Recombinant Cricetulus griseus UTP--glucose-1-phosphate uridylyltransferase (UGP2), partial

What is the primary function of UGP2 in Cricetulus griseus metabolism?

UGP2 (UTP--glucose-1-phosphate uridylyltransferase) catalyzes the formation of UDP-glucose from UTP and glucose-1-phosphate, serving as a critical junction in carbohydrate metabolism. This enzyme plays two significant roles: regulating glycogen synthesis, which impacts cellular survival under nutrient-starved conditions, and supporting protein N-glycosylation modifications. Research has identified UGP2 as a key mediator required for cell survival and proliferation, particularly in certain cancer cell types. The enzyme's dual role in both energy storage (glycogen) and protein modification pathways makes it an important metabolic regulator in Cricetulus griseus cells .

How does recombinant partial UGP2 differ from the full-length native enzyme?

Recombinant partial UGP2 contains only selected domains of the complete protein, typically preserving the catalytic core while omitting regulatory regions. This structural difference can significantly impact enzymatic properties including:

• Altered substrate binding kinetics and catalytic efficiency

• Modified regulatory responses to cellular metabolites

• Different oligomerization properties and structural stability

• Potentially enhanced expression in heterologous systems due to reduced size

What experimental approaches are most effective for studying UGP2 structure-function relationships?

Understanding structure-function relationships in UGP2 requires integrating multiple experimental approaches:

Combining these approaches allows researchers to create a comprehensive understanding of how UGP2's structural elements contribute to its dual roles in glycogen synthesis and protein N-glycosylation pathways .

What expression systems yield optimal results for recombinant partial Cricetulus griseus UGP2?

The choice of expression system significantly impacts the quality and functionality of recombinant partial UGP2. Comparative analysis reveals several viable options:

What purification challenges are specific to recombinant partial UGP2 and how can they be overcome?

Purifying recombinant partial UGP2 presents several challenges that require strategic approaches:

• Solubility issues: Partial constructs may expose hydrophobic regions normally buried in the full-length protein. Address this by:

  • Including 10-20% glycerol in purification buffers

  • Using solubility-enhancing fusion tags (MBP, SUMO)

  • Optimizing buffer ionic strength (typically 150-300 mM NaCl)

• Maintaining catalytic activity: Activity loss during purification is common. Preserve function by:

  • Including appropriate metal ions (Mg²⁺ or Mn²⁺) in all buffers

  • Adding reducing agents to protect cysteine residues

  • Keeping samples at 4°C throughout purification

  • Avoiding freeze-thaw cycles

• Preventing aggregation: Partial constructs are often prone to aggregation. Minimize this by:

  • Including low concentrations of non-ionic detergents (0.01-0.05% Triton X-100)

  • Filtering all buffers through 0.22 μm filters

  • Performing size exclusion chromatography as a final polishing step

The optimal pH range for buffer systems typically falls between 7.0-8.5, consistent with the pH range (5-10) mentioned for terminal transferase reactions using buffers such as cacodylate, Tris, HEPES, or Tricine .

How can enzymatic activity be preserved during the purification process?

Preserving UGP2 enzymatic activity throughout purification requires careful attention to multiple factors:

• Critical additives for stability:

  • Pyrophosphatase: Addition of inorganic pyrophosphatase (e.g., from Saccharomyces cerevisiae) can help maintain forward reaction direction by reducing pyrophosphate buildup

  • Metal ions: Include Mg²⁺ (5-10 mM) in all buffers as a critical cofactor

  • Glycerol: 10-15% glycerol helps maintain protein stability

  • Reducing agents: 1-5 mM DTT or β-mercaptoethanol prevents oxidation

• Temperature management strategy:

  • Maintain 4°C throughout purification

  • For critical steps, consider working in a cold room

  • Avoid extended periods at room temperature

  • Store enzymes on ice between purification steps

• Buffer optimization protocol:

  • Test pH range 7.0-8.5 to determine optimal stability

  • Include 150-300 mM NaCl to maintain solubility

  • Consider adding low concentrations of substrate analogs as stabilizers

  • Use Tris or HEPES buffer systems at 25-50 mM concentration

Implementing these strategies can significantly increase the yield of active enzyme, particularly for partial constructs that may lack stabilizing domains present in the full-length protein.

What are the most reliable assays for measuring partial UGP2 enzymatic activity?

