UCHL3 Mouse

What is UCHL3 and what are its primary functions?

UCHL3 is a deubiquitinating enzyme belonging to the ubiquitin C-terminal hydrolase (UCH) family. It primarily functions in processing pro-ubiquitin gene products and catalyzing the removal of small molecules from ubiquitin . Unlike other UCH family members, UCHL3 has dual functionality - it acts as both a ubiquitin hydrolase and a NEDD8 hydrolase, regulating the maturation of NEDD8 by cleaving its precursor forms . At the cellular level, UCHL3 plays critical roles in maintaining genome integrity through regulation of DNA double-strand break repair mechanisms .

How is UCHL3 expression regulated in response to cellular stress?

UCHL3 is phosphorylated and activated by ATM (Ataxia Telangiectasia Mutated) kinase in response to DNA damage . This activation represents a critical mechanism for the cell's DNA damage response system. Research indicates that UCHL3's deubiquitylating activity increases following cellular stress events, particularly those that induce DNA damage. This regulation appears to be tissue-specific, with certain cell types showing more pronounced changes in UCHL3 activity following stress induction .

What cellular compartments contain UCHL3 protein?

UCHL3 demonstrates compartment-specific localization depending on cell type. In mammalian sperm cells, UCHL3 is predominantly localized in the acrosome and flagella, including the mitochondrial sheath and neck region of some spermatozoa . In dividing cells, UCHL3 has been observed at kinetochores during mitosis, where it plays a role in error correction and chromosome segregation . Cytoplasmic expression is also common across various cell types, consistent with its GO terms which include cytoplasmic localization (GO:0005737) .

How does UCHL3 expression correlate with cancer progression in mouse models?

Studies reveal that UCHL3 expression is significantly elevated in multiple cancer cell lines compared to normal cells. For example, in melanoma cell lines (SK-MEL-2, MV-3, A375, and MUN2B), UCHL3 expression is markedly higher than in normal HaCat cells . This increased expression appears to support cancer cell proliferation, as UCHL3 knockdown using siRNAs significantly reduces proliferation rates in melanoma cells . Mouse models with altered UCHL3 expression show corresponding changes in tumor growth rates, suggesting that UCHL3 functions as a potential oncogene in certain contexts.

What methodologies are most effective for studying UCHL3's role in cancer therapy resistance?

Effective methodologies include:

• RNA interference approaches using validated siRNA sequences (e.g., 5′-CUGAAGAACGAGCCAGAUATT−3′ and 5′-UAUCUGGCUCGUUCUUCAGTT−3′) that have demonstrated significant knockdown efficiency .

• Pharmacological inhibition using specific UCHL3 inhibitors such as 4,5,6,7-Tetrachloro-1H-Indene-1,3(2H)-dione (TCID), which allows for temporal control of UCHL3 activity .

• Combination studies of UCHL3 inhibition with standard chemotherapeutic agents or radiation to assess sensitization effects, particularly important given that high UCHL3 expression has been associated with resistance to radiation and chemotherapy in breast cancer models .

• Cell viability assays (MTT) performed on days 2-4 after treatment to capture the full temporal window of proliferation effects .

What are the molecular mechanisms by which UCHL3 contributes to melanoma development?

UCHL3 contributes to melanoma development through several mechanisms:

• Regulation of autophagy: UCHL3 knockdown enhances autophagy, autophagosome formation, and LC3B protein expression in melanoma cells .

• NEDD8 pathway modulation: UCHL3 affects NEDD8 protein expression, with knockdown resulting in decreased NEDD8 levels and altered LC3II/LC3I ratios .

• Cell proliferation control: siRNA-mediated UCHL3 silencing significantly suppresses melanoma cell proliferation while enhancing apoptosis .

These mechanisms suggest that UCHL3 promotes melanoma progression by suppressing autophagy and apoptosis while enhancing proliferation, likely through its effects on the NEDD8 signaling pathway.

How does UCHL3 expression correlate with sperm quality parameters in mouse models?

UCHL3 expression positively correlates with multiple sperm quality parameters. Studies show significant positive correlations between UCHL3 levels and:

These data demonstrate that UCHL3 serves as a potential biomarker for sperm quality assessment, with higher UCHL3 expression associated with improved spermatozoal parameters .

What techniques are recommended for detecting UCHL3 in mouse sperm cells?

For detecting UCHL3 in mouse sperm cells, the following techniques are recommended:

• Immunofluorescence microscopy to visualize UCHL3 localization in sperm structures, particularly in the acrosome and flagella .

• Western blotting using anti-UCHL3 antibodies (recommended dilution 1:500) with α-tubulin (1:5,000) as a loading control. For protein extraction, approximately 1.0 × 10^7 spermatozoa should be lysed in 200 μL cold lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65mM DTT, 1mM PMSF, and proteinase inhibitors .

• Quantitative band density analysis using ImageJ software (rsbweb.nih.gov/ij/) for accurate comparison of UCHL3 levels between different sperm samples .

