OTUD3 Antibody

What is OTUD3 and what are its primary biochemical functions?

OTUD3 (OTU Domain Containing 3) is a deubiquitinating enzyme (DUB) that primarily hydrolyzes 'Lys-6'- and 'Lys-11'-linked polyubiquitin chains. It plays a crucial role in the ubiquitin-proteasome system (UPS), which controls protein turnover and homeostasis within cells. OTUD3 functions by removing ubiquitin molecules from proteins, thus maintaining their stability. The UPS is essential for eukaryotic life and regulates many aspects of cell physiology, with its dysfunction contributing to various human diseases including cancer, metabolic syndromes, neurodegeneration, autoimmunity, inflammatory disorders, and infection .

OTUD3 is also an important regulator of energy metabolism. Glucose and fatty acids can trigger its nuclear translocation through CBP-dependent acetylation. Once in the nucleus, OTUD3 deubiquitinates and stabilizes the nuclear receptor PPARD, regulating the expression of various genes involved in glucose and lipid metabolism and oxidative phosphorylation .

What are the recommended applications and dilutions for OTUD3 antibodies?

It is important to note that these dilutions should be optimized for each specific experimental system. The appropriate dilution may be sample-dependent, so researchers should check validation data galleries provided by manufacturers and conduct titration experiments to determine optimal conditions for their specific application .

How does OTUD3 expression vary across different tissue types and disease states?

OTUD3 exhibits a fascinating pattern of differential expression across tissues and disease states, with important implications for its role in pathogenesis:

In cancer contexts:

• Decreased expression: OTUD3 shows reduced expression in breast cancer, hepatocellular cancer, colon cancer, and cervical cancer compared to adjacent normal tissues, concomitant with reduction of PTEN abundance .

• Increased expression: Conversely, OTUD3 is significantly upregulated in human lung cancer tissues, and elevated expression correlates with poor prognosis in lung cancer patients .

• Expression in cholangiocarcinoma: OTUD3 exhibits high expression in cholangiocarcinoma tissues, correlating with poor patient prognosis .

In normal tissues:

• OTUD3 is detectable in multiple cell types including HeLa, Neuro-2a, HepG2, MCF-7, and HCT 116 cells, suggesting broad expression across tissues .

• Western blot analyses have confirmed OTUD3 expression in various cancer cell lines, including lung cancer cell lines (H1299, A549), colorectal cancer cell lines (SW480, DLD1, LoVo), and breast cancer cell lines (MCF7, MDA-MB-231) .

This differential expression pattern across tissues and disease states underscores the context-dependent role of OTUD3 in cellular processes and disease progression.

How does OTUD3 function as both tumor suppressor and oncogene depending on cellular context?

OTUD3 presents a remarkable example of context-dependent protein function in cancer biology, with dual roles that depend on the specific tissue type and molecular environment:

Tumor suppressor functions:

• In breast cancer, OTUD3 suppresses tumorigenesis by deubiquitinating and stabilizing PTEN, a critical tumor suppressor. In vivo deletion of OTUD3 promotes breast cancer development in mouse models .

• Similar tumor-suppressive roles are observed in hepatocellular cancer, colon cancer, and cervical cancer, where OTUD3 depletion promotes cell proliferation, anchorage-independent growth, and tumor metastasis .

• Mechanistically, OTUD3 depletion in these cancers results in decreased PTEN levels, leading to enhanced activation of oncogenic pathways like PI3K/AKT .

Oncogenic functions:

• In lung cancer, OTUD3 acts as an oncogene by deubiquitinating and stabilizing GRP78 (glucose-regulated protein). Deletion of OTUD3 slows down Kras G12D-driven lung adenocarcinoma initiation and progression, markedly increasing survival in mice .

• OTUD3 demonstrates a similar oncogenic role in cholangiocarcinoma by stabilizing ARID3A through deubiquitination, with GSK3β enhancing this process through phosphorylation .

• In colorectal cancer, OTUD3 stabilizes YY1 by removing K48-linked polyubiquitination at K332, K339, and K409 residues, accelerating cancer progression .

This dual functionality emphasizes the importance of considering tissue-specific substrate interactions when studying OTUD3. The enzymatic activity of OTUD3 is consistent across tissues—removing ubiquitin from proteins—but the biological outcome varies dramatically depending on which substrate proteins are stabilized in different cellular contexts .

What role does OTUD3 play in regulating metabolic pathways through deubiquitination?