Several complementary approaches can be used to accurately measure UGP2 activity:

• Spectrophotometric coupled assays: These continuous assays link UDP-glucose production to measurable changes in absorption:

  • Couple with UDP-glucose dehydrogenase to measure NADH production at 340 nm

  • Advantage: Real-time monitoring of reaction kinetics

  • Limitation: Potential interference from coupling enzyme activities

• Pyrophosphate release detection:

  • Measure inorganic pyrophosphate (PPi) released during the reaction

  • Requires pyrophosphatase to convert PPi to phosphate, which can be detected with malachite green

  • Advantage: Direct correlation with UGP2 activity

  • Limitation: Endpoint assay requiring sample processing

• HPLC-based product quantification:

  • Directly measure UDP-glucose formation by HPLC

  • Advantage: Highest specificity and direct product quantification

  • Limitation: Lower throughput, requires specialized equipment

• Radiometric assays:

  • Use ³²P-labeled UTP to track product formation

  • Advantage: Extremely sensitive detection

  • Limitation: Requires radioactive materials handling

When working with partial UGP2 constructs, validation across multiple assay formats is recommended to ensure that activity measurements reflect true enzymatic function rather than artifacts of the assay system.

How do cofactors and reaction conditions affect UGP2 catalytic activity?

UGP2 activity is highly dependent on specific reaction conditions and cofactors:

The addition of inorganic pyrophosphatase is particularly important as it prevents product inhibition and reverse reactions by hydrolyzing the pyrophosphate generated during UDP-glucose formation. This approach helps drive the reaction forward and reduces the rate of terminal transferase strand dismutation, as noted in similar enzymatic systems .

How can UGP2 kinetic parameters be accurately determined for recombinant partial constructs?

Determining accurate kinetic parameters for recombinant partial UGP2 requires careful experimental design:

• Initial velocity measurements:

  • Ensure <10% substrate conversion to maintain initial velocity conditions

  • Use sufficiently low enzyme concentrations (typically 1-10 nM)

  • Implement appropriate controls for background activity

• Substrate concentration ranges:

  • For Km determination, test substrate concentrations from 0.1× to 10× expected Km

  • Typically for UGP2: UTP (0.01-1 mM) and glucose-1-phosphate (0.05-5 mM)

  • Include at least 7-8 concentration points for reliable curve fitting

• Data analysis protocol:

  • Use non-linear regression to fit data to Michaelis-Menten equation

  • For complex kinetic patterns, consider more sophisticated models (Hill, substrate inhibition)

  • Analyze residuals to validate model fit quality

• Special considerations for partial constructs:

  • Compare kinetic parameters with full-length enzyme when possible

  • Assess potential cooperativity that might differ from full-length enzyme

  • Evaluate product inhibition patterns that might be altered in partial constructs

When reporting kinetic parameters, include detailed descriptions of assay conditions, particularly pH, temperature, and metal ion concentrations, as these significantly impact the values obtained and allow for proper comparison across studies.

How does UGP2 regulate glycogen synthesis and cell survival in nutrient-limited conditions?

UGP2 plays a crucial role in cellular adaptation to nutrient limitation through its control of glycogen metabolism:

• Metabolic regulation mechanism: UGP2 catalyzes the formation of UDP-glucose, the direct precursor for glycogen synthesis. Research indicates that UGP2 regulates glycogen synthesis which directly impacts survival in nutrient-starved conditions . This suggests UGP2 activity may be a rate-limiting step in the glycogen synthesis pathway under certain cellular conditions.

• Cellular survival pathway: Experimental evidence demonstrates that UGP2 knockdown halts tumor growth in xenograft models, indicating its essential role in cell proliferation and survival . This growth inhibition correlates with reduced Ki67 staining (a marker of cell proliferation), suggesting UGP2's role extends beyond basic metabolism to influence fundamental cellular growth programs.

• Nutrient sensing integration: UGP2 likely functions within a broader metabolic network that responds to nutrient availability. While the exact signaling mechanisms remain under investigation, UGP2 activity appears to be coordinated with cellular energy status and glucose availability to balance UDP-glucose production between immediate metabolic needs and storage as glycogen.

These findings highlight UGP2 as a potential therapeutic target, particularly in contexts where disrupting glycogen metabolism could selectively impact rapidly proliferating cells dependent on this metabolic pathway .

