Recombinant Mouse Ubiquitin thioesterase OTUB1 (Otub1)

What is OTUB1 and what are its primary functions in cellular processes?

OTUB1 (Otubain-1) is a member of the ovarian tumor (OTU) protein family of deubiquitinating enzymes (DUBs) that specifically cleaves K48-linked polyubiquitin chains. It is a 271 amino acid polypeptide with a predicted molecular weight of 32 kDa . OTUB1 is highly conserved across species, with mouse and rat orthologs showing 99% identity at the amino acid level to human OTUB1 .

The primary functions of OTUB1 include:

• Catalytic cleavage of K48-linked polyubiquitin chains, which typically mark proteins for proteasomal degradation

• Non-catalytic inhibition of ubiquitin conjugation by binding to and suppressing the activity of certain E2 ubiquitin-conjugating enzymes, including UBE2N (UBC13), UBE2D family members, and UBE2E family members

• Regulation of protein stability by preventing proteasomal degradation of target proteins

• Modulation of various signaling pathways including MAPK, ERα, EMT, RHOa, mTORC1, FOXM1, and P53

These dual mechanisms allow OTUB1 to regulate ubiquitin-dependent processes through both enzymatic and non-enzymatic mechanisms, making it a unique player in cellular protein homeostasis.

What experimental methods are commonly used to characterize OTUB1 activity?

Several methodological approaches are employed to characterize different aspects of OTUB1 activity:

• Deubiquitinating activity assays: Researchers typically use internally quenched fluorescent (IQF) substrates such as FRET-K48 diubiquitin, which contains TAMRA conjugated to one ubiquitin and a quencher to the other. Cleavage is monitored by measuring fluorescence (ex. 544 nm; em. 590 nm) over time .

• Binding affinity measurements: Isothermal Titration Calorimetry (ITC) is commonly used to measure binding affinities between OTUB1 and its interaction partners. For this purpose, researchers often use catalytically inactive OTUB1 mutants (e.g., C91S) to prevent substrate cleavage during measurements .

• Kinetic parameter determination: Standard enzyme kinetic assays determine parameters like KM and kcat. For OTUB1, researchers typically measure the cleavage of K48-linked diubiquitin substrates at various concentrations to generate Michaelis-Menten plots .

• Ubiquitination inhibition assays: To measure the non-catalytic inhibition of E2 enzymes by OTUB1, researchers monitor the autoubiquitination or substrate ubiquitination activities of E2 enzymes in the presence and absence of OTUB1 .

For accurate results, the reaction conditions (temperature, pH, salt concentration) need to be optimized for each specific application, as recommended for recombinant OTUB1 proteins .

How does mouse OTUB1 compare structurally and functionally to human OTUB1?

Mouse OTUB1 shares 99% amino acid sequence identity with human OTUB1 , suggesting high conservation of structure and function between these species. This high degree of conservation makes mouse OTUB1 an excellent model for studying the functions of human OTUB1.

Key shared characteristics include:

• Similar substrate specificity for K48-linked ubiquitin chains

• The ability to interact with and regulate the same E2 ubiquitin-conjugating enzymes

• Comparable roles in various signaling pathways

What are the kinetic parameters of OTUB1 and how are they affected by E2 enzyme binding?

OTUB1's deubiquitinating activity is significantly influenced by interaction with E2 enzymes. Detailed kinetic analysis reveals that E2 enzymes substantially enhance OTUB1's catalytic efficiency by increasing its affinity for K48-linked diubiquitin substrates.

In the absence of E2 enzymes, OTUB1 has the following kinetic parameters:

• KM: 102 μM for K48 diubiquitin

• kcat: 0.03 s−1

When bound to different E2 partners, these parameters change as follows:

These data demonstrate that different E2 enzymes vary in their ability to stimulate OTUB1 activity, with UBE2D family members being the most potent stimulators, followed by UBE2E1 and UBE2N . Interestingly, this stimulation occurs primarily through decreasing KM rather than increasing kcat, suggesting that E2 enzymes enhance OTUB1's substrate binding rather than its catalytic rate.

What is the molecular mechanism by which E2 enzymes enhance OTUB1's affinity for K48-linked diubiquitin?

The mechanism by which E2 enzymes enhance OTUB1's affinity for K48-linked diubiquitin has been elucidated through binding studies using isothermal titration calorimetry (ITC) with the catalytically inactive OTUB1 (C91S) mutant.

The improvement in binding affinity correlates well with the decrease in KM observed in enzymatic assays, confirming that E2 enzymes enhance OTUB1's catalytic efficiency by increasing its affinity for substrate .

The molecular basis for this enhancement appears to involve the promotion of folding of OTUB1's ubiquitin-binding helix, which effectively "pre-pays" the energetic cost of helix formation required for substrate binding . This structural rearrangement creates a more optimal binding site for the K48-linked diubiquitin, resulting in increased affinity and catalytic efficiency.