OTUD3 exerts significant influence on cellular metabolic pathways through its deubiquitinating activity:

mTORC1 pathway regulation:

• OTUD3 suppresses the mTORC1 signaling pathway by deubiquitinating KPTN, a component of the KICSTOR complex. The OTU domain of OTUD3 interacts with KPTN, ITFG2, and C12orf66 (components of KICSTOR) .

• Through this mechanism, OTUD3 affects cellular metabolic pool products, significantly influencing tumor cell growth and proliferation .

Metabolic changes in OTUD3-deficient cells:

• Nuclear magnetic resonance (NMR) spectroscopy comparing OTUD3-knockout and wild-type HeLa cells identified significant differences in 18 metabolites, including:

  • Increased levels of amino acids (phenylalanine, tyrosine, histidine, aspartic acid, alanine, valine, isoleucine, and leucine)

  • Elevated succinate, indicating a shift towards glycolytic metabolism (Warburg effect)

  • Higher levels of reduced glutathione, favoring maintenance of oxidative stress levels

  • Increased UDP, uridine, and uracil, suggesting enhanced RNA synthesis

  • Elevated UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, crucial for protein modification processes

Energy metabolism regulation:

• OTUD3 acts as a regulator of energy metabolism through its interaction with PPARD, a nuclear receptor involved in glucose and lipid metabolism.

• Glucose and fatty acids trigger OTUD3's nuclear translocation through CBP-dependent acetylation, where it deubiquitinates and stabilizes PPARD .

These findings demonstrate that OTUD3 can be a pivotal regulator of cellular metabolism, with its deubiquitinating activity affecting multiple metabolic pathways that support cell growth, proliferation, and stress responses .

How do post-translational modifications regulate OTUD3 activity and substrate specificity?

Post-translational modifications play critical roles in modulating OTUD3 function, affecting its activity, localization, and substrate interactions:

Phosphorylation regulation:

• GSK3β phosphorylates OTUD3 at Ser9, significantly enhancing its binding affinity to substrate proteins like ARID3A in cholangiocarcinoma. This phosphorylation leads to more efficient deubiquitination and stabilization of ARID3A, promoting cancer progression .

• Phosphorylation-dependent regulation also affects OTUD3's interaction with YY1 in colorectal cancer, highlighting the importance of this modification in determining substrate specificity .

• PLK1 has been identified as another kinase that can interact with OTUD3, suggesting additional phosphorylation events may regulate its function .

Acetylation effects:

• OTUD3 undergoes CBP-dependent acetylation in response to glucose and fatty acids, which triggers its nuclear translocation. This nutritional sensing mechanism allows OTUD3 to regulate metabolic genes through interaction with nuclear receptors like PPARD .

• Nicotine exposure can affect OTUD3 expression through PI3K/AKT/FOXO1 signaling. FOXO1 promotes OTUD3 transcription, and nicotine reduces OTUD3 promoter activity by decreasing the enrichment of FOXO1, p300, H3K27ac, and RNA polymerase II on the OTUD3 promoter .

Catalytic activity dependence:

• The catalytically inactive mutant OTUD3-C76A retains its ability to interact with substrates but fails to enhance their stability. For example, wild-type OTUD3, but not OTUD3-C76A, can stabilize YY1 protein in colorectal cancer cells .

• Similarly, wild-type OTUD3, rather than OTUD3-C76A, inhibits the polyubiquitination of substrate proteins, confirming that enzymatic activity is essential for its biological functions .

These modifications constitute a complex regulatory network that determines when, where, and which substrates OTUD3 deubiquitinates, explaining its diverse and sometimes opposing functions in different cellular contexts .

What experimental approaches should be used to study OTUD3-substrate interactions?

Investigating OTUD3-substrate interactions requires a comprehensive set of experimental techniques to establish binding, determine functional consequences, and validate physiological relevance:

Protein-protein interaction assays:

• Co-immunoprecipitation (Co-IP): Overexpress tagged OTUD3 and potential substrate proteins (e.g., OTUD3-Myc and KPTN-Flag) in cells like HEK-293T, followed by immunoprecipitation with appropriate antibodies. Perform reciprocal experiments to confirm interactions. This approach has successfully identified interactions between OTUD3 and various substrates including GRP78, YY1, KPTN, and ARID3A .

• GST pull-down assay: Purify GST-OTUD3 from E. coli BL21 (DE3) using Glutathione Sepharose 4B beads. Incubate with cell lysates containing candidate substrate proteins (e.g., Flag-YY1) and detect bound proteins via immunoblotting. This approach helps confirm direct interactions .

