Recombinant Mouse Ubiquitin carboxyl-terminal hydrolase 47 (Usp47), partial

What is the structural composition of mouse Usp47 and how does it differ from human USP47?

The mouse Usp47 gene shares approximately 89.54% similarity with the human USP47 gene. The human USP47 gene encodes a protein of 1,375 amino acids with an approximate molecular weight of 157 kDa, with two additional transcripts producing proteins of 1,287aa (~147 kDa) and 1,355aa (~155 kDa) . The largest domain is the N-terminal catalytic core (spanning residues 188-564 in humans), which contains the conserved catalytic triad composed of Cysteine, Histidine, and Aspartic acid/Asparagine residues . Like USP7, Usp47 also contains multiple UBL (ubiquitin-like) domains that enhance catalytic activity and facilitate substrate binding . Compared to human USP7, Usp47 processes Lys48- and Lys63-linked polyubiquitin chains more efficiently and exhibits specific sequence differences in the catalytic core that may enable selective targeting .

What are the standard methods for expressing and purifying recombinant mouse Usp47?

For recombinant expression of mouse Usp47, researchers typically employ bacterial expression systems using E. coli strains such as BL21(DE3) with vectors containing 6xHis or GST tags to facilitate purification. The protocol involves:

• Cloning the Usp47 cDNA into an appropriate expression vector

• Transforming the construct into E. coli

• Inducing protein expression with IPTG at lower temperatures (16-18°C) to enhance solubility

• Lysing cells using sonication in buffer containing protease inhibitors

• Purifying using affinity chromatography (Ni-NTA for His-tagged or glutathione sepharose for GST-tagged)

• Further purification via size exclusion chromatography

• Verifying purity using SDS-PAGE and western blotting

• Assessing enzymatic activity using fluorogenic ubiquitin substrates

For functional studies, many researchers also use partial constructs focusing on the catalytic domain (CD) or catalytic domain with UBL domains to address specific research questions .

How can I validate the deubiquitinating activity of recombinant mouse Usp47 in vitro?

To validate the deubiquitinating activity of recombinant mouse Usp47, researchers typically employ several complementary approaches:

• Fluorogenic substrate assay: Using ubiquitin-AMC (Ub-AMC) as a substrate to measure enzymatic activity kinetically. The assay involves monitoring the release of the fluorescent AMC group upon deubiquitination. For Usp47 CD-UBL12, reported kinetic parameters include a KM of approximately 3.0 ± 0.6 μM and kcat of 0.03 ± 0.002 s−1, resulting in a catalytic efficiency (kcat/KM) of approximately 12 × 103 M−1 s−1 .

• Di-ubiquitin chain cleavage assay: Incubating recombinant Usp47 with different di-ubiquitin chains (K48, K63, etc.) and analyzing the generation of mono-ubiquitin by SDS-PAGE and western blotting to assess chain-type specificity .

• Site-directed mutagenesis: Creating catalytically inactive mutants (C97S or C97A in the catalytic domain) as negative controls. The C97S mutation has been shown to maintain ubiquitin binding (KD = 1.1 μM ± 0.1 μM) without catalytic activity, making it useful for structural studies .

• Isothermal titration calorimetry (ITC): For measuring binding affinity between Usp47 and ubiquitin, particularly with catalytically inactive mutants .

What are the optimal conditions for studying Usp47 activity in cellular assays?

For optimal cellular assays investigating Usp47 activity, consider the following methodological approaches:

• Cell line selection: Use cell lines with moderate to high endogenous Usp47 expression. For cancer research, cell lines like HCT116 (colorectal cancer), T47D (breast cancer), or K562 (CML) have been successfully used in Usp47 studies .

• Knockdown approaches: For transient knockdown, use validated siRNA oligos targeting Usp47. Two different siRNA sequences should be tested to confirm specificity. For stable knockdown, shRNA expressed from lentiviral vectors has shown effective reduction of Usp47 levels .

• Overexpression systems: Use expression vectors with epitope tags (FLAG, HA, etc.) for immunoprecipitation and detection. Include both wild-type and catalytically inactive mutants (C97S) as controls.