How does OTUB1 interact with different E2 ubiquitin-conjugating enzymes?

OTUB1 interacts with various E2 ubiquitin-conjugating enzymes with similar affinities despite having different effects on their activities. The binding affinities between OTUB1 and different E2 enzymes have been measured using isothermal titration calorimetry (ITC):

The binding interactions with UBE2E2 and UBE2E3 could not be measured by ITC due to protein precipitation issues . Interestingly, no detectable binding was observed between OTUB1 and UBE2G2 or CDC34, which aligns with the observation that these E2 enzymes do not stimulate OTUB1's activity .

Despite the similar binding affinities, OTUB1 has distinct effects on different E2 enzymes:

• Inhibition of ubiquitin transfer: OTUB1 inhibits the ubiquitin-conjugating activities of UBE2D isoforms, UBE2N, and all three UBE2E enzymes (UBE2E1, UBE2E2, and UBE2E3) by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer .

• Differential inhibition of autoubiquitination: While OTUB1 inhibits autoubiquitination by UBE2E1 and UBE2E2, it cannot suppress autoubiquitination by UBE2E3, suggesting specific regulatory mechanisms for different E2 enzymes .

The fact that the binding affinities don't directly correlate with the ability of different E2s to stimulate OTUB1 activity suggests that additional structural or dynamic factors beyond simple binding affinity determine these functional interactions.

What role does OTUB1 play in immune evasion by cancer cells?

OTUB1 has been identified as a critical regulator of programmed death ligand 1 (PD-L1) stability, playing an important role in cancer cell immune evasion. The mechanism through which OTUB1 promotes immune evasion includes:

• Stabilization of PD-L1: OTUB1 interacts with and removes K48-linked ubiquitin chains from the PD-L1 intracellular domain, hindering its degradation through the ERAD (Endoplasmic Reticulum-Associated Degradation) pathway .

• Dependency on deubiquitinase activity: This stabilization requires OTUB1's deubiquitinating enzyme activity, demonstrating a direct enzymatic role rather than a non-catalytic scaffolding function .

• Impact on immune cell interactions: The increased PD-L1 levels resulting from OTUB1 activity enhance cancer cell binding to PD-1 on immune cells, suppressing anti-tumor immune responses .

Experimental evidence supports this role:

• Depletion of OTUB1 markedly decreases PD-L1 abundance and reduces PD-1 protein binding to tumor cell surfaces

• OTUB1 knockdown increases tumor cell sensitivity to cytotoxicity mediated by peripheral blood mononuclear cells (PBMCs)

• In vivo, OTUB1 ablation leads to increased CD8+ T cell infiltration into tumors and elevated IFN-γ levels in serum, enhancing anti-tumor immunity

• The tumor growth suppression caused by OTUB1 silencing can be reversed by PD-L1 overexpression

Clinical relevance is supported by the observation of a significant correlation between PD-L1 abundance and OTUB1 expression in human breast carcinoma . This suggests that OTUB1 could be a potential therapeutic target for cancer immunotherapy, particularly in tumors with high OTUB1 expression.

How does OTUB1 function in protecting against liver injury and bacterial hepatitis?

OTUB1 plays a critical protective role in hepatocytes during bacterial infection and acute liver injury through regulation of necroptosis pathways:

• Prevention of hepatocyte necroptosis: OTUB1 inhibits the necroptotic cell death pathway in hepatocytes, which is especially important during bacterial hepatitis (such as Listeria monocytogenes infection) and TNF-induced inflammation .

• Stabilization of c-IAP1: Mechanistically, OTUB1 reduces K48-linked polyubiquitination of cellular inhibitor of apoptosis 1 (c-IAP1), preventing its proteasomal degradation . This preserves c-IAP1 function in the cell death regulatory pathway.

• Regulation of RIPK1 ubiquitination and activation: By stabilizing c-IAP1, OTUB1 promotes K63-linked polyubiquitination of RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which inhibits RIPK1 phosphorylation and subsequent activation .

• Inhibition of necrosome formation: This ultimately prevents the formation of the RIPK1/RIPK3 necrosome complex, MLKL phosphorylation, and hepatocyte death .

• Suppression of inflammatory signaling: In addition to preventing cell death, OTUB1 also inhibits RIPK1-dependent ERK activation and TNF production in infected hepatocytes, dampening inflammatory responses .

In vivo studies have demonstrated that OTUB1 deficiency leads to increased susceptibility to Listeria monocytogenes infection and TNF-induced liver injury, with significantly higher mortality rates . Conversely, the presence of functional OTUB1 protects against these challenges, highlighting its importance as an endogenous inhibitor of hepatocyte necroptosis and a pro-survival factor during bacterial and TNF-induced inflammation.

