UCHL5 Human

What is UCHL5 and what is its primary function in human cells?

UCHL5, also known as UCH37, is a deubiquitinating enzyme (DUB) that removes ubiquitin from protein substrates. It functions primarily within the ubiquitin-proteasome system (UPS), which regulates protein degradation and is essential for cellular homeostasis. UCHL5 is notably associated with the 19S regulatory particle of the proteasome through its interaction with hRpn13/ADRM1, where it participates in substrate processing before proteolysis occurs in the 20S core particle . The enzyme plays a critical role in protein turnover, and through this function, it influences numerous cellular processes including cell cycle progression, signal transduction, and proteostasis maintenance. Research indicates that UCHL5 cannot be fully substituted by other DUBs, suggesting it has unique and essential functions despite the existence of approximately 90 other DUBs in human cells .

How is UCHL5 structurally organized and what domains are critical for its function?

UCHL5 contains multiple functional domains that facilitate its diverse activities. The protein's catalytic domain contains the active site responsible for deubiquitinating activity. It interacts with the DEUBAD domain of hRpn13/ADRM1, which not only recruits UCHL5 to the proteasome but also enhances its enzymatic activity .

When designing experiments to study UCHL5 domain functionality, researchers should consider:

• The impact of point mutations in specific domains on enzyme activity

• Domain-specific protein-protein interactions

• How structural changes affect subcellular localization

These approaches allow for detailed dissection of structure-function relationships. Notably, research has demonstrated that truncation of hRpn13 Pru domain causes it to be unable to interact with the proteasome while maintaining UCHL5 binding capacity, indicating the specificity of these domain interactions .

What experimental methods are most effective for measuring UCHL5 expression in human tissues?

Several complementary approaches should be used to comprehensively assess UCHL5 expression:

For UCHL5 detection specifically, validated ELISA kits are available with a detection range of 0.313-20 ng/ml and a sensitivity of <0.188 ng/ml . When analyzing tissue samples, researchers should establish appropriate controls and standardize sample collection protocols to ensure reproducibility. In bladder cancer research, for instance, investigators successfully used both IHC analysis of tissue microarrays and Western blotting to demonstrate elevated UCHL5 expression in cancerous tissues compared to adjacent normal tissues .

How does UCHL5 expression correlate with cancer progression and patient outcomes?

UCHL5 expression has been found to be abnormally elevated in multiple human cancer types, including cervical carcinoma, epithelial ovarian cancer, esophageal squamous cell carcinoma, lung cancer, pancreatic carcinoma, and bladder cancer . In bladder cancer specifically, analysis of TCGA and GTEx databases revealed significantly higher UCHL5 expression in cancer samples compared to normal tissues (p < 0.01) .

Clinical correlation studies demonstrate that high UCHL5 expression is associated with:

When designing studies to investigate UCHL5 in cancer, researchers should consider both expression levels and functional consequences, as well as correlations with specific immune cell populations that may influence tumor microenvironment and treatment response.

What are the key signaling pathways modulated by UCHL5 in cancer cells?

UCHL5 influences multiple signaling pathways that contribute to cancer development and progression. The primary mechanism identified thus far involves upregulation of transforming growth factor (TGF) signaling . This modulation occurs through the deubiquitinating activity of UCHL5, which can stabilize key proteins within these pathways by preventing their proteasomal degradation.

To experimentally assess UCHL5's impact on signaling pathways, researchers should:

• Employ pathway-specific reporter assays before and after UCHL5 knockdown or overexpression

• Perform phospho-proteomic analyses to identify altered phosphorylation cascades

• Use RNA-Seq to identify transcriptional changes upon UCHL5 manipulation

• Validate key findings with Western blotting for specific pathway components

The analysis should account for cell type specificity, as signaling dynamics may vary between different cancer types. For instance, in bladder cancer, UCHL5 knockdown experiments have been successfully performed using lentiviral systems with targeted shRNA sequences (e.g., ShUCHL5-1: 5′-CCGGAGCCAGTTCATGGGTTAATTTCTCGAGAAATTAACCCATGAACTGGCTTTTTTG-3′) .

How do UCHL5 inhibitors affect cancer cell proliferation and migration?

The anticancer molecule RA190, which binds covalently to both hRpn13 and UCHL5, has been shown to require hRpn13 Pru domain but not UCHL5 for its cytotoxicity . This suggests that targeting the UCHL5-hRpn13 interaction might be a promising therapeutic approach.

