • Comparison of protein stability in extract treated with USP10 inhibitors alone versus combinations with other DUB inhibitors

• Substrate validation strategy: A recommended approach involves:

  • Initial identification of potential substrates using broad DUB inhibition

  • Validation with specific inhibitors or depletion/reconstitution experiments

  • Careful analysis of substrate ubiquitination patterns under different DUB inhibition conditions

• Rescue experiments: The observation that human recombinant USP7 could broadly rescue substrate degradation in UbVS-treated Xenopus extract demonstrates the importance of including rescue experiments with recombinant USP10-B to determine substrate specificity in the context of DUB redundancy .

The research by de Poot et al. provides a powerful experimental framework for addressing DUB redundancy, showing that USP7 could rescue 16 of 20 proteins degraded in UbVS/ubiquitin-treated extracts, highlighting the extensive functional overlap among DUBs that must be accounted for in experimental design .

What are effective strategies to specifically target USP10-B activity in Xenopus systems?

Developing effective strategies to specifically target USP10-B activity in Xenopus systems requires multiple complementary approaches:

• Small molecule inhibitors:

  • While specific USP10-B inhibitors are not explicitly mentioned in the provided research, the approach used with USP7-specific inhibitor XL-188 provides a template

  • Validation of inhibitor specificity should include testing whether the compound prevents UbVS labeling of USP10-B without affecting other DUBs

  • Optimal inhibitor concentrations should be determined through dose-response experiments in Xenopus extracts

• Genetic approaches:

  • Morpholino oligonucleotides can be designed to specifically knock down USP10-B expression in Xenopus embryos

  • CRISPR/Cas9-mediated gene editing can generate USP10-B knockout or catalytically inactive mutant Xenopus models

  • Expression of dominant-negative USP10-B mutants can interfere with endogenous USP10-B function

• Immunodepletion approach:

  • Use USP10-B-specific antibodies to selectively deplete the protein from Xenopus extracts

  • Validate depletion efficiency by immunoblotting and UbVS labeling

  • Perform add-back experiments with recombinant wild-type or mutant USP10-B to confirm specificity

• Substrate-trapping mutants:

  • Generate catalytically inactive USP10-B mutants that can bind but not process substrates

  • Use these mutants as tools to identify and validate specific USP10-B substrates

  • Combine with proteomic approaches to identify trapped substrates

• Validation strategies:

  • Always confirm target engagement through activity-based probes like UbVS labeling

  • Include appropriate controls to account for redundancy among DUBs

  • Validate findings using multiple independent approaches

How can researchers accurately measure USP10-B substrate specificity in the context of redundant DUB functions?

Accurately measuring USP10-B substrate specificity in the context of redundant DUB functions requires sophisticated experimental designs:

• Comparative proteomics approach:

  • Implement TMT-based quantitative proteomics comparing protein stability under different conditions:
    a) Control (no inhibition)
    b) Broad DUB inhibition (UbVS + ubiquitin)
    c) Specific USP10-B inhibition or depletion
    d) Combinatorial inhibition of USP10-B with other DUBs

  • This approach can reveal substrates uniquely dependent on USP10-B versus those with redundant regulation

• Ubiquitin chain linkage analysis:

  • Determine which ubiquitin chain types (K48, K63, K11, etc.) USP10-B preferentially cleaves

  • Compare with the specificity profiles of other DUBs to identify unique versus overlapping activities

  • Research suggests USP10 can remove K48-linked polyubiquitin chains from Smad4 and K63-linked chains from PTEN, indicating diverse chain specificity

• Substrate validation hierarchy:

  • Tier 1: Identify candidate substrates through proteome-wide approaches

  • Tier 2: Validate direct deubiquitination using purified components in vitro

  • Tier 3: Confirm physiological relevance through in vivo studies with specific inhibition or depletion of USP10-B

  • Tier 4: Assess redundancy through combinatorial inhibition/depletion of multiple DUBs

• Temporal dynamics analysis:

  • Analyze the kinetics of substrate ubiquitination and degradation under different conditions

  • Some substrates may show different temporal dependence on specific DUBs

  • Time-course experiments can reveal primary versus compensatory DUB activities

• Domain mutant analysis:

  • Generate USP10-B mutants with alterations in specific domains

  • Determine which domains contribute to substrate specificity versus catalytic activity

  • Use domain swaps between USP10-B and other DUBs to pinpoint specificity determinants

What are the most important considerations when interpreting USP10-B experimental results across different model systems?