• Proximity Ligation Assay (PLA): Fix cells in 4% paraformaldehyde, block with 5% BSA, and incubate with antibodies against OTUD3 and substrate. Perform PLA using the Duolink In Situ PLA kit to visualize interactions in their cellular context .

Deubiquitination assays:

• In vivo ubiquitination assay: Transfect cells with plasmids expressing OTUD3 (wild-type or C76A mutant), substrate protein, and ubiquitin (preferably His-tagged for pulldown). Treat with MG132 to prevent degradation of ubiquitinated proteins. Immunoprecipitate the substrate and detect ubiquitination by immunoblotting .

• In vitro deubiquitination assay: Combine purified OTUD3 with ubiquitinated substrate proteins and analyze ubiquitination levels to determine if OTUD3 can directly deubiquitinate the substrate .

Domain mapping:

• Generate truncated constructs of OTUD3 (e.g., ΔOTU, ΔUBA, ΔTail) and perform Co-IP with substrate proteins to identify domains essential for interactions. The OTU domain has been identified as critical for several interactions .

• Create substrate mutants (e.g., YY1 mutants at K332, K339, K393, and K409) to identify specific ubiquitination sites targeted by OTUD3 .

Functional validation:

• Stability assays: Perform cycloheximide chase experiments to assess substrate protein half-life in cells with OTUD3 overexpression, knockdown, or knockout .

• Rescue experiments: In OTUD3 knockout cells, reintroduce either wild-type OTUD3 or catalytically inactive C76A mutant to determine if substrate stability can be restored .

These methodologies should be combined to provide comprehensive characterization of OTUD3-substrate interactions and their functional significance in specific cellular contexts .

What controls are essential when investigating context-dependent roles of OTUD3 in cancer?

When studying the dual roles of OTUD3 in cancer, implementing comprehensive controls is critical for valid interpretation of results:

Cellular model controls:

• Multiple cell line validation: Test findings across several cell lines from the same cancer type. For example, lung cancer studies should include multiple lines like H1299 and A549, while breast cancer studies should include lines like MCF7 and MDA-MB-231 .

• Cross-cancer comparison: Include cell lines where OTUD3 has opposing functions (e.g., breast cancer vs. lung cancer cells) to directly compare context-dependent effects .

• Normal tissue controls: Include normal cell counterparts, such as human normal bronchial epithelium HBE cells for lung cancer studies .

Expression manipulation controls:

• Activity-dependent controls: Compare effects of wild-type OTUD3 with the catalytically inactive mutant OTUD3-C76A to distinguish between scaffold and enzymatic functions .

• Domain-specific controls: Use domain deletion mutants (ΔOTU, ΔUBA, ΔTail) to identify domains critical for specific functions .

• Expression level controls: Include both knockdown/knockout and overexpression studies to establish dose-dependent effects .

Substrate-specific controls:

• Substrate measurement: Quantify levels of known context-specific OTUD3 substrates (PTEN in breast cancer, GRP78 in lung cancer, YY1 in colorectal cancer, ARID3A in cholangiocarcinoma) .

• Epistasis analysis: Perform substrate knockdown/rescue experiments in combination with OTUD3 manipulation. For example, YY1 depletion reversed OTUD3 overexpression-induced tumor progression in colorectal cancer .

Ubiquitination controls:

• Proteasome inhibition: Treat cells with MG132 to prevent degradation of ubiquitinated proteins and enable detection of ubiquitination changes .

• Ubiquitin chain specificity: Use ubiquitin mutants (K6, K11, K27, K29, K33, K48, K63) to determine the types of ubiquitin chains affected by OTUD3 .

In vivo validation:

• Cancer-specific mouse models: Use appropriate models for each cancer type, such as MMTV-PyMT for breast cancer and Kras G12D for lung cancer .

• OTUD3 knockout models: Compare effects of OTUD3 deletion across different cancer types to confirm tissue-specific roles observed in vitro .

What techniques are most effective for studying the metabolic impact of OTUD3 activity?

Investigating OTUD3's influence on cellular metabolism requires a multi-faceted approach combining several specialized techniques:

Metabolomics approaches:

• Nuclear Magnetic Resonance (NMR) spectroscopy: This technique has successfully identified significant differences in 18 metabolites between OTUD3-knockout and wild-type HeLa cells, including amino acids, succinate, glutathione, and nucleotide derivatives. NMR provides a comprehensive view of the metabolic changes induced by OTUD3 manipulation .

