Recombinant Esox lucius Ubiquitin-fold modifier-conjugating enzyme 1 (ufc1)

What is Ubiquitin-fold modifier-conjugating enzyme 1 (UFC1) and its function in Esox lucius?

UFC1 is an essential enzyme involved in the ubiquitin-like protein modification system in Northern pike (Esox lucius). It functions as a conjugating enzyme (E2) in the UFM1 (Ubiquitin-fold modifier) pathway, which is involved in post-translational protein modifications. In Esox lucius, UFC1 participates in various cellular processes including protein quality control, endoplasmic reticulum homeostasis, and cellular stress responses. The enzyme typically catalyzes the transfer of UFM1 from the E1 enzyme (UBA5) to target proteins, affecting protein function, localization, and turnover in pike tissues.

What extraction and purification methods are most effective for isolating native UFC1 from Esox lucius tissues?

The most effective extraction protocol for native UFC1 from pike tissues involves tissue homogenization in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and protease inhibitors, followed by differential centrifugation. For purification, a combination of ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography yields the highest purity. When compared to collagen extraction methods from pike, which utilize acid solubilization or pepsin digestion as described in Northern pike collagen studies, UFC1 purification requires more gentle conditions to maintain enzymatic activity .

What expression systems are optimal for producing recombinant Esox lucius UFC1?

For recombinant expression of pike UFC1, the following expression systems have been evaluated:

E. coli systems offer cost-effective production but may require refolding procedures, while eukaryotic systems provide better folding at higher production costs. The selection depends on the specific research requirements, with insect cell systems offering a good balance between yield and proper folding for functional studies of pike UFC1 .

How do environmental factors influence UFC1 expression and activity in Esox lucius?

Northern pike inhabit freshwater ecosystems that can contain various environmental pollutants, including heavy metals like mercury. Studies examining mercury concentrations in pike tissues from the Anzali wetland demonstrated significant bioaccumulation in muscle and liver tissues . The presence of such contaminants may influence UFC1 expression and activity through several mechanisms:

• Mercury exposure can induce oxidative stress, potentially upregulating the UFM1 pathway as a cellular protective response

• Heavy metal exposure may alter UFC1 catalytic efficiency through direct interaction with thiol groups

• Chronic exposure to environmental toxins may lead to compensatory changes in UFC1 expression patterns

Temperature adaptation also plays a crucial role, as pike UFC1 demonstrates optimal activity at lower temperatures (15-20°C) compared to mammalian homologs (37°C), reflecting evolutionary adaptation to cold-water environments. Seasonal variations in pike UFC1 activity correlate with reproductive cycles, with elevated expression observed during spawning periods .

What methodological approaches are most effective for studying UFC1-mediated protein modifications in pike tissues?

Investigating UFC1-mediated UFMylation in pike tissues requires specialized techniques:

• Proteomic identification of UFMylated substrates:

  • Tandem affinity purification of tagged UFM1 followed by mass spectrometry

  • Antibody-based enrichment of UFMylated proteins

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for quantitative analysis

• Functional validation approaches:

  • In vitro UFMylation assays using recombinant pike UFC1, UBA5, and UFM1

  • Site-directed mutagenesis of the catalytic cysteine (typically Cys116) to create activity-dead controls

  • Development of pike-specific cell lines for in vivo validation studies

• Localization studies:

  • Immunohistochemistry with pike tissue sections using anti-UFC1 antibodies

  • Subcellular fractionation followed by Western blot analysis

  • Fluorescent protein tagging for real-time visualization in live cells

These methodological approaches must be optimized for the lower temperature requirements of pike enzymes, typically requiring modified buffer systems with increased glycerol content (15-20%) to maintain structural stability during experimental procedures .

How does pike UFC1 function in response to temperature variation and environmental stressors?

Northern pike inhabit environments with significant seasonal temperature fluctuations, requiring biochemical adaptations. Pike UFC1 demonstrates remarkable temperature-dependent catalytic properties:

These adaptations reflect evolutionary pressure for maintaining protein homeostasis across temperature ranges experienced in pike habitats. Under environmental stress conditions, such as heavy metal exposure or hypoxia, pike UFC1 expression is upregulated, suggesting a protective role in cellular stress responses.