When evaluating UCHL5 inhibitors, researchers should employ a comprehensive panel of assays:

• Cell proliferation assays (e.g., MTT, BrdU incorporation)

• Colony formation assays to assess long-term growth effects

• Migration assays (wound healing, Transwell)

• Invasion assays using Matrigel-coated chambers

• Cell cycle analysis by flow cytometry

• Apoptosis assessment (Annexin V/PI staining, caspase activation)

Importantly, experiments should include both genetic approaches (siRNA, CRISPR) and pharmacological inhibitors to distinguish on-target from off-target effects. The methodological approach should also incorporate dose-response studies and time-course analyses to fully characterize inhibitor effects .

What genetic manipulation strategies are most effective for studying UCHL5 function?

Several genetic approaches have proven effective for investigating UCHL5 function:

For UCHL5 knockdown specifically, validated shRNA sequences targeting human UCHL5 include:

• ShUCHL5-1: 5′-CCGGAGCCAGTTCATGGGTTAATTTCTCGAGAAATTAACCCATGAACTGGCTTTTTTG-3′

• ShUCHL5-2: 5′-CCGGTGAAGGTGAAATTCGATTTAACTCGAGTTAAATCGAATTTCACCTTCATTTTTTG-3′

• ShUCHL5-3: 5′-CCGGCTGGTTGTCTAACTACCATATCTCGAGATATGGTAGTTAGACAACCAGTTTTTTG-3′

When designing genetic manipulation studies, researchers should include appropriate controls (scrambled sequences for knockdown, empty vectors for overexpression) and validate the efficiency of manipulation at both mRNA and protein levels. Additionally, rescue experiments should be performed to confirm specificity of observed phenotypes.

How can protein-protein interactions of UCHL5 be effectively characterized?

UCHL5 functions through interactions with multiple proteins, most notably hRpn13/ADRM1 and the proteasome. Comprehensive characterization of these interactions requires multiple complementary techniques:

• Co-immunoprecipitation (Co-IP): Used to validate interactions in cellular contexts

• Proximity ligation assay (PLA): Enables visualization of interactions in situ

• Yeast two-hybrid screening: Useful for identifying novel interaction partners

• Mass spectrometry-based interactomics: Provides unbiased identification of the UCHL5 interactome

• FRET/BRET analyses: Assess dynamic interactions in living cells

• Surface plasmon resonance (SPR): Determines binding kinetics and affinities

Research has demonstrated that a truncated version of hRpn13 (trRpn13) remains competent for UCHL5 binding while losing proteasome interaction capability . This finding underscores the importance of domain-specific interactions in UCHL5 function. When conducting interaction studies, researchers should account for potential conformational changes that might occur upon binding, as well as the influence of post-translational modifications on interaction dynamics.

What computational approaches can predict UCHL5 substrates and guide experimental validation?

Computational prediction of UCHL5 substrates can accelerate discovery and guide experimental design. Recommended approaches include:

• Sequence motif analysis: Identification of consensus sequences in known substrates

• Structural modeling: Docking studies to predict enzyme-substrate interactions

• Network analysis: Integration of protein-protein interaction data with ubiquitin-proteasome system components

• Machine learning approaches: Trained on known DUB-substrate pairs to predict novel substrates

The CIBERSORT computational approach, which employs support vector regression for deconvolution of cell types, has been successfully applied to analyze UCHL5 expression in relation to tumor infiltrating leukocytes . This exemplifies how computational tools can provide insights into UCHL5 function in complex cellular contexts.

For experimental validation of predicted substrates, researchers should employ:

• Ubiquitination assays before and after UCHL5 manipulation

• Protein stability assessments using cycloheximide chase experiments

• Direct deubiquitination assays with recombinant proteins

• Ubiquitin chain type-specific antibodies to determine linkage preferences

How can researchers reconcile conflicting data regarding UCHL5 function across different cancer types?

Contradictory findings regarding UCHL5's role in different cancers likely reflect context-dependent functions. To resolve such discrepancies, researchers should:

• Perform systematic meta-analyses: Integrate findings across studies, controlling for methodological variations

• Consider tissue specificity: Different cellular contexts may alter UCHL5 function

• Examine genetic background: Mutations in related genes may modify UCHL5 effects

• Assess expression levels: UCHL5 might exert concentration-dependent effects

• Evaluate isoform expression: Different splice variants may have distinct functions

When analyzing bladder cancer data from TCGA, researchers found significant associations between UCHL5 expression and immune cell infiltration patterns , suggesting that immune context may partially explain differing results across cancer types. To directly address contradictions, design experiments that systematically vary one parameter at a time while maintaining consistency in other aspects of the experimental system.

