Recombinant Human OTU domain-containing protein 7B (OTUD7B), partial

What is OTUD7B and what is its primary function?

OTUD7B, also known as Cezanne, is an ovarian tumor protein (OTU)-domain containing deubiquitinase that regulates cellular homeostasis through selective removal of ubiquitin post-translational modifications. It preferentially cleaves lysine 11-linked polyubiquitin chains, but also exhibits activity against lysine 48 and lysine 63-linked polyubiquitin chains . As a specialized protease, OTUD7B belongs to the cysteine protease subfamily of deubiquitinating enzymes (DUBs), which comprises approximately 100 human DUBs across seven distinct subfamilies .

OTUD7B functions as a critical regulator in multiple cellular pathways by targeting specific proteins for deubiquitination, thereby preventing their degradation through the ubiquitin-proteasome system or modifying their signaling activities. Its catalytic activity is essential for maintaining the balance of numerous cellular processes, including cancer progression, immune responses, and cellular homeostasis .

What are the structural characteristics of recombinant human OTUD7B protein?

Recombinant human OTUD7B protein has the following structural characteristics:

The protein contains an OTU domain that is responsible for its deubiquitinating activity, allowing it to cleave ubiquitin chains from target substrates .

How does OTUD7B affect estrogen receptor signaling in breast cancer?

OTUD7B plays a crucial role in estrogen receptor α (ERα) stabilization in breast cancer through its deubiquitinating activity. Research has revealed the following mechanisms:

• OTUD7B expression positively correlates with ERα levels in breast cancer and is associated with poor prognosis .

• Mechanistically, OTUD7B deubiquitinates ERα, preventing its degradation through the ubiquitin-proteasome pathway and thereby stabilizing ERα protein levels.

• In experimental models, depletion of OTUD7B using siRNAs significantly decreases ERα protein levels in MCF-7 and T47D breast cancer cell lines, while ectopic expression of OTUD7B upregulates ERα .

• OTUD7B promotes breast cancer cell proliferation, G1-to-S phase transition, clone formation capability, and migration through ERα stabilization:

  • Knockdown of OTUD7B decreases cell proliferation and increases G1 phase accumulation

  • Depletion of OTUD7B reduces clone formation capability in breast cancer cells

  • OTUD7B knockdown inhibits DNA synthesis as demonstrated by EdU incorporation assays

  • Loss of OTUD7B significantly decreases cell migration capacity in wound-healing assays

• Importantly, restoration of ERα expression can rescue the defects in cell proliferation, clone formation, DNA synthesis, and migration caused by OTUD7B depletion, confirming that OTUD7B's oncogenic effects in breast cancer are largely mediated through ERα stabilization .

What role does OTUD7B play in hepatocellular carcinoma (HCC)?

In contrast to its oncogenic role in breast cancer, OTUD7B functions as a tumor suppressor in hepatocellular carcinoma (HCC) through deubiquitination and stabilization of the tumor suppressor p53:

• OTUD7B is notably downregulated in HCC tissues compared to normal liver tissues.

• Mechanistically, OTUD7B removes lysine-linked polyubiquitin chains from p53, including those mediated by Mdm2, thereby preventing p53 proteasomal degradation and stabilizing p53 protein levels .

• Co-immunoprecipitation assays demonstrate that OTUD7B binds both wild-type and mutant p53 in HCC cells.

• Functionally, overexpression of OTUD7B suppresses HCC growth, while knockdown or knockout enhances tumor progression .

• The tumor-suppressive function of OTUD7B in HCC was validated through in vitro assays and xenograft models using shRNA knockdown, overexpression, and CRISPR-Cas9 knockout approaches.

This finding highlights the context-dependent nature of OTUD7B function across different cancer types, where it can act as either an oncogene or tumor suppressor depending on its primary substrate in specific tissues .

How does OTUD7B affect LSD1 function and chromatin regulation?

OTUD7B regulates lysine-specific histone demethylase 1 (LSD1) through deubiquitination, with significant implications for epigenetic regulation and cancer progression:

• OTUD7B specifically deubiquitinates LSD1 at K226/277 residues, dynamically controlling LSD1 binding partner specificity and cellular homeostasis .

• OTUD7B deficiency increases K63-linked ubiquitination of LSD1, which:

  • Disrupts LSD1/CoREST complex formation

  • Targets LSD1 for p62-mediated proteolysis

  • Impairs genome-wide LSD1 occupancy

  • Enhances methylation of H3K4/H3K9 histones

  • Profoundly impacts global gene expression

  • Abrogates breast cancer metastasis

• OTUD7B also modulates cell cycle-dependent LSD1 oscillation, ensuring proper G1/S transition.