What is the relationship between UGP2 and protein N-glycosylation pathways?

UGP2 serves as a critical link between glucose metabolism and protein N-glycosylation through its production of UDP-glucose:

• N-glycosylation dependency: Research demonstrates that UGP2 is essential for maintaining proper N-glycosylation across the proteome. Knockdown of UGP2 significantly decreases the incidence of 141 N-glycosylation modifications spread across 89 proteins, and importantly, these decreases were not explained by corresponding changes in total protein levels .

• EGFR glycosylation impact: UGP2 knockdown specifically affects glycosylation of key proteins including the epidermal growth factor receptor (EGFR). Mass spectrometry analysis identified decreased glycosylation at Asn352 of EGFR following UGP2 depletion . This site-specific modification has functional consequences, as proper EGFR glycosylation is required for efficient receptor signaling.

• Downstream signaling consequences: The altered glycosylation pattern of EGFR due to UGP2 depletion leads to impaired downstream signaling . This mechanistic link provides insight into how metabolic enzymes like UGP2 can influence cell signaling networks through post-translational modifications.

This relationship highlights the interconnected nature of cellular metabolism and protein modification pathways, with UGP2 serving as a key node connecting carbohydrate metabolism to protein function through glycosylation-dependent mechanisms.

How can CRISPR-Cas9 genome editing be applied to study UGP2 function in Cricetulus griseus cells?

CRISPR-Cas9 technology offers powerful approaches to investigate UGP2 function in its native cellular context:

• Knockout strategy design:

  • Complete gene knockout: Design guide RNAs targeting early exons of UGP2

  • Domain-specific analysis: Create in-frame deletions of specific functional domains

  • Conditional systems: Implement floxed alleles with inducible Cre expression for temporal control

• Knock-in applications:

  • Epitope tagging: Add small tags (FLAG, HA) to endogenous UGP2 for purification and localization studies

  • Fluorescent reporters: Integrate fluorescent proteins for real-time visualization of UGP2 expression and localization

  • Point mutations: Introduce specific amino acid changes to test structure-function hypotheses

• Experimental validation protocol:

  • Genomic verification: Confirm editing through PCR and sequencing of the targeted locus

  • Protein expression analysis: Validate altered expression using Western blotting

  • Functional assessment: Measure changes in UDP-glucose levels, glycogen content, and N-glycosylation patterns

• Phenotypic analysis framework:

  • Proliferation and survival: Based on previous research showing UGP2 knockdown halts tumor growth

  • Metabolic profiling: Measure glycogen synthesis and utilization under nutrient-limited conditions

  • Glycoprotein function: Assess EGFR signaling and other glycosylation-dependent pathways

These CRISPR-based approaches provide opportunities to dissect UGP2 function with precision not possible using traditional knockdown methods, allowing researchers to distinguish between structural and catalytic roles of the enzyme.

What techniques can identify UGP2 interacting partners and regulatory proteins?

Uncovering the UGP2 interactome requires combining multiple complementary approaches:

• Affinity-based methods:

  • Immunoprecipitation with UGP2-specific antibodies followed by mass spectrometry

  • Tandem affinity purification using tagged recombinant UGP2

  • Proximity labeling (BioID, APEX) to identify proteins in UGP2's vicinity

  • Advantage: Can capture physiologically relevant interactions

  • Limitation: May miss transient or weak interactions

• Library screening approaches:

  • Yeast two-hybrid screening against cDNA libraries

  • Protein fragment complementation assays

  • Phage display selection

  • Advantage: Can identify direct binary interactions

  • Limitation: Higher false positive/negative rates

• Computational prediction and validation:

  • Co-expression analysis across tissues and conditions

  • Structural modeling of potential interaction interfaces

  • Evolutionary analysis of co-conserved residues

  • Advantage: Generates testable hypotheses

  • Limitation: Requires experimental validation

The search results indicate that YAP (Yes-associated protein 1) is an important upstream activator of UGP2 expression , suggesting that transcriptional regulatory networks are critical in controlling UGP2 levels and should be a focus of interaction studies.

How is UGP2 activity regulated in response to cellular metabolic status?