When interpreting USP10-B experimental results across different model systems, researchers should consider several critical factors:

• Evolutionary conservation and divergence:

  • While many DUB-substrate interactions are conserved between Xenopus and mammals, species-specific differences may exist

  • Research shows conservation of interactions between DUBs and binding partners across species (e.g., USP10-GBP1/GBP2 and CYLD-SPATA proteins), but functional conservation should not be assumed without validation

  • Sequence comparisons between Xenopus USP10-B and mammalian orthologs should inform interpretation of cross-species findings

• Context-dependent functions:

  • USP10 functions may vary significantly between developmental stages, cell types, and physiological conditions

  • In cancer contexts, USP10 shows tissue-specific patterns of expression and correlation with immune infiltration

  • Results from Xenopus egg extract may not directly translate to adult tissues or mammalian systems

• Technical considerations in cross-species studies:

  • When using recombinant human DUBs in Xenopus systems (as demonstrated with USP7), consider potential differences in substrate recognition

  • The observation that human USP7 could rescue stability of Xenopus proteins suggests functional conservation but requires careful validation

  • Cross-reactivity of antibodies and inhibitors should be thoroughly validated

• Redundancy patterns may differ between species:

  • The extent and pattern of DUB redundancy may vary between Xenopus and mammalian systems

  • Compensatory mechanisms may differ between model organisms

  • Quantitative differences in DUB expression levels between species may affect interpretation of knockdown/inhibition studies

• Translational relevance considerations:

  • Findings regarding USP10's role in cancer-relevant processes from Xenopus studies should be validated in mammalian systems before clinical extrapolation

  • The significant diagnostic and prognostic value of USP10 in human cancers suggests important conserved functions that warrant investigation in model systems

  • Consider developmental context - Xenopus embryonic systems may best model early developmental processes rather than adult disease states

What are the most promising approaches for mapping the complete substrate profile of USP10-B in Xenopus?

Several cutting-edge approaches show promise for comprehensively mapping the USP10-B substrate profile in Xenopus:

• Integrated proteomics approaches:

  • Combine ubiquitin remnant profiling (K-ε-GG) with total proteome analysis before and after USP10-B inhibition/depletion

  • Implement TMT or SILAC-based quantitative proteomics to measure changes in both protein abundance and ubiquitination status

  • Compare profiles under conditions of single USP10-B inhibition versus broad DUB inhibition to identify primary versus redundantly-regulated substrates

• Proximity-dependent labeling:

  • Generate USP10-B fusion proteins with BioID or TurboID to identify proximal interacting proteins

  • Enrichment of biotinylated proteins followed by mass spectrometry can reveal the USP10-B "interactome"

  • This approach can identify both substrates and regulatory partners

• Substrate-trapping approaches:

  • Utilize catalytically inactive USP10-B mutants that can bind but not process substrates

  • Combine with crosslinking mass spectrometry (XL-MS) to capture transient enzyme-substrate interactions

  • This approach can identify direct substrates even in the context of redundant DUB activity

• Developmental stage-specific profiling:

  • Map USP10-B substrates across different developmental stages of Xenopus

  • Identify stage-specific versus constitutive substrates

  • Correlate with developmental phenotypes in USP10-B depletion/inhibition studies

• Comparative cross-species analysis:

  • Perform parallel substrate identification in Xenopus and mammalian systems

  • Identify evolutionarily conserved versus species-specific substrates

  • This approach can highlight fundamental versus specialized functions of USP10

The combination of these approaches, particularly when applied across different cellular contexts (normal development, DNA damage, etc.), offers the most comprehensive strategy for mapping the complete USP10-B substrate landscape.

How might functional redundancy between USP10-B and other DUBs be exploited for therapeutic applications?