• Clinical significance: Both OTUD7B and LSD1 proteins are overexpressed in high-grade or metastatic human breast cancer, and dysregulation of either protein is associated with poor survival outcomes and increased metastasis .

This research reveals OTUD7B's unique "partner-switching" role in maintaining the integrity of the LSD1/CoREST corepressor complex and regulating LSD1 turnover, highlighting another mechanism by which OTUD7B influences cancer progression.

How does OTUD7B regulate T cell activation and inflammatory responses?

OTUD7B serves as a positive regulator of T cell activation through deubiquitination of the tyrosine kinase Zap70:

• Unlike its inhibitory role in B cells (where it targets TRAF3), OTUD7B functions as a crucial positive regulator of TCR-proximal signaling in T cells.

• Mechanistically, OTUD7B:

  • Rapidly binds to Zap70 following TCR-CD28 ligation

  • Inhibits Zap70 ubiquitination

  • Prevents association of Zap70 with the phosphatase Sts1/2

  • Facilitates Zap70 phosphorylation and downstream TCR signaling

• Functional consequences of OTUD7B deficiency include:

  • Attenuated TCR-CD28-stimulated Zap70 activation

  • Impaired downstream signaling

  • Reduced T cell activation

  • Defective Th1 cell differentiation

  • Resistance to T cell-mediated autoimmunity and inflammation

• In experimental autoimmune encephalomyelitis (EAE) models, OTUD7B-deficient mice showed:

  • Profound defects in T cell recall responses

  • Reduced production of cytokines (IL-2, IFN-γ, IL-17A)

  • Decreased frequency of Th1 cells in lymphoid organs

This research establishes OTUD7B as a critical regulator of T cell-mediated immune and autoimmune responses, revealing its substrate specificity and contextual function across different immune cell types.

What is the role of OTUD7B in dendritic cell function during infection?

OTUD7B protects dendritic cells (DCs) from TNF-induced apoptosis during infection, thereby enabling efficient priming of adaptive immune responses:

• In dendritic cells, OTUD7B prevents proteasomal degradation of TRAF2 (TNF receptor-associated factor 2) following TNF stimulation.

• Mechanistically:

  • OTUD7B interacts with TRAF2 upon TNF stimulation

  • It reduces K48-linked polyubiquitin chains on TRAF2

  • This prevents proteasomal degradation of TRAF2

  • K11-linked polyubiquitination of TRAF2 remains unaffected

• Functional significance:

  • Inhibition of proteasome in TNF-stimulated bone marrow-derived dendritic cells (BMDCs) restored TRAF2 levels in OTUD7B-deficient cells

  • OTUD7B-deficient BMDCs showed enhanced K48-linked polyubiquitination of TRAF2

  • This mechanism protects dendritic cells from TNF-induced apoptosis during infection

  • Preserved DC viability leads to efficient priming of adaptive immune responses

This study highlights another context-specific role for OTUD7B in immune regulation, specifically in maintaining dendritic cell survival during inflammatory responses.

What are the most effective approaches for studying OTUD7B's deubiquitinating activity?

Several complementary approaches are effective for investigating OTUD7B's deubiquitinating activity:

• Chemoproteomics fragment screening platforms:

  • Combining activity-based protein profiling with high-throughput chemistry

  • Direct-to-biology optimization for rapid elaboration of fragment hits

  • Enables identification of novel DUB-specific compounds

  • Has successfully identified enantioselective covalent fragments for OTUD7B

• siRNA knockdown and overexpression systems:

  • siRNA libraries targeting DUBs to identify enzymes responsible for specific substrate deubiquitination

  • Nonoverlapping siRNA mixtures to minimize off-target effects

  • Ectopic expression of OTUD7B to confirm enzyme-substrate relationships

  • Rescue experiments to validate functional significance

• Ubiquitination assays:

  • Co-immunoprecipitation followed by western blotting for specific ubiquitin linkages

  • Detection of K11, K48, or K63-linked polyubiquitin chains on substrates

  • Comparison of ubiquitination levels between wild-type and OTUD7B-deficient cells

  • Proteasome inhibition to confirm proteasomal degradation mechanisms

• Functional validation in cellular and animal models:

  • Cell proliferation assays (CCK8, clone formation)

  • Cell cycle analysis

  • DNA synthesis (EdU incorporation assay)

  • Migration assays (wound-healing)

  • In vivo xenograft models

  • Disease-specific models (EAE for autoimmunity)

These methodologies provide complementary insights into OTUD7B's enzymatic activity, substrate specificity, and functional significance in various biological contexts.

How can researchers design selective inhibitors or tool compounds for OTUD7B?

