Recombinant Mouse Ubiquitin carboxyl-terminal hydrolase 40 (Usp40), partial

What is Ubiquitin carboxyl-terminal hydrolase 40 (Usp40)?

Ubiquitin carboxyl-terminal hydrolase 40 (Usp40) is a deubiquitinating enzyme belonging to the ubiquitin-specific protease (USP) family. These enzymes remove ubiquitin moieties from proteins, potentially rescuing them from degradation through the ubiquitin-proteasome system. While the HMDB database suggests Usp40 "may be catalytically inactive" , recent research demonstrates it possesses deubiquitinating functions in specific contexts, such as its ability to deubiquitinate and stabilize Claudin1 in hepatocellular carcinoma .

The methodological approach to studying Usp40 typically involves:

• Expression analysis using Western blotting, RT-qPCR, or immunohistochemistry

• Functional studies using gene knockdown or knockout approaches

• Substrate identification through co-immunoprecipitation and deubiquitination assays

• Phenotypic characterization in cellular and animal models

What are the alternative names and designations for Usp40?

Usp40 is known by several alternative names in scientific literature, which is important to consider when conducting comprehensive literature searches:

In mouse models, additional gene aliases include B230215L03Rik and C730029K03 .

What experimental approaches can verify Usp40 catalytic activity?

Despite the HMDB annotation that Usp40 "may be catalytically inactive" , experimental verification of its deubiquitinating activity requires:

• In vitro deubiquitination assays:

  • Purified recombinant Usp40 protein incubated with ubiquitinated substrates

  • Analysis of ubiquitin chain removal using Western blotting

  • Controls should include catalytically inactive mutants (typically Cys→Ser mutations in the catalytic domain)

• Cellular ubiquitination assays:

  • Monitoring substrate ubiquitination levels in cells with Usp40 overexpression or knockdown

  • Pulse-chase experiments to assess protein stability

  • Ubiquitin remnant profiling to identify potential substrates

• Domain-function analysis:

  • Structure-based mutagenesis of catalytic residues

  • Functional complementation with wild-type vs. mutant Usp40

Research has demonstrated that USP40 deubiquitinates Claudin1, providing evidence for its catalytic activity in specific contexts .

How can CRISPR/Cas9 be optimized for targeting mouse Usp40?

Designing effective CRISPR/Cas9 experiments for mouse Usp40 requires careful consideration of several factors:

• Guide RNA selection: The Zhang laboratory at the Broad Institute has designed gRNAs that uniquely target the Usp40 gene in the mouse genome . Key considerations include:

  • Target at least two different exons to increase knockout efficiency

  • Select gRNAs with minimal predicted off-target effects

  • Confirm that gRNAs target functionally important domains

• Delivery optimization:

  • For cell lines: Lentiviral vectors containing the complete gRNA expression cassette

  • For in vivo applications: AAV delivery or direct injection of Cas9-gRNA RNPs

  • For embryonic modifications: Microinjection into zygotes

• Validation strategy:

  • Genomic verification: PCR amplification and sequencing of the target region

  • Protein elimination: Western blotting to confirm knockout

  • Functional validation: Assays specific to Usp40's known activities

When ordering gRNA clones, researchers receive sequence-verified plasmids containing all elements required for gRNA expression and genome binding, including the U6 promoter, spacer sequence, gRNA scaffold, and terminator .

What are the optimal methods for generating Usp40 knockout mouse models?

Based on published research, several approaches have been successfully used to generate Usp40 knockout mouse models:

• Global knockout strategy:

  • The CRISPR/Cas9 system has been employed to generate USP40 global knockout (USP40−/−) mice

  • This approach enables studying systemic effects of Usp40 deficiency

• Conditional knockout approach:

  • Generation of USP40-Loxp mice followed by crossing with tissue-specific Cre lines

  • Example: Endothelial-specific knockout created by crossing USP40floxp/floxp mice with endothelial-specific Cre lines (Tek-Cre or Cdh5-Cre)

  • Allows investigation of tissue-specific Usp40 functions

• Validation requirements:

  • Genotyping: PCR-based confirmation of genomic modifications

  • Expression analysis: Western blotting and RT-qPCR to confirm absence of Usp40

  • Functional assessment: Phenotypic evaluation relevant to Usp40's known roles

• Rescue experiments:

  • Reintroduction of Usp40 using lentiviral vectors expressing human USP40 cDNA

  • Confirms phenotypic specificity and rules out off-target effects

Interestingly, while USP40 knockout in zebrafish produces observable phenotypes, some studies report no apparent phenotypic changes in USP40 knockout mice , highlighting potential species-specific functions or compensatory mechanisms.