• RT-qPCR for mRNA expression analysis, particularly useful for comparing UCHL3 expression across different developmental stages or treatment conditions .

What is the role of UCHL3 in spermatogenesis and meiotic differentiation?

UCHL3 plays crucial roles in apoptosis of germ cells and the meiotic differentiation of spermatocytes into spermatids . Its deubiquitinating activity appears to regulate protein turnover during critical stages of sperm development. In abnormal or asthenozoospermic samples, UCHL3 levels are significantly reduced (0.57-fold of normal) or absent in the acrosomeless, small or abnormal acrosome and in shorter or distorted tails . This indicates that UCHL3 may be essential for proper acrosome formation and flagellar development during spermatogenesis, potentially through regulation of protein quality control mechanisms in developing sperm cells.

How does UCHL3 regulate chromosome segregation during mitosis?

UCHL3 plays a critical role in regulating the fidelity of chromosome segregation during mitosis through its deubiquitylating activity . Specifically:

• UCHL3 contributes to error correction (EC) at kinetochores, which is essential for proper chromosome alignment and segregation.

• UCHL3 depletion using siRNA or inhibition with TCID results in nuclear morphology defects, including multilobed nuclei and multinucleation, suggesting chromosome segregation errors .

• Live cell imaging using H2B-mCherry as a histone marker reveals that UCHL3 regulates the fidelity of chromosome segregation rather than the timing of the process .

• UCHL3's deubiquitylating activity functionally separates error correction from spindle assembly checkpoint (SAC) signaling during mitosis .

What phenotypes are observed in UCHL3-deficient mouse cells during cell division?

UCHL3-deficient mouse cells exhibit several distinctive phenotypes during cell division:

• Increased frequency of irregular, multilobed nuclei, indicative of defective chromosome segregation .

• Higher rates of multinucleation, suggesting cytokinesis failures or severe chromosome segregation errors .

• Nuclear atypia similar to that observed when Aurora B (a key mitotic regulator) is downregulated .

• These defects appear to be specific to UCHL3 within the UCH family, as downregulation of UCHL1 does not produce similar phenotypes .

These phenotypes collectively demonstrate that UCHL3 is critical for maintaining euploidy and genomic stability in mammalian cells.

How does UCHL3 interact with the DNA damage repair machinery?

UCHL3 interacts with the DNA damage repair machinery through several mechanisms:

• UCHL3 is phosphorylated and activated by ATM kinase in response to DNA damage, positioning it as a downstream effector in the DNA damage response pathway .

• It plays a role in regulating the major pathway for repair of DNA double-strand breaks, thereby maintaining genome integrity .

• UCHL3 contributes to DNA repair by ubiquitylating TDP1 (Tyrosyl-DNA phosphodiesterase 1), a protein involved in resolving DNA-protein crosslinks .

• Disruption of TDP1 regulation by UCHL3 can lead to neurological disease and altered responses to chemotherapy in cancer .

This interaction with DNA repair machinery explains why UCHL3 deficiency results in genomic instability and suggests potential therapeutic applications in cancer treatment.

What are the most effective antibodies and protocols for Western blotting of UCHL3 in mouse tissues?

For Western blotting of UCHL3 in mouse tissues, the following approach is recommended:

• Antibody selection:

  • Mouse anti-UCHL3 monoclonal antibody (clone AB04/3F5) has demonstrated high specificity

  • Anti-UCHL3 rabbit polyclonal antibody (1:500 dilution; Bioss, Beijing, China) has been successfully used in reproductive biology studies

  • For loading control, anti-α-tubulin rabbit monoclonal antibody (1:5,000; ab52866, Abcam) is recommended

• Protein extraction protocol:

  • For tissue samples: Homogenize in cold lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65mM DTT, 1mM PMSF, and proteinase inhibitor cocktail

  • For cell lines: Standard RIPA buffer supplemented with phosphatase and protease inhibitors

• Western blot conditions:

  • Load 50 μg protein per lane

  • Use 1:1000 dilution for PrecisionAb monoclonal antibodies

  • UCHL3 should be detected as a band of approximately 25 kDa in mammalian samples

  • Secondary antibody: HRP-labelled goat anti-rabbit IgG (1:5,000)

• Quantification:

  • Band density should be quantified using ImageJ software for accurate comparison

What siRNA sequences have been validated for effective UCHL3 knockdown in mouse models?

Several siRNA sequences have been validated for effective UCHL3 knockdown:

• UCHL3 (human) siRNA-1:

  • Forward: 5′-GAACAGAAGAGGAAGAAAATT−3′

  • Reverse: 5′-UUUUCUUCCUCUUCUGUUCTT−3′

• UCHL3 (human) siRNA-2 (demonstrated highest knockdown efficiency):

  • Forward: 5′-CUGAAGAACGAGCCAGAUATT−3′

  • Reverse: 5′-UAUCUGGCUCGUUCUUCAGTT−3′

• UCHL3 (human) siRNA-3:

  • Forward: 5′-UGGAACAAUUGGACUGAUUTT−3′

  • Reverse: 5′-AAUCAGUCCAAUUGUUCCATT−3′

For negative control transfections, the following sequences have been used:

• NC forward: 5′-UGACCUACAACUUCUAUGGTT−3′

• NC reverse: 5′-UUCUCCGAACGUGUCACGUTT−3′

Transfection protocols should be optimized for each cell type, with verification of knockdown efficiency by RT-qPCR and Western blotting.