Designing selective inhibitors or tool compounds for OTUD7B presents challenges but recent advances offer promising strategies:

• Chemoproteomics-based approaches:

  • Activity-based protein profiling to identify DUB-specific hit matter

  • Fragment screening platforms that combine protein profiling with high-throughput chemistry

  • Direct-to-biology optimization for rapid fragment hit elaboration

  • This approach has successfully identified an enantioselective covalent fragment for OTUD7B

• Structural considerations for selectivity:

  • Understanding that many DUB catalytic pockets are structurally similar, requiring careful design

  • Exploiting unique structural features of OTUD7B's OTU domain

  • Considering substrate-binding regions beyond the catalytic site

  • Focusing on enantioselectivity to enhance specificity

• Validation methodologies:

  • Chemoproteomics to confirm target engagement

  • Biochemical DUB activity assays to determine potency and selectivity

  • Testing against panels of related DUBs to ensure specificity

  • Cellular assays to confirm functional effects

• Challenges in development:

  • Translation of DUB inhibitors into clinically-relevant compounds has been slow

  • Need for enhanced understanding of OTUD7B's context-dependent roles

  • Balancing potency with selectivity across the DUB family

  • Addressing potential off-target effects

These approaches can guide researchers in developing selective tool compounds for OTUD7B, which are critical for better understanding its cellular functions and therapeutic potential in various diseases.

What is known about OTUD7B's role in acute myeloid leukemia (AML)?

OTUD7B exhibits tumor-suppressive properties in acute myeloid leukemia (AML), with notable implications for patient outcomes:

• Expression patterns:

  • OTUD7B expression is significantly lower in primary leukemia cells from all types of AML patients compared to normal controls

  • Similarly reduced expression is observed in bone marrow, liver, and spleen of M2-type AML mice

  • Low OTUD7B expression correlates with shorter survival time in AML patients

• Functional effects in AML cells:

  • Overexpression of OTUD7B in AML cell lines (HL60 and Kasumi1) significantly:

    • Inhibits cell viability

    • Decreases the percentage of cells in S phase

    • Impairs cell cycle progression

• Molecular mechanisms:

  • OTUD7B significantly inhibits the phosphorylation of AKT and mTOR in AML cells

  • AKT1 overexpression partially reverses the inhibitory effect of OTUD7B on cell growth

  • This suggests OTUD7B's tumor-suppressive effect in AML may be mediated through inhibition of the AKT/mTOR signaling pathway

• Clinical significance:

  • The survival time of AML patients with low OTUD7B expression is significantly shorter

  • OTUD7B expression levels may serve as a prognostic marker in AML

  • OTUD7B could represent a potential therapeutic target for AML treatment

These findings establish OTUD7B as a tumor suppressor in AML, contrasting with its oncogenic role in certain solid tumors like breast cancer, and highlight the context-dependent nature of OTUD7B function in different malignancies.

How do researchers reconcile the seemingly contradictory roles of OTUD7B across different cancer types?

The apparently contradictory roles of OTUD7B across different cancer types represent a fascinating research challenge that can be addressed through several approaches:

• Substrate specificity analysis:

  • OTUD7B exhibits distinct substrate preferences in different cellular contexts:

    • In breast cancer: primarily deubiquitinates ERα, promoting cancer progression

    • In HCC: targets p53, functioning as a tumor suppressor

    • In AML: inhibits the AKT/mTOR pathway, suppressing leukemia cell growth

  • Comprehensive substrate identification in each cancer type using proteomics approaches can help clarify these context-specific functions

• Cellular context and microenvironment:

  • Tissue-specific expression patterns of OTUD7B and its substrates

  • Differential availability of binding partners or regulatory proteins

  • Varying ubiquitin chain preferences depending on cellular context (K11 vs. K48 vs. K63)

  • Impact of the tumor microenvironment on OTUD7B function

• Integrated multi-omics approaches:

  • Combining transcriptomics, proteomics, and ubiquitinomics data

  • Correlating OTUD7B levels with global ubiquitination patterns

  • Analyzing oncogenic pathway activation states in relation to OTUD7B expression

  • Examining genetic and epigenetic regulation of OTUD7B across cancer types

• Methodological considerations:

  • Using consistent experimental systems to enable direct comparisons

  • Developing conditional knockout models to study tissue-specific effects

  • Employing selective inhibitors or activators to manipulate OTUD7B activity

  • Utilizing patient-derived xenografts to maintain tumor heterogeneity

This comprehensive approach can help resolve the paradoxical roles of OTUD7B and potentially identify cancer types where OTUD7B inhibition or activation might offer therapeutic benefits.

What are the latest advancements in identifying novel OTUD7B substrates and their functional significance?

