OTUB1 Human

What is OTUB1 and what are its primary functions in human cells?

OTUB1 is a member of the OTU (ovarian tumor) superfamily of cysteine proteases that functions as a deubiquitinating enzyme (DUB). Located at locus 11q13.1, it is expressed in various human tissues, with particularly high expression in the brain . OTUB1 serves as a highly specific ubiquitin iso-peptidase that cleaves ubiquitin from branched poly-ubiquitin chains but not from ubiquitinated substrates .

OTUB1 possesses two distinct activities:

• Canonical activity: Direct deubiquitination through its cysteine protease function

• Non-canonical activity: Inhibition of ubiquitination by binding to ubiquitin-charged E2 enzymes, preventing ubiquitin transfer onto substrates

Through these mechanisms, OTUB1 regulates multiple cellular pathways including MAPK signaling, DNA damage response, immune response, and cell death pathways such as necroptosis .

How does OTUB1 differ structurally and functionally from other deubiquitinating enzymes?

OTUB1 has several distinctive features compared to other DUBs:

• Chain-type specificity: OTUB1 preferentially cleaves K48-linked polyubiquitin chains over K63-linked chains, unlike many other DUBs that show different preferences or less specificity .

• Unique regulatory mechanism: Structural analysis reveals that OTUB1 has differences in accessibility to the active site and surface properties compared to its close homologue OTUB2, suggesting variations in regulatory mechanisms .

• Non-canonical activity: Unlike most DUBs that function solely through catalytic activity, OTUB1 can inhibit ubiquitination independent of its catalytic function by directly binding to E2 ubiquitin-conjugating enzymes .

• Substrate recognition: OTUB1 has a narrow P1' site that correlates with its ability to preferentially cleave K48-linked ubiquitin chains .

• Conformational changes: OTUB1 undergoes significant conformational changes upon binding ubiquitin at its distal site, which are essential for both its canonical and non-canonical activities .

What is the significance of OTUB1's substrate specificity toward different ubiquitin chain types?

OTUB1's preference for K48-linked polyubiquitin chains over K63-linked chains has important functional implications:

• Proteasomal degradation regulation: K48-linked chains typically target proteins for proteasomal degradation. OTUB1's specificity allows it to selectively rescue proteins from degradation, as demonstrated by its ability to stabilize proteins like c-IAP1 and SLC7A11 .

• Signaling pathway modulation: By selectively removing K48-linked chains, OTUB1 can regulate the stability of signaling components while potentially leaving regulatory K63-linked chains intact, providing a nuanced control mechanism for signaling pathways .

• Experimental determination: This specificity can be assessed biochemically using purified components. Research has shown that when tested in vitro with different chain types, OTUB1 displays significant hydrolytic activity toward K48-linked chains but minimal activity toward K63-linked chains .

• Structural basis: Mutational analysis suggests that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave K48-linked ubiquitin chains .

What are the major signaling pathways regulated by OTUB1?

OTUB1 regulates multiple signaling pathways critical for cell survival, proliferation, and disease progression:

• MAPK pathway: OTUB1 stabilizes RAS proteins and enhances MAPK signaling, particularly in tumors with wild-type KRAS, where high OTUB1 levels correlate with increased ERK1/2 phosphorylation .

• p53 pathway: OTUB1 modulates p53 stability and activity through deubiquitination, affecting cell cycle arrest and apoptosis responses .

• Necroptosis pathway: OTUB1 prevents hepatocyte necroptosis by stabilizing c-IAP1. In OTUB1-deficient hepatocytes, c-IAP1 degradation results in increased RIPK1 phosphorylation, RIPK1/RIPK3 necrosome formation, and MLKL phosphorylation, leading to necroptotic cell death .

• TNF signaling: OTUB1 deficiency induces RIPK1-dependent ERK activation and TNF production in Listeria-infected hepatocytes .

• Ferroptosis regulation: In glioma cells, OTUB1 stabilizes SLC7A11 protein (a key suppressor of ferroptosis) by directly interacting with it and preventing its degradation .

• Additional cancer-associated pathways: OTUB1 also regulates ERα, epithelial-mesenchymal transition (EMT), RHOa, mTORC1, and FOXM1 signaling to promote tumor cell survival .

What experimental approaches can be used to distinguish between OTUB1's canonical and non-canonical activities?