How can contradictory findings about Usp40 function across species be reconciled?

The search results reveal intriguing contradictions in Usp40 function across different model systems:

• Species-specific differences:

  • Zebrafish knockdown models show clear phenotypic changes

  • USP40 knockout mice show no apparent phenotypic changes in some studies

  • Human cell studies demonstrate specific functions in endothelial and cancer cells

• Methodological approaches to reconcile differences:

  • Comparative genomics: Analyze sequence conservation and divergence across species

  • Cross-species complementation: Test if human USP40 can rescue phenotypes in mouse models

  • Substrate conservation analysis: Identify whether Usp40 substrates (e.g., Claudin1) are conserved across species

  • Multi-omics comparisons: Perform parallel transcriptomic and proteomic analyses in different species

• Experimental considerations:

  • Use consistent methodologies across species

  • Evaluate both baseline and stressed conditions (e.g., LPS challenge)

  • Consider developmental timing of Usp40 function

  • Assess potential redundancy with other deubiquitinating enzymes

Table: Comparison of Usp40 Function Across Species

What is known about Usp40's role in disease pathogenesis?

Research has identified several disease contexts where Usp40 plays significant roles:

• Hepatocellular Carcinoma (HCC):

  • USP40 mRNA and protein are elevated in HCC compared to normal tissues

  • USP40 overexpression correlates with tumor stage and predicts poor prognosis in HCC patients

  • Functional studies reveal that:

    • Knockdown of USP40 inhibits HCC cell proliferation, migration, and stemness

    • Overexpression of USP40 promotes these malignant characteristics

    • USP40 regulates stemness-related proteins including c-Myc and KLF4

• Endothelial dysfunction and inflammation:

  • USP40 attenuates LPS- or thrombin-induced human lung microvascular endothelial cell barrier disruption

  • USP40 reduces LPS-induced endothelial inflammatory responses, including ICAM1 expression and neutrophil-endothelial interactions

  • This suggests a protective role against vascular inflammation

The contrasting roles in cancer (promoting progression) versus inflammation (protective) highlight the context-dependent nature of Usp40 function, which has important implications for therapeutic targeting.

What are the known substrates and molecular interactions of Usp40?

From the available research, one well-characterized substrate of USP40 is Claudin1:

• Claudin1 as a USP40 substrate:

  • Co-immunoprecipitation demonstrates direct interaction between USP40 and Claudin1

  • Immunofluorescence confirms co-localization in the cytoplasm of HCC cells

  • USP40 regulates Claudin1 at the post-translational level without affecting its mRNA expression

  • USP40 knockdown decreases Claudin1 protein levels while overexpression increases them

  • USP40 and Claudin1 protein expression show positive correlation in HCC specimens

• Functional significance:

  • Claudin1 is a critical tight junction protein involved in cell-cell adhesion

  • In HCC, elevated Claudin1 may contribute to cancer cell proliferation and migration

  • In endothelial cells, regulation of tight junction proteins like Claudin1 could explain USP40's role in barrier function

• Methodological approaches for identifying additional substrates:

  • Ubiquitin proteomics comparing wild-type and Usp40 knockout cells

  • Stability profiling of candidate proteins

  • Pathway analysis based on phenotypic changes in Usp40 models

How does Usp40 regulate endothelial barrier function?