What are the optimal conditions for measuring OTUB1 deubiquitinating activity in vitro?

When designing in vitro assays to measure OTUB1 deubiquitinating activity, researchers should consider the following optimized conditions based on published protocols:

• Substrate selection: Internally quenched fluorescent (IQF) K48-linked diubiquitin substrates, such as FRET-K48 diubiquitin with TAMRA conjugated to one ubiquitin and a quencher to the other, provide sensitive and real-time measurement capabilities .

• Reaction conditions:

  • Temperature: 30°C is commonly used for optimal enzyme activity

  • Buffer: 25 mM HEPES, pH 7.5, 150 mM NaCl, and 0.5 mM TCEP-HCl

  • Reaction volume: 30 μL is sufficient for microplate-based assays

• Detection method: Fluorescence measurements (excitation 544 nm; emission 590 nm) taken every 5 seconds over 30 minutes using a plate reader (such as POLARStar Omega) with appropriate gain settings (e.g., 1900) based on the fluorescence from fully digested substrate .

• Standard curve preparation: Generate a standard curve by completely digesting FRET-K48 diubiquitin at concentrations ranging from 25-500 nM with 50 nM OTUB1 for 1 hour at 30°C. Plot fluorescence (AU) against concentration of cleaved substrate and fit to a linear equation to obtain the conversion factor (AU- μM−1) .

• Enzyme concentration: Use 50-100 nM of purified recombinant OTUB1 as a starting point, adjusting based on activity levels.

• Controls: Include negative controls (no enzyme) and positive controls (pre-digested substrate) to validate assay performance.

For studying the effects of E2 enzymes on OTUB1 activity, include the E2 enzyme at saturating concentrations (typically 5-10 times the Kd value) to ensure complete complex formation with OTUB1. These conditions will need to be optimized for each specific application as noted for commercial recombinant OTUB1 proteins .

What approaches should be used to analyze contradictory data regarding OTUB1's role in different cancer types?

OTUB1 has been implicated in various cancer-associated pathways, but its effects may be context-dependent, sometimes showing seemingly contradictory roles. When analyzing such contradictory data, researchers should consider the following approaches:

• Cellular context analysis: Examine the specific cellular context in which OTUB1 was studied, including:

  • Cell type and tissue origin

  • Genetic background (mutations in key signaling pathways)

  • Microenvironmental factors

• Pathway-specific effects: OTUB1 regulates multiple pathways including MAPK, ERα, EMT, RHOa, mTORC1, FOXM1, and P53 . Consider which specific pathway was investigated in each study and how these pathways interact in different contexts.

• Dual mechanism consideration: Remember that OTUB1 has both catalytic (DUB activity) and non-catalytic (E2 enzyme inhibition) functions . Determine which function was predominant in each experimental setting.

• Substrate specificity: Identify the specific ubiquitinated proteins being regulated by OTUB1 in each study, as different substrates may lead to different phenotypic outcomes.

• Methodological comparison: Compare experimental methods, including:

  • In vitro versus in vivo studies

  • Overexpression versus knockdown/knockout approaches

  • Acute versus chronic alterations in OTUB1 levels

• Integrated analysis: Perform meta-analyses of multiple studies, looking for patterns that might explain apparent contradictions.

• Validation experiments: Design experiments that directly test competing hypotheses under identical conditions.

For example, while most studies support an oncogenic role for OTUB1, particularly in tumor migration, invasion, and metastasis, some studies demonstrate that OTUB1 potentiates growth-suppressive pathways such as P53 and TGFβ . This suggests that OTUB1's contribution to cancer development and progression may depend on the tumor type or cellular context, requiring careful consideration of these factors when designing experiments and interpreting results.

What are the most promising therapeutic approaches targeting OTUB1 in cancer and inflammatory diseases?

Based on the current understanding of OTUB1's functions, several promising therapeutic approaches could be developed:

• Small molecule inhibitors of OTUB1 DUB activity: Developing specific inhibitors that block OTUB1's catalytic site could restore normal degradation of cancer-promoting proteins stabilized by OTUB1, such as PD-L1 .

• Disruptors of OTUB1-E2 interactions: Compounds that prevent OTUB1 from binding to specific E2 enzymes could selectively modulate its non-catalytic functions without affecting its DUB activity.

• Combination therapy with immune checkpoint inhibitors: Since OTUB1 stabilizes PD-L1, combining OTUB1 inhibitors with existing anti-PD-1/PD-L1 antibodies could enhance cancer immunotherapy responses, particularly in tumors with high OTUB1 expression .

• Tissue-specific OTUB1 modulators: For inflammatory conditions like bacterial hepatitis, enhancing OTUB1 activity specifically in hepatocytes could protect against excessive necroptosis and inflammation .