Distinguishing between OTUB1's canonical deubiquitinase activity and non-canonical E2 inhibition requires specialized experimental approaches:

• Catalytic-dead mutants: Generate OTUB1 C91S mutants that lack deubiquitinase activity but retain the ability to bind E2 enzymes. Compare effects of wild-type versus catalytic-dead OTUB1 on substrate ubiquitination to identify non-canonical roles .

• In vitro ubiquitination assays: Reconstitute ubiquitination reactions with purified E1, E2 (such as UBC13), E3 ligase, and substrate, with or without OTUB1. Pre-incubation of E2~Ub with OTUB1 before adding E3 and substrate can reveal non-canonical inhibition .

• Structural approaches: Use X-ray crystallography, cryo-EM, or hydrogen-deuterium exchange mass spectrometry to analyze OTUB1 binding to E2 enzymes versus substrates. The 1.8 Å resolution structure of C. elegans OTUB1 bound to human UBC13 provides insight into non-canonical mechanisms .

• N-terminal truncation mutants: The N-terminal helix of OTUB1 is critical for non-canonical activity. Truncation mutants can help distinguish between the two activities, as the N-terminus contributes to donor ubiquitin recognition .

• Ubiquitin-based active-site probes: OTUB1 and OTUB2 show differential reactivity toward ubiquitin-based active-site probes carrying various C-terminal modifications (vinyl methyl ester, 2-chloroethyl, or 2-bromoethyl groups), which can be exploited to study canonical activity specifically .

How does OTUB1 contribute to cancer progression and metastasis at the molecular level?

OTUB1 promotes cancer progression through multiple molecular mechanisms:

• RAS/MAPK pathway enhancement: In wild-type KRAS tumors, OTUB1 stabilizes RAS proteins, leading to increased ERK1/2 phosphorylation. Copy number gain of OTUB1 occurs early in lung tumor development and is mutually exclusive with KRAS mutations, suggesting OTUB1 amplification may be an alternative mechanism for activating this pathway .

• Cancer stem cell maintenance: In glioma, OTUB1 promotes cancer stemness by stabilizing SLC7A11, a key suppressor of ferroptosis. Experimental evidence shows that OTUB1 knockdown significantly reduces sphere-formation ability, stemness marker expression, and ALDH activity in glioma cells .

• EMT promotion: OTUB1 regulates epithelial-mesenchymal transition, which is crucial for tumor invasion and metastasis .

• Clinical correlation: High OTUB1 expression is associated with aggressive clinical disease and poorer patient survival across multiple cancer types. In lung cancer patients with wild-type RAS tumors, moderate OTUB1 levels correlate with significantly poorer survival .

• Experimental validation approaches:

  • Immunohistochemical analysis of human tumors to correlate OTUB1 levels with pathway activation markers

  • Patient survival analysis stratified by OTUB1 expression and oncogene status

  • In vitro invasion and migration assays with OTUB1 knockdown/overexpression

  • Animal models to assess OTUB1's impact on metastasis and tumor growth

What is the role of OTUB1 in preventing hepatocyte necroptosis and what experimental models best study this function?

OTUB1 is a critical regulator of hepatocyte necroptosis, a form of programmed cell death:

• Molecular mechanism: OTUB1 reduces K48-linked polyubiquitination of cellular inhibitor of apoptosis 1 (c-IAP1), preventing its degradation. In the absence of OTUB1, c-IAP1 degradation leads to reduced K63-linked polyubiquitination of RIPK1, increased RIPK1 phosphorylation, RIPK1/RIPK3 necrosome formation, MLKL phosphorylation, and ultimately hepatocyte death .

• Experimental models:

  • In vivo:

    • OTUB1 liver parenchymal cell-specific knockout (OTUB1 LPC-KO) mice

    • Listeria monocytogenes infection model

    • D-Galactosamine/TNF-induced sterile inflammation model

    • Rescue experiments using necrostatin-1s (RIPK1 inhibitor) or MLKL deletion

  • In vitro:

    • Human OTUB1-deficient HepG2 cells

    • Primary hepatocytes isolated from OTUB1 LPC-KO mice

    • Co-immunoprecipitation to detect necrosome formation

• Phenotypic readouts:

  • Alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release

  • Survival analysis

  • MLKL and RIPK phosphorylation status

  • TNF production

  • ERK activation

This research demonstrates that OTUB1 deficiency specifically enhances necroptosis but not apoptosis in hepatocytes, identifying OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival during bacterial hepatitis and TNF challenge.

What are the known interacting partners of OTUB1 and how can these interactions be studied?