• Mass spectrometry-based metabolomics: Complements NMR by offering higher sensitivity for detecting low-abundance metabolites and can be used for targeted analysis of specific metabolic pathways affected by OTUD3.

Signaling pathway analysis:

• mTORC1 pathway monitoring: Assess phosphorylation status of downstream mTORC1 targets (S6K, 4E-BP1) in response to OTUD3 manipulation. This approach revealed that OTUD3 suppresses mTORC1 signaling by deubiquitinating KPTN, a component of the KICSTOR complex .

• Western blotting for key metabolic regulators: Monitor levels of PPARD and other metabolic enzymes regulated by OTUD3-dependent deubiquitination .

Functional metabolic assays:

• Cell proliferation assays: Crystal violet staining and CCK8 cell proliferation assays have demonstrated that OTUD3 has the capacity to inhibit tumor cell growth and proliferation by downregulating the mTOR signaling pathway .

• Metabolic flux analysis: Use isotope-labeled nutrients to track metabolic pathway activities and determine how OTUD3 influences metabolic flux distribution.

• Seahorse XF analysis: Measure oxygen consumption rate and extracellular acidification rate to assess mitochondrial respiration and glycolysis in cells with altered OTUD3 expression.

Substrate-focused approaches:

• KICSTOR complex analysis: Investigate interactions between OTUD3 and KICSTOR components (KPTN, ITFG2, C12orf66) and how these interactions affect nutrient sensing and mTORC1 regulation .

• PPARD activity assays: Assess PPARD-mediated transcription of metabolic genes in the presence or absence of OTUD3 to understand its impact on metabolic gene expression.

These techniques provide complementary information about how OTUD3 affects cellular metabolism, enabling researchers to build a comprehensive understanding of its metabolic functions. The choice of specific techniques should be guided by the particular metabolic pathways or processes of interest .

What strategies can optimize detection of endogenous OTUD3 using different antibodies?

Optimizing detection of endogenous OTUD3 requires careful selection of antibodies and methodological refinements based on the specific application:

Western blot optimization:

• Antibody selection: Polyclonal antibodies (e.g., 29622-1-AP) work well at dilutions of 1:1000-1:4000, while recombinant antibodies (e.g., 84717-1-RR) provide higher specificity at dilutions of 1:5000-1:50000 .

• Expected molecular weight: OTUD3 has a calculated molecular weight of 45 kDa but is often detected around 43 kDa. Include positive control lysates (HeLa, HCT116, HepG2, MCF-7 cells) where OTUD3 expression has been confirmed .

• Sample preparation: Include protease inhibitors in lysis buffers to prevent degradation. For studying phosphorylated forms of OTUD3, include phosphatase inhibitors.

• Validation controls: Include OTUD3 knockout samples (generated using CRISPR/Cas9) as negative controls to confirm antibody specificity .

Immunohistochemistry considerations:

• Antigen retrieval: For optimal results, use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 .

• Antibody dilution: Start with 1:50-1:500 dilution range for IHC applications .

• Positive tissue controls: Mouse cerebellum tissue has been validated for OTUD3 IHC .

• Fixation methods: Use 4% paraformaldehyde fixation for 10 minutes when preparing samples for proximity ligation assays or immunofluorescence .

Co-immunoprecipitation strategies:

• Antibody efficiency: Some antibodies may work better for immunoprecipitation than detection. Test multiple antibodies for their ability to pull down endogenous OTUD3.

• Validation approach: Perform reciprocal co-immunoprecipitation (e.g., pull down with anti-KPTN and detect OTUD3, then pull down with anti-OTUD3 and detect KPTN) to confirm interactions .

General considerations:

• Tissue-specific expression: Be aware that OTUD3 expression varies significantly across tissue types, with higher expression in lung cancer tissues compared to breast, hepatocellular, colon, and cervical cancers .

• Titration requirement: It is strongly recommended to titrate antibodies in each testing system to obtain optimal results, as the appropriate dilution may be sample-dependent .

• Storage conditions: Store antibodies at -20°C for stability. For 20μl sizes, note that they may contain 0.1% BSA .

By implementing these optimization strategies, researchers can achieve reliable detection of endogenous OTUD3 across various experimental systems and applications .

What is known about OTUD3 inhibitors and their potential therapeutic applications?

Recent research has identified promising OTUD3 inhibitors with therapeutic potential, particularly for cancer treatment:

OTUDin3 inhibitor development:

• A small-molecule inhibitor of OTUD3 (named OTUDin3) was discovered through computer-aided virtual screening and validated through biological experimental verification .