How can UCHL3 enzyme activity be accurately measured in tissue samples?

UCHL3 enzyme activity in tissue samples can be accurately measured through several approaches:

• Deubiquitinating enzyme activity assays:

  • Using synthetic substrates such as ubiquitin-AMC (7-amido-4-methylcoumarin) that release a fluorescent signal upon cleavage

  • Monitoring the hydrolysis of polyubiquitin chains using purified K48- or K63-linked ubiquitin chains followed by Western blot analysis

• NEDD8 hydrolase activity assays:

  • Given UCHL3's dual role as a ubiquitin and NEDD8 hydrolase, measuring NEDD8 processing provides additional functional insights

  • This can be assessed by monitoring the conversion of NEDD8 precursors to mature NEDD8 forms

• Correlation analysis:

  • Statistical correlation between UCHL3 protein levels and deubiquitinating activity can be evaluated using Spearman's correlation coefficient

  • In sperm studies, correlations between UCHL3 activity and functional parameters (count, motility) provide functional insights

• Pharmacological inhibition:

  • Using specific UCHL3 inhibitors like TCID as a negative control to confirm assay specificity

How might UCHL3 modulation be exploited therapeutically in cancer treatment strategies?

UCHL3 modulation shows significant therapeutic potential based on several research findings:

• Differential sensitivity: High UCHL3 expression renders breast cancer cells resistant to radiation and chemotherapy, while low expression sensitizes cells to these treatments . This suggests that UCHL3 inhibition could be an effective strategy to overcome therapy resistance.

• Melanoma applications: UCHL3 knockdown suppresses proliferation and enhances apoptosis in melanoma cells , indicating that UCHL3 inhibitors might be effective anti-melanoma agents.

• Combination therapy approaches: Given UCHL3's role in DNA repair via TDP1 ubiquitylation , combining UCHL3 inhibitors with DNA-damaging agents may create synthetic lethality in cancer cells.

• Target specificity considerations: The development of highly specific UCHL3 inhibitors (like TCID) that don't affect other UCH family members would be crucial for minimizing off-target effects .

• Biomarker potential: UCHL3 expression levels could serve as predictive biomarkers for therapy response, enabling personalized treatment approaches.

What is the relationship between UCHL3 and other deubiquitinating enzymes in maintaining cellular homeostasis?

The relationship between UCHL3 and other deubiquitinating enzymes involves:

• Functional redundancy vs. specificity: While UCHL3 shares structural similarities with other UCH family members (UCHL1, BAP1, and UCHL5), it has unique functions that appear non-redundant, particularly in mitosis and genome integrity maintenance .

• Differential substrate specificity: Unlike other UCH family members, UCHL3 possesses dual specificity for both ubiquitin and NEDD8 , suggesting it operates at the intersection of these two post-translational modification pathways.

• Regulatory network interactions: UCHL3 likely functions within a network of deubiquitinating enzymes that collectively maintain the balance of ubiquitinated proteins in cells. Disruption of UCHL3 may cause compensatory changes in other DUBs.

• Tissue-specific cooperation: In specialized contexts like spermatogenesis, UCHL3 may cooperate with other testis-specific DUBs to regulate protein turnover during meiotic differentiation .

• Pathway-specific roles: In DNA damage repair, UCHL3 appears to have distinct functions from other DUBs involved in the same pathway, as demonstrated by the specific nuclear abnormalities observed upon UCHL3 depletion .

What epigenetic mechanisms regulate UCHL3 expression across different tissue types?

While current research on epigenetic regulation of UCHL3 is limited, several potential mechanisms warrant investigation:

• Tissue-specific expression patterns: UCHL3 shows differential expression across tissues, with particularly notable expression in melanoma cells and reproductive tissues , suggesting tissue-specific regulatory mechanisms.

• Developmental regulation: UCHL3's role in spermatogenesis implies developmental regulation of its expression, potentially through epigenetic mechanisms that control temporal expression patterns.

• Stress-responsive regulation: UCHL3 is activated in response to DNA damage , suggesting stress-responsive regulatory elements in its promoter that might be epigenetically controlled.

• Cancer-associated dysregulation: The elevated expression of UCHL3 in multiple cancer cell lines points to potential epigenetic dysregulation in malignant transformation, possibly involving promoter hypomethylation or altered histone modifications.

• Therapeutic implications: Understanding the epigenetic regulation of UCHL3 could reveal opportunities for indirect modulation of its expression through epigenetic therapies, potentially offering alternative approaches to direct enzyme inhibition.