• Activity measurement: Assess global ubiquitination levels using anti-ubiquitin antibodies after Usp47 perturbation. For substrate-specific effects, immunoprecipitate suspected target proteins and probe for ubiquitination.

• Cellular assays: Measure proliferation (MTT, BrdU), apoptosis (Annexin V/PI staining, PARP cleavage), cell cycle distribution (PI staining), and DNA damage repair (γH2AX foci, comet assay) as functional readouts. Usp47 knockdown has been shown to significantly increase the apoptotic index and the percentage of cells in G2/M phase .

• Drug sensitivity assays: Combine Usp47 modulation with therapeutic agents like chemotherapeutics to assess synergistic effects. Usp47 depletion has been demonstrated to markedly increase the cytotoxic effects of chemotherapeutic agents, confirmed by activation of caspase 3, caspase 7, and PARP cleavage .

How can I generate and validate Usp47 knockout mouse models for cancer research?

Generating and validating Usp47 knockout mouse models involves several critical steps:

• Knockout strategy selection:

  • Conventional knockout: Targeting critical exons using CRISPR-Cas9 or gene trap strategies

  • Conditional knockout: Using Cre-loxP system for tissue-specific or inducible deletion

  • Hypomorphic models: Gene trap insertions that reduce but don't eliminate expression

• Generation approach:

  • CRISPR-Cas9: Design guide RNAs targeting early exons of Usp47

  • Gene trap: Use embryonic stem cell lines with trap vector insertions (like the RRJ301 line mentioned in the literature)

• Validation methods:

  • Genotyping: PCR-based strategies to identify homozygous, heterozygous, and wild-type mice

  • Expression analysis: Quantitative RT-PCR using primers covering untranslated regions and coding sequences

  • Protein verification: Western blotting of tissues and derived MEFs to confirm protein reduction/absence

  • Functional validation: Assess cellular phenotypes such as proliferation and apoptosis sensitivity

• Cancer research applications:

  • Transplantation models: Use Usp47-deficient cells in xenograft or syngeneic models

  • Crossing with cancer-prone models: Breed with established cancer models (e.g., BCR-ABL for CML)

  • Drug response: Assess therapy response in Usp47-deficient tumors

Note that complete Usp47 knockout might not be viable, as studies have shown that hypomorphic alleles result in reduced but not abolished expression. In one study, mice homozygous for a hypomorphic Usp47 allele were viable and fertile, but showed increased sensitivity to ultraviolet-induced cell death in derived MEFs .

What are the recommended protocols for investigating Usp47-protein interactions in cancer cells?

For investigating Usp47-protein interactions in cancer cells, the following methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against endogenous Usp47 or epitope-tagged Usp47 constructs

  • Include appropriate controls (IgG, catalytically inactive mutants)

  • Perform reciprocal Co-IPs when possible

  • Analyze interaction under various conditions (e.g., ribosomal stress for RPS2 interaction)

  • Consider crosslinking for transient interactions

• Proximity-based approaches:

  • BioID or TurboID: Fusion of biotin ligase to Usp47 to identify proximal proteins

  • APEX2: Peroxidase-based proximity labeling for capturing rapid interactions

  • These methods are particularly useful for detecting weak or transient interactions

• Domain mapping:

  • Generate truncation mutants to identify interaction domains

  • For Usp47, the catalytic core domain (CD) mediates interactions with proteins like β-TrCP, SATB1, and YAP

  • The UBL domains are involved in binding substrates like RPS2

• Binding motif analysis:

  • Investigate specific binding motifs, such as the novel DEGxxxE motif in Usp47 that mimics phosphoserine for β-TrCP binding

  • Compare with typical binding motifs (e.g., DSGxxS for β-TrCP) to understand substrate specificity

• Functional validation:

  • Mutagenesis of key residues in the interaction interface

  • Competition assays with peptides derived from binding regions

  • In vitro deubiquitination assays with purified components

These approaches have led to the identification of several Usp47 interaction partners, including β-TrCP, SATB1, RPS2, RPL11, YB-1, and various components of DNA damage repair pathways, contributing to our understanding of Usp47's role in cancer biology .