OTUB1 interacts with various proteins to execute its diverse cellular functions:

• Known interaction partners:

  • E2 ubiquitin-conjugating enzymes: UBC13/UEV1a complex - mediates non-canonical inhibition of ubiquitination

  • c-IAP1: Cellular inhibitor of apoptosis protein 1 - OTUB1 reduces its K48-linked polyubiquitination

  • SLC7A11: A key suppressor of ferroptosis - stabilized by OTUB1 in glioma cells

  • FUS: Fusion involved in t(12;6) in malignant liposarcoma - suggesting roles in RNA processing

  • RACK1: Receptor for activated kinase 1 - suggesting roles in cell adhesion/morphology

• Experimental approaches to study interactions:

  • Co-immunoprecipitation and MS/MS: Identify interaction partners by pulldown followed by tandem mass spectrometry

  • Proximity-based labeling: BioID or APEX2 fusion proteins to identify proximal interactors in living cells

  • Yeast two-hybrid screening: Identify direct protein-protein interactions

  • In vitro binding assays: Using purified recombinant proteins to characterize direct interactions

  • Surface plasmon resonance: Determine binding affinities (e.g., KD of 7.04 μM for human OTUB1 binding to UBC13)

  • Crystallography: Determine atomic-level details of interactions (as demonstrated by the 1.8 Å resolution structure of OTUB1 bound to UBC13)

  • Deletion/mutation mapping: Identify critical interaction domains/residues

• Correlation studies in clinical samples: Positive correlation between OTUB1 and SLC7A11 expression in clinical glioma samples supports their functional relationship .

How can OTUB1's role in glioma cell stemness be experimentally validated?

Validating OTUB1's role in glioma cell stemness requires multiple complementary approaches:

• Expression analysis:

  • Analyze OTUB1 expression in glioma tissues versus adjacent normal tissues using immunohistochemistry and Western blotting

  • Correlate expression with patient survival data from multiple datasets

  • Assess co-expression with stemness markers in patient-derived glioma samples

• Loss-of-function studies:

  • Generate OTUB1 knockdown in glioma cell lines using siRNA or shRNA

  • Create CRISPR-Cas9 mediated knockout cell lines

  • Assess effects on:

    • Sphere-formation ability (a key indicator of stemness)

    • Expression of stemness markers

    • ALDH activity measurement (enzymatic marker of cancer stem cells)

• Mechanism validation:

  • Examine OTUB1-SLC7A11 interaction using co-immunoprecipitation

  • Measure SLC7A11 protein stability in OTUB1 knockdown versus control cells

  • Perform ubiquitination assays to assess SLC7A11 ubiquitination status

  • Conduct rescue experiments to determine if ectopic SLC7A11 expression can reverse the effects of OTUB1 knockdown

• Ferroptosis assessment:

  • Measure ferroptosis markers in OTUB1 knockdown cells

  • Test sensitivity to ferroptosis inducers

  • Use ferroptosis inhibitors to rescue OTUB1 knockdown phenotypes

• In vivo validation:

  • Orthotopic xenograft models using OTUB1 knockdown versus control glioma cells

  • Patient-derived xenograft models

  • Assessment of tumor initiation ability, growth kinetics, and stemness marker expression

These approaches have revealed that OTUB1 stabilizes SLC7A11 protein via direct interaction, preventing ferroptosis and promoting stemness in glioma cells .

What methods are most effective for studying OTUB1's deubiquitinating activity in vitro and in cells?

Studying OTUB1's deubiquitinating activity requires specialized techniques:

• In vitro enzymatic assays:

  • Di-ubiquitin cleavage assay: Using synthetically linked K48 or K63 di-ubiquitin substrates to measure chain-type specificity

  • Polyubiquitin chain disassembly: Incubating OTUB1 with K48/K63-linked polyubiquitin chains and monitoring chain dismantling by Western blot

  • Fluorogenic substrates: Using Ub-AMC (ubiquitin-7-amino-4-methylcoumarin) to measure catalytic activity in real-time

  • Ubiquitin-based active site probes: K48 or K63-linked di-ubiquitin probes with reactive groups (vinyl methyl ester, 2-chloroethyl, 2-bromoethyl) at the C-terminus

• Cellular assays:

  • Ubiquitination state analysis: Examining target protein ubiquitination under OTUB1 overexpression or knockdown conditions

  • Cycloheximide chase: Measuring protein stability changes when OTUB1 is modulated

  • Tandem ubiquitin binding entity (TUBE) pulldown: Enriching for ubiquitinated proteins followed by Western blot for specific targets

  • Proximity ligation assay: Visualizing OTUB1-substrate interactions in situ

• Technical considerations:

  • Controls: Use catalytically inactive OTUB1 C91S mutant as negative control

  • Physiological relevance: Maintain physiological pH and temperature conditions

  • Chain-type verification: Use linkage-specific antibodies to verify chain types

  • Expression systems: Bacterial expression systems for recombinant protein may require optimization of folding conditions

• Advanced approaches:

  • CRISPR-Cas9 gene editing: Generate cells with endogenous OTUB1 mutations to study specific aspects of function

  • Ubiquitin replacement strategies: Replace endogenous ubiquitin with mutants lacking specific lysine residues to examine linkage specificity

  • Quantitative proteomics: Identify global changes in ubiquitination patterns using techniques like SILAC or TMT labeling

What are the challenges in developing specific inhibitors for OTUB1?

Developing specific inhibitors for OTUB1 presents several challenges:

• Structural considerations:

  • Active site accessibility: Differences in accessibility to the active site between OTUB1 and other DUBs like OTUB2 make selective targeting challenging

  • Conformational changes: OTUB1 undergoes significant conformational changes upon ubiquitin binding, with three regions of the OTU domain remodeling . Inhibitors may need to account for these dynamic structural elements

  • Multiple functional domains: Inhibitors might need to target either the catalytic site (for canonical activity) or E2-binding interfaces (for non-canonical activity)

• Selectivity challenges:

  • DUB family similarity: The OTU domain is conserved across the OTU family of DUBs, making selective inhibition difficult

  • Non-canonical activity: Traditional active site inhibitors wouldn't block OTUB1's non-canonical activity

  • Ubiquitin binding pockets: The ubiquitin-binding surfaces are relatively conserved across DUBs

• Experimental approaches:

  • High-throughput screening: Utilizing in vitro deubiquitination assays with fluorogenic substrates

  • Fragment-based drug discovery: Identifying small molecular fragments that bind to OTUB1

  • Structure-based design: Using the crystal structures of OTUB1 (e.g., PDB: 2ZFY) to design specific inhibitors

  • Allosteric inhibitors: Targeting non-catalytic regions that regulate OTUB1 activity

  • Protein-protein interaction disruptors: Compounds that prevent OTUB1-E2 interactions for non-canonical activity

• Validation strategies:

  • Cellular thermal shift assay (CETSA): Confirm target engagement in cells

  • Selectivity profiling: Test against panels of DUBs to ensure specificity

  • Phenotypic assays: Verify that inhibitors recapitulate OTUB1 knockout phenotypes

  • Target validation: Use OTUB1 knockout or knockdown models to confirm biological relevance of inhibition

What are the current conflicting findings regarding OTUB1's role in cancer development?

Research on OTUB1's role in cancer presents some apparent contradictions:

• Pro-oncogenic versus tumor-suppressive roles:

  • Pro-oncogenic evidence: Most studies support an oncogenic role for OTUB1, especially in tumor migration, invasion, and metastasis. High OTUB1 expression correlates with aggressive clinical disease and poorer patient survival across multiple cancer types .

  • Tumor-suppressive evidence: Some studies demonstrate OTUB1 potentiates growth-suppressive pathways such as p53 and TGFβ, suggesting possible tumor-suppressive functions in specific contexts .

• Contextual dependencies:

  • Tumor type-dependent effects: The contribution of OTUB1 to cancer development and progression may vary based on tumor type .

  • Genetic background interactions: In lung cancer, OTUB1 copy number gain is mutually exclusive with KRAS mutational status, suggesting different roles depending on genetic context .

  • Expression level nuances: In lung cancer patients with wild-type RAS tumors, moderate (not high) OTUB1 levels correlate with significantly poorer survival .

• Methodological approaches to resolve contradictions:

  • Context-specific analysis: Stratify studies by cancer type, genetic background, and tumor stage

  • Multi-omics integration: Combine genomic, transcriptomic, proteomic data to understand context-dependent effects

  • Pathway-specific assessment: Examine OTUB1's effects on specific pathways in different cellular contexts

  • In vivo models: Use conditional knockout/knockin animal models to examine tissue-specific effects

  • Patient stratification: Analyze clinical outcomes based on both OTUB1 expression and other genetic markers

• Potential explanations:

  • Substrate specificity: Different OTUB1 substrates may predominate in different cellular contexts

  • Non-canonical versus canonical activity: The balance between these activities may vary by context

  • Interaction partners: Different binding partners may direct OTUB1 to different functions

  • Post-translational modifications: OTUB1 itself may be differently regulated in various contexts

Understanding these contextual dependencies is critical for developing OTUB1-targeted therapeutic strategies.