• The development process involved targeting the deubiquitinating activity of OTUD3, recognizing its role in stabilizing oncoproteins in certain cancer types .

Mechanism of action:

• OTUDin3 specifically inhibits the deubiquitinating activity of OTUD3, preventing it from removing ubiquitin from substrate proteins .

• In non-small-cell lung cancer (NSCLC), this inhibition prevents OTUD3 from stabilizing oncoprotein GRP78, leading to its degradation through the ubiquitin-proteasome pathway .

Therapeutic effects in NSCLC:

• OTUDin3 exhibited pronounced antiproliferative and proapoptotic effects in NSCLC cell lines .

• In vivo studies demonstrated that OTUDin3 efficaciously inhibited growth of lung cancer xenografts in mice, validating its potential as a therapeutic agent .

Context-dependent considerations:

• The therapeutic potential of OTUD3 inhibition must be evaluated in a cancer-specific context, given OTUD3's dual role as both oncogene and tumor suppressor .

• In cancers where OTUD3 functions as an oncogene (e.g., lung cancer, cholangiocarcinoma), inhibition could be beneficial .

• In cancers where OTUD3 acts as a tumor suppressor (e.g., breast cancer, hepatocellular cancer), inhibition might potentially promote tumor growth and should be approached with caution .

Future directions:

• Development of more selective OTUD3 inhibitors with improved pharmacokinetic properties.

• Investigation of combination therapies that might enhance efficacy while mitigating potential adverse effects.

• Exploration of context-specific delivery methods to target inhibition to tissues where OTUD3 functions as an oncogene.

The discovery of OTUDin3 represents a significant step toward targeting the ubiquitin-proteasome system for cancer therapy, particularly for NSCLC where OTUD3 plays an oncogenic role .

How can researchers effectively evaluate OTUD3 activity in response to experimental treatments?

Evaluating OTUD3 activity in response to experimental treatments requires a multi-dimensional approach focusing on enzymatic function, substrate stabilization, and downstream effects:

Direct enzymatic activity assays:

• In vitro deubiquitination assays: Measure OTUD3's ability to cleave ubiquitin chains from substrates in the presence of inhibitors or other treatments. Compare activity of wild-type OTUD3 versus catalytically inactive mutant OTUD3-C76A as controls .

• Ubiquitin chain specificity analysis: Determine how treatments affect OTUD3's preference for different ubiquitin linkages (Lys-6, Lys-11, K48, etc.) using purified ubiquitin chains and recombinant OTUD3 .

Substrate stabilization assessment:

• Protein stability assays: Perform cycloheximide chase experiments to measure half-lives of known OTUD3 substrates (PTEN, GRP78, YY1, ARID3A, KPTN) in the presence or absence of treatments .

• Ubiquitination status monitoring: Conduct immunoprecipitation of substrate proteins followed by ubiquitin immunoblotting to assess changes in ubiquitination levels after treatment .

• Context-specific substrate analysis: Based on the cellular context, focus on relevant substrates (e.g., GRP78 in lung cancer, PTEN in breast cancer) .

Downstream pathway evaluation:

• mTORC1 signaling: Monitor phosphorylation of S6K and 4E-BP1 to assess mTORC1 pathway activity when targeting the OTUD3-KPTN interaction .

• Metabolite profiling: Use NMR spectroscopy to detect changes in metabolic profiles in response to OTUD3 modulation .

• Cell proliferation and apoptosis: Assess antiproliferative and proapoptotic effects using crystal violet staining, CCK8 assays, and apoptosis markers to evaluate functional consequences of OTUD3 inhibition .

OTUD3 post-translational modifications:

• Phosphorylation analysis: Use phospho-specific antibodies to detect changes in OTUD3 phosphorylation status (e.g., at Ser9) in response to treatments targeting kinases like GSK3β .

• Nuclear translocation: Monitor OTUD3's subcellular localization after metabolic treatments that affect CBP-dependent acetylation .

In vivo evaluation:

• Xenograft models: Assess tumor growth in mice treated with OTUD3 inhibitors or other experimental agents .

• Patient-derived samples: Analyze OTUD3 activity and substrate levels in patient samples before and after treatment when possible.

• Cancer-specific modeling: Use appropriate cancer models based on OTUD3's role in that specific cancer type (e.g., Kras G12D for lung cancer, MMTV-PyMT for breast cancer) .

By combining these approaches, researchers can comprehensively evaluate how experimental treatments affect OTUD3 activity and determine whether observed effects align with the expected molecular mechanisms of action .