How does Usp47 contribute to cancer development and resistance mechanisms?

Usp47 contributes to cancer development and resistance mechanisms through multiple pathways:

• Regulation of DNA damage repair:

  • Usp47 deubiquitinates and stabilizes DNA polymerase β (Polβ), enhancing base excision repair

  • In CML, Usp47 stabilizes Y-box binding protein 1 (YB-1), contributing to DNA damage repair and TKI resistance

  • Knockdown of Usp47 increases sensitivity to DNA-damaging agents in multiple cancer types

• Modulation of the p53 pathway:

  • Usp47 deubiquitinates ribosomal proteins RPS2 and RPL11, regulating their interaction with MDM2

  • Under normal conditions, Usp47 prevents RPS2-mediated inhibition of MDM2, suppressing p53 activation

  • Under ribosomal stress, Usp47 knockdown enhances p53 stabilization, inhibiting cell proliferation in a p53-dependent manner

• Wnt/β-catenin signaling activation:

  • Usp47 deubiquitinates β-catenin, preventing its degradation and promoting Wnt signaling

  • Knockdown of Usp47 increases β-catenin ubiquitination and degradation, inhibiting proliferation in lung and prostate cancer cells

• Regulation of epithelial-mesenchymal transition (EMT):

  • In colorectal cancer, Usp47 deubiquitinates Snail and SATB1, promoting EMT and metastasis

  • Usp47 can also stabilize E-cadherin in normal conditions, but under hypoxic conditions or during EMT, its binding to Snail and SATB1 is enhanced

• Cancer stem cell maintenance:

  • Usp47 promotes CRC development by maintaining the stemness of colorectal cancer stem cells

  • In CML, Usp47 is essential for the survival of Lin−Sca1+c-Kit+ leukemia stem/progenitor cells

• Chemoresistance mechanisms:

  • In gastric cancer, Usp47 activates the NF-κB signaling pathway by promoting RelA nuclear translocation, conferring resistance to camptothecin and etoposide

  • In CML, Usp47 is highly upregulated compared to normal bone marrow CD34+ cells and contributes to TKI resistance

These mechanisms highlight Usp47 as a potential target for cancer therapy, particularly in overcoming resistance to conventional treatments.

What are the current approaches for developing selective inhibitors of Usp47 and how can their efficacy be evaluated?

Current approaches for developing selective Usp47 inhibitors and evaluating their efficacy include:

• Inhibitor design strategies:

  • Structure-based design targeting unique features of the Usp47 catalytic domain

  • Analysis of sequence differences between Usp47 and homologous USPs, particularly USP7

  • Focus on the specific sequence of Usp47 at 431-485aa, which differs from USP7 and USP40

  • Targeting unique substrate-binding motifs, such as the "DEGxxxE" motif for β-TrCP binding

  • Development of allosteric inhibitors that prevent catalytic triad rearrangement

• Known inhibitors and compounds:

  • P22077: Originally developed as a USP7/USP47 dual inhibitor, has shown efficacy against CML cells with or without TKI resistance

  • FT671 and FT827: While these target USP7 specifically, studying their mechanisms provides insights for developing Usp47-specific inhibitors

• In vitro efficacy evaluation:

  • Enzymatic assays using Ub-AMC or di-ubiquitin substrates to determine IC50 values

  • Thermal shift assays to confirm direct binding

  • Structural studies using X-ray crystallography or cryo-EM to visualize inhibitor binding

  • Target engagement assays in cell lysates

• Cellular assays for efficacy:

  • Assessment of substrate stabilization (e.g., DNA polymerase β, YB-1, β-catenin)

  • Cell viability and proliferation assays in cancer cell lines

  • Combination studies with chemotherapeutics or targeted therapies

  • Examination of pathway modulation (p53, Wnt, DNA damage repair)

• In vivo evaluation models:

  • Xenograft models using cancer cell lines with known Usp47 dependence

  • Patient-derived xenograft (PDX) models for translational relevance

  • Genetically engineered mouse models of cancer (e.g., BCR-ABL-induced CML)

  • Assessment of inhibitor effects on cancer stem/progenitor cells

For example, the inhibitor P22077 has demonstrated efficacy in eliminating leukemia stem/progenitor cells in CML mice while exhibiting low toxicity to normal peripheral blood mononuclear cells, suggesting that selective Usp47 inhibition can achieve a therapeutic window .