UGP2 activity is dynamically regulated to coordinate UDP-glucose production with cellular needs:

• Transcriptional regulation:

  • YAP-dependent expression: Research identifies YAP as an important upstream activator of UGP2 expression

  • This mechanism links UGP2 levels to YAP-regulated growth and proliferation pathways

  • Potential coordination with Hippo pathway signaling that controls YAP activity

• Allosteric regulation:

  • While specific allosteric regulators of UGP2 are not detailed in the search results, metabolic enzymes typically respond to:

  • Energy charge indicators (ATP/AMP ratio)

  • Substrate and product concentrations

  • Redox status of the cell

• Post-translational modifications:

  • Potential phosphorylation, acetylation, or other modifications that modulate enzyme activity

  • These modifications likely serve as rapid response mechanisms to changing metabolic conditions

• Compartmentalization and protein-protein interactions:

  • Changes in subcellular localization may direct UDP-glucose toward different metabolic fates

  • Interaction with different protein complexes could channel activity toward glycogen synthesis versus glycoprotein production

Understanding these regulatory mechanisms provides insight into how UGP2 integrates into broader cellular response networks that coordinate metabolism with growth, survival, and stress responses.

What is the relationship between UGP2 and YAP signaling pathways?

The connection between UGP2 and YAP signaling represents an important link between metabolism and growth control:

• Transcriptional regulation axis: The search results explicitly identify YAP as an important upstream activator of UGP2 expression . This places UGP2 as a downstream effector in the YAP-controlled transcriptional program, which typically promotes cell growth, proliferation, and survival.

• Metabolic feedback mechanism: Data shown in the search results suggest UGP2 functions as an effector of YAP activity to regulate growth and metabolism in pancreatic ductal adenocarcinoma (PDAC) cells . This indicates a potential bidirectional relationship where UGP2 not only responds to YAP activation but may also feed back to influence YAP activity through metabolic pathways.

• Functional significance in proliferation: The dual role of UGP2 in both glycogen synthesis and protein N-glycosylation suggests it coordinates multiple metabolic processes downstream of YAP to support the anabolic demands of cell growth and division. This coordination may be particularly important in rapidly proliferating cells where both energy storage and proper protein modification are essential.

• Therapeutic implications: The YAP-UGP2 axis represents a potential intervention point where disrupting either component could impact the other's function. Since UGP2 knockdown halts tumor growth in xenograft models , targeting this pathway might offer therapeutic approaches for cancers with hyperactive YAP signaling.

This relationship highlights how signaling pathways and metabolic enzymes are integrated to coordinate cellular growth with appropriate metabolic support.

Why might recombinant partial UGP2 show low enzymatic activity, and how can this be resolved?

Low enzymatic activity in recombinant partial UGP2 preparations can result from multiple factors:

• Structural integrity issues:

  • Problem: Improper folding due to truncation at non-optimal domain boundaries

  • Solution: Redesign constructs based on structural predictions or homology models

  • Diagnostic approach: Circular dichroism spectroscopy to assess secondary structure content

• Expression system limitations:

  • Problem: Lack of essential post-translational modifications

  • Solution: Switch from prokaryotic to eukaryotic expression systems

  • Diagnostic approach: Compare activity of protein expressed in different systems

• Purification-related problems:

  • Problem: Essential cofactors removed during purification

  • Solution: Include Mg²⁺ or Mn²⁺ (5-10 mM) in all buffers

  • Diagnostic approach: Activity rescue experiments with different metal ions

• Suboptimal reaction conditions:

  • Problem: Inhibitory buffer components or suboptimal pH

  • Solution: Systematically test buffers (cacodylate, Tris, HEPES) at various pH values (5-10)

  • Diagnostic approach: pH-activity profile determination

• Pyrophosphate accumulation:

  • Problem: Product inhibition by pyrophosphate

  • Solution: Add inorganic pyrophosphatase from Saccharomyces cerevisiae to reaction mixtures

  • Diagnostic approach: Activity measurements with/without pyrophosphatase

Drawing from terminal transferase enzyme optimization, buffer systems with pH between 5-10 (particularly cacodylate or Tris) supplemented with appropriate salts (Na⁺, K⁺, Mg²⁺, Mn²⁺) and inorganic pyrophosphatase can significantly improve enzymatic performance .

What approaches can resolve protein aggregation issues with recombinant UGP2?