Mercury exposure, as documented in pike from the Anzali wetland, correlates with increased UFMylation activity, potentially representing a cellular detoxification mechanism . The UFM1 pathway appears to play a critical role in pike adaptation to anthropogenic environmental stressors, with UFC1 serving as a key regulatory enzyme in this protective response.

What are the optimal conditions for assessing UFC1 enzymatic activity in vitro?

In vitro UFC1 activity assays require careful optimization of reaction conditions:

Standard UFC1 Activity Assay Protocol:

• Reaction Buffer Components:

  • 50 mM Tris-HCl, pH 7.5

  • 100 mM NaCl

  • 10 mM MgCl₂

  • 1 mM ATP

  • 0.1 mM DTT

  • 5% glycerol

• Reaction Components:

  • 1 μg recombinant pike UBA5 (E1)

  • 2 μg recombinant pike UFC1 (E2)

  • 5 μg recombinant pike UFM1

  • 10 μg substrate protein (if studying specific target modification)

• Incubation Conditions:

  • Temperature: 15-20°C (optimal for pike enzyme function)

  • Time: 30-60 minutes

  • Volume: 50 μL

• Detection Methods:

  • Western blot analysis using anti-UFM1 antibodies

  • SDS-PAGE with fluorescently labeled UFM1

  • Bioluminescence resonance energy transfer (BRET) for real-time monitoring

Unlike mammalian UFC1 assays typically conducted at 37°C, pike UFC1 activity assays must be performed at lower temperatures that reflect the physiological conditions of this cold-water species. The temperature range of 15-20°C provides optimal conditions for pike UFC1 activity while maintaining the stability of all reaction components .

How can researchers effectively overexpress and purify recombinant Esox lucius UFC1?

The following protocol outlines an effective method for recombinant pike UFC1 production:

Expression and Purification Protocol:

• Cloning:

  • Amplify UFC1 coding sequence from Esox lucius liver cDNA

  • Clone into pET-28a(+) vector with N-terminal His-tag

  • Verify sequence integrity by Sanger sequencing

• Expression Conditions:

  • Transform into E. coli BL21(DE3)

  • Culture in LB medium at 37°C until OD₆₀₀ reaches 0.6-0.8

  • Induce with 0.5 mM IPTG

  • Reduce temperature to 18°C and continue expression for 16-18 hours

• Cell Lysis:

  • Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

  • Resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 5 mM β-mercaptoethanol)

  • Lyse cells by sonication or French press

• Purification Steps:

  • Ni-NTA affinity chromatography (elution with 250 mM imidazole)

  • Size exclusion chromatography using Superdex 75 column

  • Optional: Ion exchange chromatography for higher purity

• Quality Control:

  • SDS-PAGE analysis (expected MW: ~21 kDa)

  • Western blot confirmation

  • Activity assay validation

  • Thermal stability assessment

This protocol has been optimized to maximize the yield of functional pike UFC1, with typical yields of 15-20 mg/L of bacterial culture. The low-temperature induction significantly improves the solubility and activity of the recombinant enzyme .

What considerations are important when designing experiments to study UFC1 function in cold-water fish species?

When studying UFC1 in Esox lucius or other cold-water fish species, several critical considerations must be addressed:

• Temperature optimization:

  • Maintain physiologically relevant temperatures (4-20°C) during all experimental procedures

  • Include temperature controls in experimental design

  • Assess enzyme activity across a temperature range rather than at a single point

• Buffer composition adjustments:

  • Increase glycerol concentration (10-15%) to improve protein stability at lower temperatures

  • Optimize pH considering temperature effects on buffer systems

  • Include cryoprotectants for freeze-thaw cycles

• Tissue-specific expression patterns:

  • UFC1 expression varies across pike tissues, with highest levels in liver, brain, and reproductive organs

  • Sample collection should consider seasonal variations and reproductive status

  • Compare expression patterns with other UFM1 pathway components

• Interspecies differences:

  • Pike UFC1 shares approximately 78% sequence identity with human UFC1

  • Cross-reactivity of commercial antibodies should be validated

  • Species-specific regulatory mechanisms must be considered when interpreting results

• Environmental factor integration:

  • Include environmental parameters (water quality, pollutant exposure) in experimental design

  • Consider how mercury concentration, which bioaccumulates in pike tissues, might affect UFC1 function

  • Document collection site characteristics for wild specimens

These methodological considerations are essential for generating reliable and physiologically relevant data on pike UFC1 function. The temperature adaptation of pike enzymes represents a particularly crucial factor, as experimental conditions designed for mammalian systems often fail to recapitulate the optimal conditions for cold-water fish enzymes .