Understanding the functional redundancy between USP10-B and other DUBs reveals several potential therapeutic strategies:

• Combination DUB inhibition approaches:

  • Research showing that USP7 can rescue multiple substrates destabilized under broad DUB inhibition suggests that combinatorial targeting may overcome functional redundancy

  • Simultaneous inhibition of USP10 with redundant DUBs (particularly USP7) may provide synergistic effects in therapeutic contexts

  • This approach could potentially lower required doses of individual inhibitors, reducing off-target effects

• Targeting DUB-substrate interfaces:

  • Rather than inhibiting catalytic activity, developing compounds that disrupt specific DUB-substrate interactions could provide greater specificity

  • This approach could overcome redundancy by targeting unique binding interfaces rather than conserved catalytic domains

  • Structural studies of USP10-B with various substrates would be needed to enable this approach

• Context-specific inhibition strategies:

  • The finding that USP10 expression correlates with immune cell infiltration in multiple cancers suggests potential for combination with immunotherapy approaches

  • USP10 inhibition might enhance immune recognition in tumors with high USP10 expression

  • The association between USP10 expression and tumor mutation burden suggests potential biomarkers for identifying contexts where USP10 inhibition might be most effective

• Exploiting synthetic lethality:

  • Identify contexts where redundant DUBs compensate for each other

  • Target USP10 in contexts where redundant DUBs are already compromised

  • This approach could provide cancer-specific therapeutic windows

• Developmental context considerations:

  • Research in Xenopus systems provides insights into the developmental roles of USP10-B

  • Understanding the developmental context of DUB redundancy could inform potential toxicity profiles of therapeutic approaches

  • Embryonic phenotypes from USP10-B inhibition in Xenopus could predict developmental toxicities

What are the key unresolved questions regarding USP10-B function in Xenopus developmental biology?

Several critical questions regarding USP10-B function in Xenopus developmental biology remain unresolved:

• Developmental expression patterns:

  • What is the spatial and temporal expression pattern of USP10-B throughout Xenopus development?

  • How does USP10-B expression correlate with critical developmental transitions?

  • Are there tissue-specific isoforms or post-translational modifications of USP10-B during development?

• Developmental phenotypes:

  • What are the consequences of USP10-B depletion or inhibition at different developmental stages?

  • Are there specific developmental processes that are particularly sensitive to USP10-B function?

  • How do phenotypes of USP10-B disruption compare with those of other DUBs, particularly those with redundant functions?

• Substrate dynamics during development:

  • Does the substrate profile of USP10-B change during different developmental stages?

  • Are there development-specific substrates that are not recognized in other contexts?

  • How does the balance between redundant and specific DUB functions change during development?

• Cellular signaling pathway integration:

  • How does USP10-B function integrate with known developmental signaling pathways (Wnt, Notch, BMP, etc.)?

  • Does USP10-B regulate key developmental transcription factors or morphogens?

  • How does the p53-regulatory function of USP10-B contribute to normal development versus stress responses?

• Evolutionary considerations:

  • How conserved is USP10 function between amphibian and mammalian development?

  • Are there species-specific adaptations in USP10 function that reflect different developmental strategies?

  • Can Xenopus USP10-B studies inform understanding of human developmental disorders?

• Technical challenges:

  • What are the most effective approaches for studying USP10-B function in the context of whole Xenopus embryos versus extract systems?

  • How can the redundancy between USP10-B and other DUBs be effectively addressed in developmental studies?

  • What are the best tools and reagents needed to advance understanding of USP10-B in developmental contexts?

Addressing these questions will require integration of biochemical approaches (as demonstrated in the extract studies) with developmental biology techniques specific to the Xenopus model system .

How conserved are USP10 functions between Xenopus and mammalian systems?

Based on the available research, several lines of evidence suggest substantial conservation of USP10 functions between Xenopus and mammalian systems:

• Substrate conservation:

  • The p53 regulatory function appears conserved, with USP10 serving as a key deubiquitinating enzyme for p53 in both mammalian systems and likely in Xenopus

  • Similar interactions with binding partners have been observed across species, such as SPATA proteins with CYLD and GBP1/GBP2 with USP10, suggesting conservation of protein-protein interaction networks

• Catalytic mechanism conservation:

  • The fundamental catalytic mechanism involving cysteine protease activity to cleave ubiquitin from substrates appears conserved across species

  • The ability of human recombinant DUBs (like USP7) to rescue substrate degradation in Xenopus extracts suggests functional conservation of catalytic mechanisms

• Structural conservation:

  • The USP domain structure and organization appears broadly conserved across species

  • The sequence conservation supports similar three-dimensional structures and substrate recognition mechanisms

• Functional redundancy patterns:

  • The observation of DUB redundancy in Xenopus extracts mirrors findings in mammalian systems

  • Similar patterns of overlapping substrate specificity have been observed across species

• Regulatory mechanisms:

  • Subcellular localization changes in response to cellular stresses (such as DNA damage) appear to be a conserved regulatory mechanism

  • Similar post-translational modifications likely regulate USP10 activity across species

While substantial conservation exists, species-specific differences may include:

• Developmental timing and tissue-specific expression patterns

• Subtle differences in substrate recognition specificity

• Variations in regulatory mechanisms and interacting partners

• Potentially different compensation mechanisms when USP10 function is compromised

The significant conservation observed suggests Xenopus studies can provide valuable insights into fundamental USP10 functions relevant to human biology and disease .