What statistical approaches are most appropriate for analyzing UCHL5 expression data in clinical samples?

Analysis of UCHL5 expression in clinical samples requires robust statistical methods:

Sample size calculation is crucial prior to study initiation to ensure adequate statistical power. Additionally, researchers should account for multiple testing correction (e.g., Bonferroni, FDR) when performing genome-wide or proteome-wide analyses in relation to UCHL5.

How should researchers interpret changes in proteasome-bound ubiquitinated proteins after UCHL5 manipulation?

Alterations in proteasome-bound ubiquitinated proteins following UCHL5 manipulation provide insights into its functional significance. When interpreting such changes, researchers should consider:

• Substrate specificity: Determine whether changes are global or substrate-specific

• Ubiquitin chain topology: Analyze specific linkage types (K48, K63, etc.) affected

• Temporal dynamics: Assess acute versus chronic effects of UCHL5 manipulation

• Compensatory mechanisms: Evaluate potential upregulation of other DUBs

Research has shown that deletion of UCHL5 from HCT116 cells causes increased levels of ubiquitinated proteins in whole-cell extracts and at proteasomes, suggesting that other DUBs cannot fully compensate for UCHL5 activity despite the existence of approximately 90 DUBs in human cells . This finding highlights the non-redundant role of UCHL5 in the ubiquitin-proteasome system.

When designing experiments to assess proteasome-bound ubiquitinated proteins, researchers should:

• Include appropriate controls (e.g., proteasome inhibitors as positive controls)

• Combine biochemical fractionation with immunoblotting or mass spectrometry

• Consider both steady-state levels and flux through the ubiquitin-proteasome system

• Account for potential changes in proteasome activity or assembly

What are the most promising therapeutic approaches targeting UCHL5 for cancer treatment?

Several therapeutic strategies targeting UCHL5 show promise for cancer treatment:

• Small molecule inhibitors: Compounds that directly inhibit UCHL5 catalytic activity

• Protein-protein interaction disruptors: Molecules that prevent UCHL5-hRpn13 binding

• Degraders: PROTAC-type compounds that induce UCHL5 degradation

• Combination therapies: UCHL5 inhibitors with established cancer treatments

The anticancer molecule RA190, which binds covalently to both hRpn13 and UCHL5, has shown that it requires the hRpn13 Pru domain but not UCHL5 for its cytotoxicity . This suggests that targeting the UCHL5-hRpn13 interaction might be more effective than targeting UCHL5 alone.

When designing therapeutic studies, researchers should evaluate:

• Target selectivity against other DUBs

• Pharmacokinetic properties

• Resistance mechanisms

• Biomarkers of response

How can single-cell technologies advance our understanding of UCHL5 function in heterogeneous tissues?

Single-cell approaches offer unprecedented insights into UCHL5 biology in complex tissues:

When applying these technologies to UCHL5 research, investigators should:

• Validate findings across multiple platforms

• Integrate data types for comprehensive understanding

• Develop computational pipelines specific to ubiquitin-proteasome system components

• Consider temporal dynamics in addition to spatial heterogeneity

The CIBERSORT approach used in bladder cancer research to estimate immune cell infiltration levels could be extended to single-cell resolution, providing more precise mapping of UCHL5 expression patterns in relation to the tumor microenvironment.

What approaches can identify the complete substrate profile of UCHL5 in different cellular contexts?

Comprehensive substrate identification requires integrated proteomics approaches:

• Ubiquitin remnant profiling: Identification of ubiquitinated proteins after UCHL5 manipulation

• Protein stability profiling: Global protein half-life measurements

• BioID/TurboID proximity labeling: Identification of proteins in close proximity to UCHL5

• Crosslinking mass spectrometry: Capturing transient enzyme-substrate interactions

• SILAC-based quantitative proteomics: Measuring changes in the ubiquitinome after UCHL5 inhibition

To account for cellular context, these approaches should be applied across:

• Different cell types

• Various stress conditions

• Developmental stages

• Disease states

Integration of substrate identification data with structural information and functional validation will provide a comprehensive understanding of UCHL5 biology and guide the development of targeted therapeutic approaches.