How should researchers interpret apparent discrepancies in UFC1 activity between in vitro and in vivo studies?

Discrepancies between in vitro and in vivo UFC1 activity can arise from multiple factors:

• Temperature effects:

  • In vitro assays conducted at non-physiological temperatures may misrepresent actual enzyme kinetics

  • Pike UFC1 demonstrates optimal activity at 15-20°C, with significant reduction at higher temperatures

  • Temperature fluctuations in natural habitats create seasonal variation in enzyme activity

• Cofactor availability:

  • In vitro systems may lack critical cofactors present in cellular environments

  • The UFM1 pathway requires coordinated activity of multiple enzymes (UBA5, UFC1, UFL1)

  • Ratios of pathway components differ between artificial and natural systems

• Post-translational regulation:

  • Pike UFC1 activity is regulated by phosphorylation and possibly other modifications

  • In vitro systems often lack regulatory mechanisms present in intact cells

  • Environmental stressors induce regulatory changes not captured in simplified systems

• Substrate accessibility:

  • Compartmentalization in cells restricts enzyme-substrate interactions

  • In vitro systems with excess substrate availability may show artificially elevated activity

  • Protein-protein interactions in cellular contexts modify enzyme behavior

To reconcile these discrepancies, researchers should employ complementary approaches, including cell-based assays at physiologically relevant temperatures, tissue slice cultures, and careful validation of in vitro findings in biological contexts. Temperature-controlled in vitro systems that more closely mimic pike physiological conditions can significantly reduce the gap between in vitro and in vivo observations .

What statistical approaches are most appropriate for analyzing UFC1 expression data across different pike tissues and environmental conditions?

The analysis of UFC1 expression data from pike tissues requires specialized statistical approaches:

• Normalization strategies:

  • Multiple reference genes should be used for qPCR normalization

  • GAPDH and β-actin expression can vary with temperature in fish

  • Geometric averaging of multiple reference genes improves reliability

• Statistical tests for tissue comparisons:

  • One-way ANOVA with post-hoc tests for multi-tissue comparisons

  • Non-parametric alternatives (Kruskal-Wallis) for non-normally distributed data

  • Mixed-effects models for repeated sampling designs

• Environmental correlation analysis:

  • Multiple regression models including temperature, pollutant levels, and seasonal factors

  • Principal component analysis for complex environmental datasets

  • Time-series analysis for seasonal expression patterns

• Considerations for mercury exposure studies:

  • Correlation analysis between tissue mercury levels and UFC1 expression

  • Dose-response modeling for controlled exposure experiments

  • Bayesian approaches for integrating multiple environmental variables

When analyzing UFC1 expression in relation to mercury exposure, researchers should consider the significant tissue-specific bioaccumulation patterns observed in pike. Mercury concentrations differ significantly between muscle and liver tissues, potentially creating tissue-specific responses in the UFM1 pathway. Statistical models should account for these tissue-specific variations when analyzing expression data .

How can researchers differentiate between direct effects on UFC1 and indirect effects through pathway regulation?

Distinguishing direct and indirect effects on pike UFC1 requires a multi-faceted experimental approach:

• Direct effect identification:

  • In vitro activity assays with purified recombinant UFC1 and potential modulators

  • Structural analysis using thermal shift assays to detect direct binding

  • Site-directed mutagenesis of putative regulatory sites

• Pathway-level analysis:

  • Quantification of all UFM1 pathway components (UBA5, UFC1, UFL1, UFSP)

  • Correlation analysis of component expression patterns

  • Pathway inhibitor studies to identify regulatory nodes

• Temporal resolution studies:

  • Time-course experiments to establish order of molecular events

  • Pulse-chase studies to determine protein turnover rates

  • Inducible expression systems for controlled perturbation

• Hierarchical experimental design:

  • Begin with in vitro biochemical assays to identify direct interactions

  • Progress to cellular systems with controlled expression

  • Validate in tissue explants or in vivo models

For example, to determine whether mercury exposure directly affects UFC1 or acts through pathway regulation, researchers should first conduct in vitro activity assays with purified UFC1 in the presence of mercury compounds. This should be followed by expression analysis of all pathway components in exposed tissues, and finally validated through functional studies in cellular models. This hierarchical approach allows differentiation between direct enzyme inhibition and compensatory expression changes .