Recent advancements in identifying novel OTUD7B substrates have employed sophisticated methodologies to expand our understanding of its functional roles:

• Proteomics-based substrate identification:

  • Mass spectrometry screening of immunoprecipitates from various cell types has identified multiple OTUD7B-binding proteins

  • Comparative ubiquitinome analysis between wild-type and OTUD7B-deficient cells

  • Recently identified substrates include:

    • p53 in hepatocellular carcinoma

    • Zap70 in T cells

    • TRAF2 in dendritic cells

    • LSD1 in breast cancer

• Validation of substrate deubiquitination:

  • Co-immunoprecipitation assays with endogenous, ectopic, and mutant forms of OTUD7B and potential substrates

  • Assessment of polyubiquitination levels using linkage-specific antibodies

  • Proteasome inhibition experiments to confirm degradation mechanisms

  • In vitro deubiquitination assays with purified components

• Functional significance elucidation:

  • Rescue experiments overexpressing substrates in OTUD7B-deficient backgrounds

  • Analysis of substrate phosphorylation and other post-translational modifications

  • Examination of protein-protein interactions affected by OTUD7B-mediated deubiquitination

  • Genome-wide occupancy studies for chromatin-associated substrates (e.g., LSD1)

• Physiological contexts:

  • Cell cycle-dependent regulation (OTUD7B modulates cell cycle-dependent LSD1 oscillation)

  • Immune signaling contexts (OTUD7B regulates TCR signaling through Zap70)

  • Cancer progression models (multiple substrates with context-dependent outcomes)

  • Inflammatory responses (protection of dendritic cells from TNF-induced apoptosis)

These advancements highlight the diverse functional roles of OTUD7B through its expanding repertoire of substrates, offering new insights into its therapeutic potential across various disease contexts.

What are the most promising therapeutic applications targeting OTUD7B in disease?

Based on current research, several promising therapeutic applications targeting OTUD7B are emerging:

• Breast cancer treatment:

  • Inhibiting OTUD7B could reduce ERα stability in hormone-positive breast cancers

  • This approach may complement existing endocrine therapies or address resistance mechanisms

  • Combined targeting of OTUD7B and LSD1 pathways might be particularly effective for metastatic disease

• Hepatocellular carcinoma therapy:

  • Activating or stabilizing OTUD7B could enhance p53 function in HCC

  • This strategy may reactivate tumor suppression mechanisms

  • Particularly valuable in p53 wild-type tumors where ubiquitin-mediated degradation drives oncogenesis

• Autoimmune disease management:

  • Targeting OTUD7B in T cells could attenuate pathogenic T cell responses

  • Potentially beneficial in T cell-mediated autoimmune conditions

  • Demonstrated efficacy in experimental autoimmune encephalomyelitis models

• Acute myeloid leukemia therapy:

  • Restoring OTUD7B expression or function could inhibit AKT/mTOR signaling

  • May sensitize AML cells to existing therapies

  • Prognostic value in stratifying patients for targeted interventions

• Tool development priorities:

  • Selective OTUD7B inhibitors for breast cancer and autoimmunity

  • OTUD7B stabilizers or activators for HCC and AML

  • Context-specific delivery systems to target OTUD7B modulation to specific tissues

The development of effective OTUD7B-targeting therapeutics will require careful consideration of its context-dependent roles and substrate specificity across different diseases. The recent identification of enantioselective covalent fragments for OTUD7B represents a significant step toward this goal .

What technological advances are needed to better characterize OTUD7B function in complex biological systems?

Advancing our understanding of OTUD7B function in complex biological systems requires several technological innovations:

• Improved structural biology approaches:

  • Cryo-EM structures of OTUD7B in complex with various substrates

  • Structural analysis of OTUD7B bound to different ubiquitin chain types

  • Time-resolved structural studies to capture the dynamics of deubiquitination

  • Computational modeling of substrate binding specificity

• Enhanced in vivo imaging techniques:

  • Live-cell imaging of OTUD7B-substrate interactions

  • Biosensors for real-time monitoring of deubiquitinating activity

  • Spatiotemporal analysis of OTUD7B localization and function

  • In vivo imaging of ubiquitination dynamics in animal models

• Advanced genetic models:

  • Tissue-specific and inducible OTUD7B knockout/knockin systems

  • CRISPR-based screens to identify synthetic lethal interactions

  • Patient-derived organoids to study OTUD7B in human disease contexts

  • Humanized mouse models for immunological studies

• Single-cell analysis technologies:

  • Single-cell proteomics to examine OTUD7B expression and substrate levels

  • Single-cell ubiquitinomics to capture heterogeneity in deubiquitination events

  • Spatial transcriptomics/proteomics to map OTUD7B function within tissues

  • Integration with single-cell genomics data to link genetic variation to function

• Selective chemical tools:

  • Development of highly selective OTUD7B inhibitors and activators

  • Activity-based probes for monitoring OTUD7B engagement in vivo

  • Degraders (PROTACs) targeting OTUD7B for temporal control

  • Targeted delivery systems for tissue-specific modulation