• Substrate-selective approaches: Developing compounds that specifically disrupt OTUB1's interaction with particular substrates (like c-IAP1 or PD-L1) could allow for targeted intervention in specific pathways without disrupting all OTUB1 functions.

Future research should focus on:

• Structural studies to better understand OTUB1's interaction surfaces

• Development of high-throughput screening assays for OTUB1 inhibitors

• In vivo validation of OTUB1 as a therapeutic target in relevant disease models

• Biomarker development to identify patients most likely to benefit from OTUB1-targeted therapies

These approaches hold promise for addressing both cancer and inflammatory conditions where OTUB1 plays a significant role in disease pathogenesis.

What technical challenges need to be addressed in studying OTUB1's complex regulatory network?

Studying OTUB1's complex regulatory network presents several technical challenges that researchers must address:

• Distinguishing catalytic from non-catalytic functions: Since OTUB1 has both deubiquitinating activity and non-catalytic E2 enzyme inhibition functions, designing experiments that can distinguish between these activities is challenging. Using catalytically inactive mutants (e.g., C91S) alongside wild-type OTUB1 can help separate these functions .

• Identifying physiologically relevant substrates: While OTUB1 shows specificity for K48-linked polyubiquitin chains in vitro, identifying its true physiological substrates remains challenging. Advanced proteomics approaches coupling ubiquitin remnant profiling with OTUB1 manipulation are needed to comprehensively identify OTUB1 substrates.

• E2 enzyme specificity and redundancy: OTUB1 interacts with multiple E2 enzymes with similar affinities , making it difficult to dissect specific E2-dependent functions. Systematic approaches using multiple E2 knockdowns/knockouts may be required to fully understand the functional significance of each interaction.

• Context-dependent activities: OTUB1's functions appear to be highly context-dependent, with different effects observed in different cell types or disease states . This necessitates studying OTUB1 in multiple cellular contexts and validating findings across different models.

• Temporal dynamics of deubiquitination: Standard biochemical assays often fail to capture the dynamic nature of ubiquitination/deubiquitination cycles. Real-time imaging techniques and biosensors for ubiquitination may help address this limitation.

• Structural complexity of ubiquitin chains: The structural diversity of polyubiquitin chains makes it challenging to study specific chain types. Developing better tools for generating and detecting specific ubiquitin chain types is essential.

• Integration with other post-translational modifications: Ubiquitination often interplays with other modifications like phosphorylation. Multiomics approaches combining ubiquitinomics, phosphoproteomics, and other techniques are needed to fully understand these complex networks.

Addressing these challenges will require interdisciplinary approaches combining biochemistry, structural biology, cellular biology, and systems biology, as well as the development of new technologies for studying dynamic protein modifications in living cells.

What quality control measures are essential when working with recombinant OTUB1 preparations?

When working with recombinant OTUB1 preparations, researchers should implement the following quality control measures to ensure reliable and reproducible results:

• Purity assessment: Verify the purity of recombinant OTUB1 using SDS-PAGE and/or HPLC. Commercial preparations should have >90% purity; for critical applications, higher purity may be required.

• Activity validation: Confirm enzymatic activity using standard substrates like FRET-K48 diubiquitin. Compare activity to established benchmarks or previous preparations to ensure consistency.

• Stability testing: Monitor stability over time and through freeze-thaw cycles using activity assays. OTUB1 typically requires reducing agents (like DTT or TCEP) to maintain the catalytic cysteine in a reduced state.

• Proper storage conditions: Store recombinant OTUB1 according to manufacturer recommendations, typically at -80°C for long-term storage and with minimal freeze-thaw cycles. Working aliquots can often be stored at -20°C.

• Buffer compatibility: Ensure the storage buffer is compatible with planned experimental conditions. Consider buffer exchange if necessary, particularly for sensitive applications.

• Structural integrity: For critical applications, circular dichroism (CD) spectroscopy can verify proper protein folding.

• Catalytic site integrity: Use site-directed mutants (e.g., C91S) as negative controls to confirm that observed activities are due to OTUB1's catalytic function rather than contaminants.

• Endotoxin testing: For cell-based or in vivo applications, verify that recombinant preparations are endotoxin-free to prevent confounding inflammatory responses.

• Batch consistency: Maintain records of different batches and their specific activities. When possible, use the same batch for comparative experiments.

• Reconstitution documentation: Carefully document reconstitution procedures as recommended for commercial preparations . Use calibrated calculations to ensure accurate concentrations.

These quality control measures will help ensure that experimental outcomes reflect true OTUB1 biology rather than artifacts from variable protein quality or handling.

How should researchers integrate OTUB1 findings from mouse models into human disease research?

When translating OTUB1 research from mouse models to human disease applications, researchers should consider the following best practices: