Recombinant Mouse Ubiquitin carboxyl-terminal hydrolase CYLD (Cyld), partial

What is the structural composition of recombinant mouse CYLD and how does it compare to native CYLD?

Recombinant mouse CYLD (partial form) typically contains the amino acid range 579-952, representing the catalytic domain of the protein. The partial recombinant protein has a theoretical molecular weight of approximately 50.6 kDa when expressed in E. coli systems. For research applications, the recombinant protein is commonly engineered with an N-terminal 10xHis tag and C-terminal Myc tag to facilitate detection and purification .

The crystal structure analysis reveals that CYLD, like other ubiquitin carboxyl-terminal hydrolases (UCHs), contains a catalytic triad consisting of cysteine, histidine, and aspartate residues in the active site. This catalytic domain adopts a conformation that allows specific recognition and processing of ubiquitin chains. The partial recombinant form maintains the essential catalytic activity while eliminating domains not required for the deubiquitinating function.

How does mouse CYLD function as a deubiquitinating enzyme in molecular signaling pathways?

Mouse CYLD functions as a specialized deubiquitinating enzyme that selectively removes ubiquitin chains from target proteins, thereby regulating protein stability and modulating multiple cellular signaling pathways. The primary mechanism of action involves:

• Recognition of specific ubiquitin chain linkages (particularly K63-linked chains)

• Hydrolysis of the isopeptide bond between ubiquitin molecules or between ubiquitin and substrate proteins

• Consequent alteration of protein stability or signaling capacity

One of the most well-characterized functions of CYLD is negative regulation of NF-κB signaling through deubiquitination of key pathway components such as TRAF2, TRAF6, and NEMO. This deubiquitination prevents sustained pathway activation, thereby controlling inflammatory responses. Additionally, CYLD influences other cellular processes including cell cycle progression, apoptosis, and immune responses through its deubiquitinating activity on various substrates .

What are the optimal expression systems for producing functional recombinant mouse CYLD, and what yields can be expected?

While multiple expression systems can be used to produce recombinant mouse CYLD, E. coli remains the most commonly employed platform due to its simplicity and cost-effectiveness . For researchers seeking higher yields or post-translational modifications, alternatives include:

Based on similar UCH family proteins, when expression is optimized in P. pastoris with the appropriate induction conditions (pH 6.0 in BMMY/methanol medium), yields of up to 210 mg/L can be achieved for UCH family proteins . For E. coli expression systems, optimizing growth temperature (typically 16-25°C post-induction), IPTG concentration (0.1-0.5 mM), and culture density at induction (OD600 of 0.6-0.8) can significantly improve the yield of soluble, active CYLD.

What purification strategy provides the highest purity and activity for recombinant mouse CYLD?

A multi-step purification strategy is recommended to obtain highly pure and active recombinant mouse CYLD:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary purification step, exploiting the N-terminal His tag. Optimization of imidazole concentration in washing buffers (20-50 mM) is critical to remove non-specific binding proteins while retaining CYLD .

• Intermediate Purification: Size exclusion chromatography (SEC) effectively separates aggregates and contaminants of different molecular weights.

• Polishing Step: Ion exchange chromatography (IEX) can be employed as a final polishing step to achieve >95% purity.

• Activity Preservation: Throughout purification, maintain reducing conditions (typically 1-5 mM DTT or 0.5-2 mM TCEP) to preserve the catalytic thiol group. Additionally, including 10% glycerol in storage buffers and flash-freezing aliquots helps maintain enzymatic activity during long-term storage.

The final purified protein should be assessed for both purity (by SDS-PAGE and Western blot) and enzymatic activity using a deubiquitination assay with appropriate substrates. Typical yields after complete purification range from 5-15 mg of active protein per liter of bacterial culture.

How can the deubiquitinating activity of recombinant mouse CYLD be accurately measured in vitro?

Multiple methodological approaches can be employed to assess the deubiquitinating activity of recombinant mouse CYLD:

• Fluorogenic Ubiquitin Substrate Assay: Using ubiquitin-AMC (7-amino-4-methylcoumarin) as a substrate, where cleavage of the ubiquitin-AMC bond by active CYLD releases fluorescent AMC that can be quantified. This assay provides real-time kinetic data but only measures general DUB activity.

• Chain-Specific Deubiquitination Assay: Using purified K63-linked polyubiquitin chains (CYLD's preferred substrate) followed by SDS-PAGE analysis to visualize the pattern of ubiquitin chain disassembly over time. This method more accurately reflects CYLD's natural substrate specificity.

• Mass Spectrometry-Based Assay: Employing mass spectrometry to analyze the products of deubiquitination reactions provides precise information about the cleavage sites and specificity. Similar approaches have been used successfully with UCH enzymes to confirm their precise cleavage of ubiquitin fusion proteins .

For activity confirmation, ubiquitin fusion proteins can serve as substrates, with cleavage products analyzed by techniques such as Tricine-SDS-PAGE and ESI-MS. This approach has been validated for UCH family enzymes, demonstrating their ability to precisely recognize and cleave at the carboxyl terminus of ubiquitin .

What are the optimal buffer conditions and reaction parameters for studying CYLD enzymatic activity?

The optimization of reaction conditions is crucial for accurate assessment of CYLD enzymatic activity:

When planning deubiquitination assays, it's important to note that CYLD shows preference for K63-linked polyubiquitin chains, with limited activity against K48-linked chains. Additionally, including deubiquitinase inhibitors such as N-ethylmaleimide (NEM) in control reactions can confirm the specificity of observed activity.

For kinetic analysis, determining Km and kcat values requires testing a range of substrate concentrations (typically 0.1-10× Km) and measuring initial reaction velocities under steady-state conditions. These parameters provide valuable insights into CYLD's catalytic efficiency and substrate preference.

How does CYLD dysregulation contribute to inflammatory disorders and cancer pathogenesis?

CYLD dysregulation has been implicated in various pathological conditions through several mechanistic pathways:

• Inflammatory Disorders: CYLD negatively regulates NF-κB signaling, which is a master regulator of inflammation. Loss of CYLD function leads to enhanced and prolonged NF-κB activation, resulting in excessive inflammatory responses and contributing to conditions such as inflammatory bowel disease and autoimmune disorders . The mechanism involves CYLD's deubiquitination of TRAF proteins and IKK complex components, which normally activate NF-κB.

• Cancer Development: CYLD functions as a tumor suppressor in multiple tissue types. Its downregulation or inactivation promotes:

  • Cell proliferation through enhanced cyclin D1 expression

  • Resistance to apoptosis via increased anti-apoptotic protein expression

  • Metastatic potential through altered cell adhesion and migration

  • Enhanced angiogenesis via VEGF upregulation

These effects stem from CYLD's role in regulating not only NF-κB but also other signaling pathways including Wnt/β-catenin, JNK, and p38 MAPK pathways.

• Fibrotic Conditions: Similar to other deubiquitinating enzymes like UCHL5, CYLD may influence TGF-β signaling components, potentially affecting fibrotic processes in conditions such as idiopathic pulmonary fibrosis . UCHL5, another member of the UCH family, has been shown to de-ubiquitinate Smad2 and Smad3, stabilizing these proteins and promoting TGF-β1-induced expression of profibrotic proteins.

Understanding these pathological mechanisms provides the foundation for developing targeted therapeutic approaches aimed at restoring normal CYLD function or compensating for its loss.

What experimental models are most appropriate for studying CYLD function in specific disease contexts?

Various experimental models have been developed to study CYLD's role in disease pathogenesis:

For inflammatory disorders, myeloid-specific conditional CYLD knockout models are particularly valuable, as they allow assessment of CYLD's role in immune cell function without confounding effects on other tissues. For cancer studies, xenograft models using CYLD-manipulated cell lines provide insights into tumor growth and metastasis.

When designing experiments, it's essential to consider the specific ubiquitin chain types (K63 vs. K48) relevant to the pathway being studied, as CYLD's selectivity for K63-linked chains influences which cellular processes will be affected by its manipulation.

What techniques can be used to identify novel substrates and interaction partners of CYLD in different cellular contexts?

Identifying the complete interactome of CYLD requires sophisticated methodological approaches:

• Proximity-Based Labeling: BioID or APEX2 fusion proteins with CYLD allow biotinylation of proximal proteins, which can be purified and identified by mass spectrometry. This technique captures both stable and transient interactions in living cells.

• Ubiquitinome Analysis: Quantitative proteomics comparing ubiquitination patterns in CYLD-expressing versus CYLD-deficient cells identifies substrates based on differential ubiquitination. This typically involves:

  • Tandem ubiquitin binding entity (TUBE) pulldown to enrich ubiquitinated proteins

  • Stable isotope labeling with amino acids in cell culture (SILAC) for quantitative comparison

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

• Genetic Screens: CRISPR-based screens can identify synthetic lethal or synthetic viable interactions with CYLD, revealing functional relationships not detectable by direct protein interaction studies.

• Structural Biology Approaches: Hydrogen-deuterium exchange mass spectrometry (HDX-MS) or crosslinking mass spectrometry (XL-MS) can map interaction interfaces and conformational changes upon substrate binding. UCH family enzymes are known to undergo significant conformational changes upon substrate binding, transitioning from inactive to active states .

When implementing these approaches, researchers should consider tissue-specific or context-dependent interactions, as CYLD may have different substrate preferences in different cell types or under various cellular stresses.

How can structural analysis of CYLD inform the development of specific inhibitors or activators for therapeutic applications?

Structural biology approaches provide critical insights for rational drug design targeting CYLD:

• Active Site Targeting: X-ray crystallography and cryo-electron microscopy structures of CYLD's catalytic domain reveal the spatial arrangement of the catalytic triad (cysteine, histidine, and aspartate) that is essential for deubiquitinating activity. Similar to other UCH enzymes, CYLD likely has an unstructured loop that restricts access to the active site, presenting opportunities for selective inhibitor design .

• Allosteric Modulation: Nuclear magnetic resonance (NMR) spectroscopy can identify allosteric sites where small molecule binding induces conformational changes affecting enzymatic activity. UCH family enzymes are known to exist in active and inactive conformations, with significant structural rearrangements occurring upon ubiquitin binding .

• Structure-Activity Relationship (SAR) Studies: Virtual screening combined with enzymatic assays can identify lead compounds that selectively inhibit or activate CYLD. Molecular dynamics simulations can then predict how structural modifications might improve specificity or pharmacokinetic properties.

• Protein-Protein Interaction (PPI) Targeting: Structural characterization of CYLD-substrate complexes identifies interfaces that could be targeted to selectively disrupt specific CYLD interactions while preserving others, potentially reducing side effects.

The development of selective CYLD-targeting compounds requires careful consideration of specificity, as the UCH family shares structural similarities in the catalytic domain. Focusing on unique structural features of CYLD and targeting non-catalytic domains involved in substrate selection could increase specificity.

What are the common pitfalls in working with recombinant CYLD and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant CYLD:

• Low Solubility and Aggregation:

  • Problem: CYLD can form insoluble aggregates during expression and purification.

  • Solution: Express at lower temperatures (16-20°C), include solubility enhancers like sorbitol (0.5-1 M) or arginine (50-100 mM) in buffers, and consider fusion tags like SUMO or MBP that enhance solubility.

• Loss of Enzymatic Activity:

  • Problem: The catalytic cysteine residue is susceptible to oxidation, resulting in activity loss.

  • Solution: Maintain reducing conditions throughout purification and storage (1-5 mM DTT or 0.5-2 mM TCEP), minimize freeze-thaw cycles, and consider adding protease inhibitors to prevent autolysis.

• Non-specific DUB Activity in Assays:

  • Problem: Cell lysates contain multiple DUBs that can confound CYLD-specific activity measurements.

  • Solution: Use selective inhibitors of other DUB classes in assays, employ immunoprecipitation to isolate CYLD complexes, and include appropriate negative controls (catalytically inactive CYLD mutants).

• Substrate Specificity Challenges:

  • Problem: Distinguishing CYLD activity from other DUBs with overlapping specificity.

  • Solution: Use defined chain linkage-specific ubiquitin substrates (K63-linked for CYLD), perform comparative assays with other purified DUBs, and validate results using CYLD knockout controls.

• Variable Expression Levels:

  • Problem: Inconsistent yields between batches.

  • Solution: Standardize growth conditions, optimize codon usage for expression system, and consider inducible expression systems with tightly controlled promoters.

How can experimental design be optimized when using CYLD to study ubiquitin-dependent signaling pathways?

Robust experimental design for studying CYLD function requires careful consideration of several factors:

• Appropriate Controls:

  • Catalytically inactive CYLD mutant (C601A) to distinguish enzymatic versus scaffolding functions

  • Domain deletion mutants to map interaction regions

  • Chain linkage specificity controls (K48 vs. K63 polyubiquitin) to confirm substrate selectivity

• Temporal Considerations:

  • Ubiquitin-mediated signaling is dynamic, requiring time-course analyses

  • Synchronize cells when studying cell cycle-dependent processes

  • Use inducible systems (Tet-On/Off) for temporal control of CYLD expression

• Cellular Context:

  • Different cell types may express different CYLD substrates or regulatory proteins

  • Primary cells versus cell lines may show different CYLD-dependent phenotypes

  • Consider tissue-specific conditional knockout models for in vivo studies

• Readout Selection:

  • Direct measures of ubiquitination status using linkage-specific antibodies

  • Downstream signaling events (NF-κB activation, MAP kinase phosphorylation)

  • Functional outcomes (cell proliferation, apoptosis, inflammatory cytokine production)

  • Combine multiple readouts for comprehensive pathway analysis

• Data Analysis Approaches:

  • Quantitative analysis of ubiquitination levels (Western blot densitometry or mass spectrometry)

  • Kinetic modeling of deubiquitination reactions

  • Network analysis to understand system-wide effects of CYLD manipulation

By carefully considering these experimental design elements, researchers can generate more reliable and interpretable data on CYLD's role in ubiquitin-dependent signaling pathways.