How can conflicting evidence about Usp47's role in different cancer types be reconciled?

Reconciling conflicting evidence about Usp47's role in different cancer types requires careful consideration of context-dependent factors:

• Cell type and tissue specificity:

  • Usp47 may have different substrate preferences in different cell types

  • The abundance of Usp47 relative to its substrates varies across tissues

  • The expression levels of competing DUBs differ between cancer types

  • Example: While Usp47 is generally oncogenic, its effects may be more pronounced in tissues with high expression, such as breast cancer compared to other tissues

• Substrate competition and availability:

  • In different contexts, Usp47 may preferentially bind to different substrates

  • The E-cadherin/Snail paradox demonstrates this: Usp47 can deubiquitinate and stabilize E-cadherin in normal conditions, but under hypoxia or during EMT, it preferentially binds Snail, indirectly reducing E-cadherin

  • This suggests a "substrate switching" mechanism dependent on cellular state

• Signaling network status:

  • The activation state of pathways like p53, Wnt, and NF-κB influences Usp47's impact

  • In p53-wild-type cells, Usp47 knockdown inhibits proliferation by activating p53, but this effect is absent in p53-knockout cells

  • In gastric cancer, Usp47's effect on drug resistance depends on the activation of NF-κB signaling

• Experimental design considerations:

  • Acute vs. chronic Usp47 depletion may have different effects

  • Complete knockout vs. partial knockdown can yield different phenotypes

  • The hypomorphic Usp47 mouse model showed viability despite reduced expression

  • Different domains of Usp47 may mediate distinct functions

• Methodological approach for resolving conflicts:

  • Perform tissue-specific and inducible knockout studies

  • Use domain-specific mutations to separate different functions

  • Conduct proteome-wide substrate identification across different cancer types

  • Develop markers to assess which Usp47 functions are active in specific contexts

This understanding can guide personalized therapeutic approaches targeting Usp47, as its inhibition may be more effective in certain cancer types or in combination with specific therapies based on the molecular context.

Clinical and Translational Aspects

Combination strategies utilizing Usp47 inhibition with existing cancer therapies offer promising approaches to overcome resistance:

• Combination with tyrosine kinase inhibitors (TKIs) in CML:

  • Usp47 inhibition via P22077 showed cytotoxicity to CML cells with or without TKI resistance

  • Mechanistically, Usp47 knockdown significantly inhibited both BCR-ABL-induced CML and BCR-ABL T315I-induced CML in mice, addressing resistance to imatinib and second-generation TKIs

  • The combination targets leukemia stem/progenitor cells that are often resistant to TKI monotherapy

• Sensitization to DNA-damaging agents:

  • Usp47 depletion increases sensitivity to ultraviolet radiation in mouse embryonic fibroblasts

  • Knockdown of Usp47 significantly enhances the cytotoxic effects of chemotherapeutic agents, with activation of caspase 3, caspase 7, and PARP cleavage

  • This synergy is based on Usp47's role in DNA repair through deubiquitination of DNA polymerase β

• Overcoming NF-κB-mediated resistance in gastric cancer:

  • Usp47 knockdown promotes drug sensitivity in camptothecin and etoposide-resistant gastric cell lines

  • The mechanism involves disruption of Usp47-mediated NF-κB signaling activation

  • This strategy specifically addresses acquired resistance rather than intrinsic resistance

• Targeting p53-dependent pathways:

  • In p53 wild-type cancers, Usp47 inhibition leads to p53 stabilization through the ribosomal protein-MDM2 axis