What insights from Xenopus USP10-B studies might have therapeutic relevance for human cancers?

Several key insights from Xenopus USP10-B studies have potential therapeutic relevance for human cancers:

• DUB redundancy implications:

  • The finding that USP10-B likely functions redundantly with other DUBs in Xenopus systems suggests that combined inhibition strategies may be necessary for effective cancer therapy

  • Single DUB inhibition might be insufficient due to compensation by redundant enzymes

  • This insight could inform combination therapy approaches targeting multiple DUBs simultaneously

• Substrate identification:

  • Studies in Xenopus provide a system to identify evolutionarily conserved USP10 substrates

  • These substrates may represent core cancer-relevant targets that could be exploited therapeutically

  • Proteins destabilized upon USP10 inhibition in Xenopus systems may represent potential tumor suppressors regulated by USP10 in human cancers

• USP10-p53 axis:

  • The conservation of p53 regulation by USP10 suggests this axis remains a promising therapeutic target

  • In cancers with wild-type p53, inhibiting USP10 might lead to p53 destabilization

  • In cancers with mutant p53, USP10 inhibition might reduce levels of oncogenic mutant p53

• Diagnostic and prognostic relevance:

  • Human studies show significant diagnostic and prognostic value of USP10 across multiple cancer types

  • The high AUC values for USP10 as a diagnostic marker (ranging from 0.714 to 1.000 depending on cancer type) suggest potential clinical utility

  • The correlation between USP10 expression and poor prognosis in multiple cancers suggests it may be a viable therapeutic target

• Immune infiltration correlations:

  • The significant correlation between USP10 expression and immune cell infiltration in multiple cancers suggests potential immunomodulatory roles

  • This insight opens possibilities for combining USP10 inhibition with immunotherapy approaches

  • Understanding how USP10 affects the tumor immune microenvironment could inform personalized therapeutic strategies

• Tumor mutation burden associations:

  • The association between USP10 expression and tumor mutation burden suggests potential biomarkers for identifying patients who might benefit most from USP10-targeted therapies

  • This could enable more precise patient selection for clinical trials of USP10 inhibitors

These insights demonstrate how fundamental research in model systems like Xenopus can inform translational approaches to cancer therapy .

What methodological advances in Xenopus USP10-B research could be applied to studying other DUBs across model systems?

Several innovative methodological approaches from Xenopus USP10-B research could be broadly applied to study other DUBs across different model systems:

• Comprehensive DUB profiling strategy:

  • The approach of using UbVS labeling to simultaneously detect multiple active DUBs provides a powerful tool for profiling DUB activity across various conditions and models

  • This technique could be adapted to study tissue-specific or developmental stage-specific DUB activity profiles in other model organisms

• Functional redundancy analysis framework:

  • The experimental design comparing broad DUB inhibition with specific inhibition, coupled with rescue experiments, provides an excellent framework for dissecting functional redundancy

  • This approach could be applied to study redundancy among DUBs in mammalian cells, zebrafish, Drosophila, and other models

• Substrate identification pipeline:

  • The strategy of using TMT-based quantitative proteomics following DUB inhibition/depletion provides a systematic approach for identifying DUB substrates

  • This pipeline could be adapted for substrate discovery for other DUB family members across model systems

• Chemical biology approaches:

  • The use of specific small molecule inhibitors (like the USP7 inhibitor XL-188) in combination with broad inhibitors (UbVS) demonstrates an effective approach for dissecting individual DUB functions

  • Similar approaches could be applied to study other DUB family members, particularly those with redundant functions

• Cross-species validation strategy:

  • The demonstration that human recombinant DUBs can rescue function in Xenopus extracts provides a powerful approach for cross-species validation

  • This approach could be expanded to systematically compare DUB functions across evolutionary diverse systems

• Integration with clinical data:

  • The correlation analysis between USP10 expression, immune infiltration, and clinical outcomes provides a template for translating basic DUB research to clinical relevance

  • Similar approaches could connect fundamental studies in model organisms to human disease contexts for other DUB family members

• Activity-based probe development:

  • The use of HA-tagged UbVS provides a template for developing increasingly specific activity-based probes

  • This approach could be expanded to develop probes with selectivity for specific DUB subfamilies or individual enzymes across model systems

These methodological advances collectively provide a comprehensive toolkit that could accelerate research on the broader DUB family across diverse model systems .