The search results indicate that USP40 plays an important role in maintaining endothelial integrity:

• Protective effects in endothelial cells:

  • USP40 attenuates LPS- or thrombin-induced human lung microvascular endothelial cell barrier disruption

  • USP40 reduces LPS-induced inflammatory responses, including:

    • ICAM1 expression (important for leukocyte adhesion)

    • Neutrophil-endothelial cell interactions

• Potential molecular mechanisms:

  • Regulation of tight junction proteins, potentially including Claudin1

  • Deubiquitination of key barrier-regulating proteins

  • Modulation of inflammatory signaling pathways

• Experimental approaches to study this function:

  • Endothelial-specific knockout models (using Tek-Cre or Cdh5-Cre)

  • In vitro barrier function assays (TEER, permeability, immunofluorescence)

  • Inflammatory stimulation with LPS or thrombin

• Translational significance:

  • Potential therapeutic target for vascular inflammatory conditions

  • Relevance to acute lung injury, sepsis, and other conditions with endothelial dysfunction

What are the best experimental controls when studying recombinant mouse Usp40?

When designing experiments with recombinant mouse Usp40, appropriate controls are essential for valid interpretation:

• For protein expression studies:

  • Positive control: Tissues known to express Usp40 (e.g., liver based on HCC studies)

  • Negative control: Tissues from verified Usp40 knockout mice

  • Loading control: Housekeeping proteins (β-actin, GAPDH) for Western blots

• For functional studies:

  • Catalytically inactive mutant: Cysteine→Serine mutation in the catalytic domain

  • Domain deletion mutants: To map specific function-structure relationships

  • Empty vector controls: For overexpression studies

• For CRISPR/Cas9 experiments:

  • Non-targeting gRNA controls

  • Single guide vs. multiple guide approaches to control for off-target effects

  • Rescue experiments with wild-type Usp40 to confirm specificity

• For animal studies:

  • Wild-type littermates

  • Cre-only controls (for conditional knockout models)

  • Tissue-specific and global knockout comparisons

How can researchers validate antibodies for mouse Usp40 studies?

Antibody validation is crucial for reliable Usp40 research. A systematic approach includes:

• Initial selection criteria:

  • Verify species reactivity (mouse-specific or cross-reactive antibodies)

  • Check application compatibility (Western blot, IHC, ELISA)

  • Review validation data provided by manufacturers

• Experimental validation:

  • Positive control: Overexpression of tagged Usp40 in cell lines

  • Negative control: Lysates from verified Usp40 knockout cells or tissues

  • Peptide competition: Pre-incubation with immunizing peptide should eliminate signal

• Multi-method confirmation:

  • Concordance between different antibodies targeting different epitopes

  • Correlation between protein detection and mRNA expression

  • Consistency across different detection methods (Western blot, IHC, immunofluorescence)

Based on the search results, several validated antibodies are available for mouse Usp40 detection, including:

• Antibodies with reactivity to mouse, human, and rat for Western blot, ELISA, and IHC applications

• Mouse-specific antibodies validated for Western blotting

What are the methodological considerations for studying Usp40 catalytic activity?

Investigating the catalytic activity of Usp40 requires specific methodological considerations, especially given the uncertainty about its enzymatic status :

• In vitro deubiquitination assays:

  • Substrate selection: Using confirmed substrates like Claudin1 rather than generic ubiquitin chains

  • Reaction conditions: Testing various pH, salt concentrations, and cofactors

  • Controls: Including known active DUBs and catalytically inactive Usp40 mutants

• Cellular approaches:

  • Ubiquitin chain analysis: Evaluating changes in K48, K63, and other ubiquitin linkages on substrates

  • Protein stability assays: Cycloheximide chase experiments with Usp40 modulation

  • Ubiquitin mutant studies: Using linkage-specific ubiquitin mutants to determine chain preferences

• Structural considerations:

  • Domain analysis: Identifying and characterizing the catalytic triad and ubiquitin binding regions

  • Molecular dynamics simulations: Predicting substrate binding and catalytic mechanisms

  • Structural alignments with well-characterized USP family members

• Activity modulation:

  • Identifying potential activators or inhibitors

  • Testing posttranslational modifications that might regulate activity

  • Investigating binding partners that might enhance or suppress catalytic function