  • Combination with MDM2 inhibitors could potentially enhance p53 activation

  • This approach would be specifically effective in p53 wild-type tumors

• Addressing Wnt/β-catenin-dependent resistance:

  • Usp47 inhibition reduces β-catenin stability, suppressing Wnt signaling

  • Combination with other Wnt pathway inhibitors could provide synergistic effects

  • This strategy may be particularly effective in Wnt-dependent cancers like colorectal cancer

• Experimental design for combination therapy studies:

  • Use matrix dose-response studies to identify synergistic combinations

  • Assess sequence-dependent effects (e.g., Usp47 inhibition before or after chemotherapy)

  • Evaluate long-term effects on resistance development with repeated treatment cycles

  • Consider cancer stem cell targeting as a readout for effective combinations

These combination approaches leverage Usp47's involvement in multiple cancer pathways to overcome resistance mechanisms, potentially expanding the therapeutic window and improving patient outcomes.

What biomarkers could predict sensitivity to Usp47-targeted therapies in cancer patients?

Several biomarkers could potentially predict sensitivity to Usp47-targeted therapies in cancer patients:

• Expression-based biomarkers:

  • High Usp47 expression levels in tumor tissue relative to matched normal tissue

  • Elevated Usp47 protein expression detected by immunohistochemistry

  • Increased Usp47 mRNA expression (detected via RT-PCR or RNA-seq)

  • Expression ratio of Usp47 to other competing DUBs that target the same substrates

• Substrate accumulation markers:

  • Elevated levels of known Usp47 substrates such as:

    • DNA polymerase β

    • β-catenin

    • YB-1 (Y-box binding protein 1)

    • SATB1

  • The ratio of ubiquitinated to non-ubiquitinated forms of these substrates

• Pathway activation indicators:

  • p53 pathway status: Usp47 inhibition shows stronger effects in p53 wild-type settings

  • DNA damage repair pathway activity: High dependency indicates potential sensitivity

  • Wnt/β-catenin pathway activation: Tumors with active Wnt signaling may be more dependent on Usp47

  • NF-κB activation status: Particularly relevant for gastric cancers with chemoresistance

• Genetic and molecular markers:

  • Mutations or deletions in competing DUBs that may increase Usp47 dependency

  • Genomic instability signatures that suggest reliance on DNA repair pathways

  • BCR-ABL fusion (particularly T315I mutation) in CML patients

  • Specific cancer stem cell markers in CML (Lin−Sca1+c-Kit+) that indicate potential sensitivity to Usp47 inhibition

• Functional assays:

  • Ex vivo drug sensitivity testing of patient-derived cells

  • Measurement of apoptotic response to Usp47 inhibitors in circulating tumor cells

  • Assessment of DNA damage accumulation following exposure to Usp47 inhibitors

Predictive biomarker panel example for CML patients:

These biomarkers would help identify patients most likely to benefit from Usp47-targeted therapies, enabling a more personalized approach to cancer treatment.

What structural studies are needed to better understand Usp47's substrate specificity and develop selective inhibitors?

Several critical structural studies are needed to advance our understanding of Usp47's substrate specificity and facilitate selective inhibitor development:

• Full-length Usp47 structure determination:

  • While partial structures exist, the complete three-dimensional structure of Usp47 remains undetermined

  • Cryo-EM would be particularly suitable for capturing the full-length protein's flexible domains

  • This would reveal interdomain interactions that regulate activity and substrate recognition

• Catalytic domain comparative analysis:

  • High-resolution crystal structures of the Usp47 catalytic domain in both apo and ubiquitin-bound states

  • Current structures at 3.0 Å resolution provide initial insights, but higher resolution is needed

  • Detailed comparison with USP7 catalytic domain to identify selective binding pockets

  • Focus on the unique sequence at 431-485aa that differs from USP7 and USP40

• UBL domain functional studies:

  • Crystal structures of Usp47 UBL domains individually and in complex with the catalytic domain

  • Investigation of how UBL domains enhance DUB activity, as they increase USP7 activity by 100-fold

  • Mapping the interactions between UBL domains and specific substrates

• Substrate complex structures:

  • Co-crystal structures with various substrates (DNA polymerase β, β-catenin, YB-1, etc.)