What are the most critical unanswered questions about USP10-B that should guide future Xenopus research?

Based on the current state of knowledge, several critical questions about USP10-B should guide future Xenopus research:

• Complete substrate landscape identification:

  • What is the full complement of USP10-B substrates in Xenopus?

  • How does this substrate profile change during development or in response to cellular stresses?

  • Which substrates are uniquely dependent on USP10-B versus those with redundant regulation?

• Mechanistic understanding of redundancy:

  • What are the molecular determinants that govern functional redundancy between USP10-B and other DUBs?

  • Is redundancy a result of overlapping substrate recognition or independent targeting of the same substrates?

  • Can specific domains or motifs in USP10-B be identified that confer unique versus redundant functions?

• Developmental roles:

  • What are the specific developmental processes regulated by USP10-B in Xenopus?

  • How does USP10-B function integrate with known developmental signaling pathways?

  • What phenotypes result from USP10-B disruption at different developmental stages?

• Regulatory mechanisms:

  • How is USP10-B activity regulated during development and in response to cellular stresses?

  • What post-translational modifications control USP10-B function?

  • What protein-protein interactions modulate USP10-B activity or substrate specificity?

• Evolutionary conservation analysis:

  • Which aspects of USP10 function are most strongly conserved between Xenopus and mammals?

  • Are there species-specific adaptations in USP10 function?

  • Can evolutionary conservation patterns identify the most fundamental USP10 functions?

• Technological development needs:

  • What tools and resources are needed to better study USP10-B in Xenopus?

  • How can specificity be achieved when targeting USP10-B in the context of redundant DUBs?

  • What are the most effective approaches for tissue-specific or stage-specific manipulation of USP10-B function?

Addressing these questions will require interdisciplinary approaches combining biochemistry, developmental biology, genetics, and systems biology perspectives .

How should researchers approach the challenge of DUB redundancy when studying USP10-B function?

Researchers should employ a multi-faceted strategic approach to address the challenge of DUB redundancy when studying USP10-B function:

• Hierarchical inhibition approach:

  • Begin with broad DUB inhibition (UbVS) to identify the complete set of potentially regulated substrates

  • Progress to specific USP10-B inhibition/depletion to identify unique dependencies

  • Employ combinatorial inhibition strategies targeting USP10-B alongside potentially redundant DUBs

  • This tiered approach can systematically unmask functions hidden by redundancy

• Substrate-specific analysis:

  • For each identified substrate, perform targeted analysis of ubiquitination patterns

  • Determine which ubiquitin chain linkages are present and which DUBs act upon them

  • Assess whether multiple DUBs act on different ubiquitin chains on the same substrate

  • This approach can reveal substrate-specific redundancy mechanisms

• Quantitative redundancy assessment:

  • Develop quantitative measures of redundancy based on substrate stabilization under different conditions

  • Compare the extent of substrate rescue by individual DUBs versus combinations

  • Establish mathematical models of DUB redundancy networks

  • This approach can move beyond binary (redundant/non-redundant) classifications

• Context-dependent redundancy mapping:

  • Systematically assess redundancy across different developmental stages

  • Evaluate redundancy under different cellular stress conditions (DNA damage, proteotoxic stress, etc.)

  • Determine if redundancy patterns shift in different cellular compartments

  • This approach can reveal conditional redundancy that might be exploited experimentally

• Genetic compensation analysis:

  • Assess whether long-term USP10-B depletion leads to compensatory upregulation of redundant DUBs

  • Compare acute versus chronic inhibition/depletion phenotypes

  • Use inducible systems to bypass potential developmental compensation

  • This approach can distinguish between immediate redundancy versus adaptive compensation

• Cross-species validation:

  • Compare redundancy patterns between Xenopus and mammalian systems

  • Identify evolutionarily conserved versus divergent redundancy networks

  • Use conservation as a guide to prioritize the most fundamental redundancy relationships

This comprehensive approach acknowledges redundancy as a central feature rather than an experimental obstacle, potentially revealing biological insights about the evolution and organization of the ubiquitin system .