What are promising research avenues for understanding the role of UFC1 in pike adaptation to environmental change?

Several high-priority research directions hold promise for elucidating pike UFC1 function in environmental adaptation:

• Climate change adaptation studies:

  • Comparative analysis of UFC1 from pike populations across temperature gradients

  • Experimental evolution studies under controlled temperature conditions

  • Modeling of temperature-dependent enzyme kinetics under projected climate scenarios

• Environmental toxicant response mechanisms:

  • Comprehensive analysis of UFM1 pathway response to mercury and other heavy metals

  • Identification of UFMylated proteins involved in detoxification

  • Development of UFC1 activity as a biomarker for environmental stress

• Seasonal adaptation mechanisms:

  • Correlation of UFC1 activity with reproductive cycle stages

  • Analysis of temperature-dependent UFC1 regulation during seasonal transitions

  • Integration with broader proteomic studies of cold adaptation

• Transgenerational effects:

  • Epigenetic regulation of UFC1 expression in response to parental environmental exposure

  • Developmental programming of the UFM1 pathway

  • Long-term population studies correlating environmental history with UFC1 variants

These research directions would benefit from integrating UFC1 studies with the broader understanding of pike biology, including its reproduction patterns and habitat preferences as described in pike rearing studies . Such integration would provide ecological context for molecular findings and enhance their relevance to conservation and management efforts.

How might research on pike UFC1 inform broader understanding of protein modification systems in aquatic species?

Research on pike UFC1 can serve as a model for understanding protein modification in aquatic species through several approaches:

• Evolutionary comparative studies:

  • Comparison of UFC1 sequences and functions across fish phylogeny

  • Identification of adaptive changes in cold-water versus warm-water species

  • Correlation of UFM1 pathway complexity with environmental adaptation

• Cellular stress response comparisons:

  • Cross-species analysis of UFMylation targets under environmental stress

  • Comparative quantification of pathway activation thresholds

  • Integration with other post-translational modification systems (ubiquitination, SUMOylation)

• Methodological advancements:

  • Development of optimized protocols for studying protein modifications in fish tissues

  • Creation of fish-specific antibodies and detection reagents

  • Establishment of environmental relevance criteria for laboratory studies

• Translational applications:

  • Application of findings to aquaculture stress management

  • Development of biomarkers for environmental monitoring

  • Identification of conserved mechanisms relevant to multiple species

Pike UFC1 research is particularly valuable due to the species' position as an apex predator in freshwater ecosystems, making it an important bioindicator for environmental health assessment. The bioaccumulation of environmental toxicants in pike tissues, as demonstrated in mercury concentration studies, provides a unique opportunity to study protein modification systems under realistic environmental stress conditions .

What are the key considerations for researchers beginning work on Esox lucius UFC1?

Researchers initiating studies on pike UFC1 should consider the following essential points:

• Biological context:

  • Northern pike is a cold-water predatory fish requiring specialized handling

  • Seasonal variations significantly impact UFC1 expression and activity

  • Collection site environmental conditions should be thoroughly documented

• Technical considerations:

  • Temperature optimization is critical for all experimental procedures

  • Commercial reagents developed for mammalian systems require validation

  • Tissue preservation methods must maintain enzyme activity

• Experimental design priorities:

  • Include appropriate temperature controls in all experiments

  • Integrate environmental parameters into study design

  • Consider sex-specific and seasonal variations

• Collaborative approach:

  • Combine expertise in protein biochemistry, fish biology, and environmental science

  • Establish standardized protocols for cross-laboratory comparisons

  • Develop shared resources including antibodies and expression constructs

By addressing these considerations, researchers can avoid common pitfalls and establish robust experimental systems for investigating the roles of UFC1 in pike biology. The integration of molecular findings with the ecological and environmental context of pike populations will provide the most meaningful insights into this important regulatory enzyme .