  • Identification of substrate-specific binding interfaces beyond the catalytic site

  • Analysis of the "DEGxxxE" motif that mediates β-TrCP binding

• Inhibitor binding studies:

  • Structure of Usp47 in complex with P22077 and other inhibitors

  • Mapping the conformational changes induced by inhibitor binding

  • Identification of allosteric sites that could be exploited for selective inhibition

• Dynamic structural analyses:

  • Molecular dynamics simulations to understand conformational flexibility

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • NMR studies of domain interactions and substrate recognition

• Methodological considerations:

  • Use of catalytically inactive mutants (C97S) that maintain ubiquitin binding (KD = 1.1 μM ± 0.1 μM)

  • Truncation constructs to overcome crystallization challenges

  • Nanobodies or other crystallization chaperones to stabilize flexible regions

These structural studies would provide crucial insights into Usp47's mechanism of action and enable structure-based design of selective inhibitors with therapeutic potential.

How do post-translational modifications regulate Usp47 activity and substrate selection?

Post-translational modifications (PTMs) likely play crucial roles in regulating Usp47 activity and substrate selection, though this area remains understudied:

• Phosphorylation:

  • Potential phosphorylation sites in Usp47 can be predicted using bioinformatics tools

  • Phosphorylation may alter enzyme activity, as observed with other USPs

  • Kinases involved in DNA damage response (ATM, ATR, CHK1/2) may phosphorylate Usp47 to regulate its DNA repair functions

  • Cell cycle-dependent kinases might phosphorylate Usp47 to control substrate preference in different phases

• Ubiquitination:

  • Auto-regulation through self-deubiquitination is possible

  • E3 ligases like SMURF2 (which opposes Usp47 in SATB1 regulation) may target Usp47 itself

  • Different ubiquitin chain linkages (K48, K63) on Usp47 could alter its stability or function

  • Methodological approach: Mass spectrometry analysis of immunoprecipitated Usp47 to identify ubiquitination sites

• SUMOylation:

  • SUMOylation often regulates nuclear localization and protein-protein interactions

  • As Usp47 interacts with nuclear proteins like SATB1, SUMOylation may control these interactions

  • Experimental approach: In vitro SUMOylation assays with purified components followed by activity tests

• Acetylation:

  • Acetylation might regulate Usp47 localization or substrate recognition

  • Deacetylases like HDACs or Sirtuins could modulate Usp47 activity

  • Techniques: Acetylome analysis in cells with Usp47 modulation

• PTM-dependent regulation mechanisms:

  • Conformational changes affecting catalytic triad alignment

  • Altered interactions between catalytic domain and UBL domains

  • Modified substrate recognition surfaces

  • Changes in subcellular localization

  • Regulation of protein-protein interactions with cofactors

• Context-dependent PTM patterns:

  • Different cellular stresses (DNA damage, hypoxia, nutrient deprivation) likely induce distinct PTM patterns

  • Cell type-specific PTM profiles may explain differential functions across tissues

  • PTMs could explain the substrate switching phenomenon observed with E-cadherin/Snail

• Experimental approaches to study PTM regulation:

  • Site-directed mutagenesis of predicted PTM sites

  • Phospho-mimetic and phospho-deficient mutants

  • Proximity labeling to identify modifying enzymes

  • In vitro reconstitution of PTM-dependent activity

  • Temporal analysis of PTMs during cell cycle or stress response

Understanding the PTM landscape of Usp47 would provide insights into its regulation and potentially reveal new therapeutic opportunities through manipulation of these modifications.

What are the emerging roles of Usp47 beyond cancer that warrant investigation?

Beyond cancer, Usp47 has emerging roles in several biological processes and disease contexts that warrant further investigation:

These emerging areas represent exciting new frontiers for Usp47 research, potentially expanding its significance beyond cancer and revealing new therapeutic opportunities across multiple disease contexts.