How might OTUB1 serve as a therapeutic target in cancer and inflammatory diseases?

OTUB1's involvement in multiple pathological processes suggests therapeutic potential:

• Cancer targeting strategies:

  • Direct inhibition: Developing small molecule inhibitors targeting OTUB1's catalytic activity or non-canonical functions

  • Context-specific approaches: Targeting OTUB1 in specific genetic contexts, such as wild-type KRAS tumors with OTUB1 overexpression

  • Combination therapies: Pairing OTUB1 inhibition with existing therapies, particularly for cancers with stemness features

  • Ferroptosis induction: In gliomas, OTUB1 inhibition could sensitize cells to ferroptosis by destabilizing SLC7A11

• Inflammatory disease applications:

  • Hepatoprotective strategies: OTUB1 activation or mimicry could protect against liver injury by preventing necroptosis

  • Pathway-specific modulation: Targeting OTUB1's regulation of the RIPK1-dependent necroptosis pathway specifically

  • Cell death pathway switching: Using OTUB1 modulation to switch between apoptotic and necroptotic cell death modes

• Biomarker potential:

  • Prognostic marker: OTUB1 expression levels as indicators of patient prognosis in various cancers

  • Predictive biomarker: Potential marker for response to therapies targeting RAS/MAPK pathway or ferroptosis inducers

• Technical challenges:

  • Target selectivity: Achieving specificity among DUB family members

  • Tissue-specific delivery: Targeting OTUB1 in disease tissues while sparing essential functions

  • Activity monitoring: Developing assays to measure OTUB1 inhibition in vivo

• Methodological approaches:

  • High-throughput screening: For small molecule inhibitors or activators

  • Structure-based drug design: Using solved crystal structures to design inhibitors

  • PROTAC approach: Targeted protein degradation of OTUB1

  • Substrate-specific intervention: Targeting specific OTUB1-substrate interactions rather than OTUB1 itself

These diverse approaches highlight the potential of OTUB1 as a therapeutic target across multiple disease contexts, with the specific strategy dependent on disease type and molecular context.

What novel experimental techniques could advance our understanding of OTUB1 function?

Advancing OTUB1 research requires innovative experimental approaches:

• Advanced structural biology techniques:

  • Cryo-electron microscopy: Visualize OTUB1 complexes with substrates and interacting proteins

  • Hydrogen-deuterium exchange mass spectrometry: Map conformational changes upon binding

  • Single-molecule FRET: Study real-time dynamics of OTUB1-substrate interactions

  • AlphaFold2 and other AI prediction tools: Model OTUB1 interactions with novel partners

• Genome editing technologies:

  • Domain-specific CRISPR editing: Create precise mutations in endogenous OTUB1 to dissect domain functions

  • Base editing: Introduce specific amino acid changes without double-strand breaks

  • Prime editing: Make specific edits to study regulatory elements

  • CRISPR screens: Identify synthetic lethal interactions with OTUB1 deficiency

• Proteomics approaches:

  • Global ubiquitinome analysis: Quantify changes in ubiquitination patterns upon OTUB1 modulation

  • Interaction proteomics: BioID or APEX2 proximity labeling to map the OTUB1 interactome

  • Targeted proteomics: Monitor specific OTUB1 substrates and their ubiquitination states

  • Degradomics: Study protein turnover rates affected by OTUB1

• Live-cell imaging techniques:

  • FRET/BRET biosensors: Monitor OTUB1 activity in real-time

  • Optogenetics: Control OTUB1 activity with light to study temporal effects

  • Live-cell ubiquitination reporters: Track deubiquitination events in single cells

• Translational research approaches:

  • Patient-derived organoids: Study OTUB1 function in more physiologically relevant systems

  • Humanized mouse models: Better recapitulate human OTUB1 biology

  • Multi-omics integration: Combine genomic, transcriptomic, and proteomic data from patient samples

These innovative approaches could reveal new aspects of OTUB1 biology, from molecular mechanisms to disease relevance, and potentially identify novel therapeutic strategies.

Molecular Interactions and Mechanisms of OTUB1

OTUB1 Structure-Function Relationships