Protein aggregation presents a common challenge when working with recombinant partial UGP2:

• During expression phase:

  • Lower induction temperature to 16-18°C

  • Reduce inducer concentration and extend expression time

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use solubility-enhancing fusion partners (MBP, SUMO, Thioredoxin)

• During purification phase:

  • Include mild solubilizing agents (0.1% Triton X-100, 0.5-1.0 M urea)

  • Add stabilizing osmolytes (0.5 M trehalose, 0.5-1.0 M arginine)

  • Maintain reducing environment with fresh DTT or β-mercaptoethanol

  • Include 10-20% glycerol in all buffers

• Storage considerations:

  • Store at higher protein concentrations (>1 mg/mL) to prevent surface adsorption

  • Avoid freeze-thaw cycles by preparing single-use aliquots

  • Consider protein stabilizing additives (BSA, glycerol, sucrose)

• Analytical monitoring tools:

  • Dynamic light scattering to detect early aggregation

  • Size exclusion chromatography to quantify monomeric protein

  • Thermal shift assays to identify stabilizing buffer conditions

When designing recombinant partial UGP2 constructs, consider that N-terminal truncations that maintain catalytic activity have been successful in related enzymes , suggesting similar approaches might improve UGP2 solubility.

How can inconsistent results between different batches of recombinant UGP2 be addressed?

Batch-to-batch variation is a common challenge when working with recombinant enzymes:

• Standardized production protocol:

  • Document every step of expression and purification in detail

  • Control cell growth density before induction (typically OD₆₀₀ = 0.6-0.8)

  • Standardize induction time, temperature, and inducer concentration

  • Use the same purification equipment and column matrices

• Quality control metrics:

  • Implement specific activity measurements for each batch

  • Perform SDS-PAGE and Western blot analysis to assess purity and integrity

  • Use size exclusion chromatography to verify oligomeric state

  • Measure protein concentration using multiple methods (Bradford, BCA, A₂₈₀)

• Storage standardization:

  • Prepare standard buffer formulations with precise pH verification

  • Aliquot and flash-freeze all preparations identically

  • Store all batches at the same temperature (-80°C preferred)

  • Track and limit freeze-thaw cycles

• Comprehensive batch validation:

  • Test each batch with standard substrate concentrations

  • Determine Km and Vmax for key substrates

  • Verify response to known activators or inhibitors

  • Assess thermal stability profile

For enzymatic assays, including inorganic pyrophosphatase can improve reproducibility by consistently driving the reaction forward and preventing variable levels of product inhibition . Similarly, maintaining standardized pH and metal ion concentrations is crucial for consistent activity measurements.

How might emerging technologies advance our understanding of UGP2 function?

Several cutting-edge technologies show promise for deeper insights into UGP2 biology:

• Cryo-electron microscopy:

  • Potential application: Determine high-resolution structures of UGP2 in different conformational states

  • Advantage: Minimal sample requirements and no crystallization needed

  • Impact: Could reveal substrate binding mechanisms and conformational changes during catalysis

• Single-molecule enzymology:

  • Potential application: Directly observe individual UGP2 molecules during catalysis

  • Advantage: Reveals heterogeneity in enzyme behavior masked in bulk measurements

  • Impact: Could discover intermediate states and rare catalytic events

• Spatially-resolved metabolomics:

  • Potential application: Map UDP-glucose production and utilization within cellular compartments

  • Advantage: Connects UGP2 activity to specific metabolic outcomes with spatial context

  • Impact: Could reveal how UGP2-produced UDP-glucose is directed to different metabolic fates

• Alpha-fold and related AI protein structure prediction:

  • Potential application: Generate accurate structural models of UGP2 from different species

  • Advantage: Rapidly test structural hypotheses without experimental structure determination

  • Impact: Could accelerate structure-based drug design targeting UGP2

These technologies, combined with established biochemical and cellular approaches, promise to provide a more comprehensive understanding of UGP2's roles in glycogen synthesis and protein N-glycosylation .

What are the most promising applications of UGP2 in biotechnology and medicine?

UGP2's central role in UDP-glucose production positions it for several potential applications:

• Therapeutic target development:

  • Rationale: UGP2 knockdown halts tumor growth in xenograft models

  • Approach: Structure-based design of small molecule UGP2 inhibitors

  • Challenge: Achieving selectivity to avoid disrupting normal cellular metabolism

  • Potential application: Treatment of cancers dependent on enhanced glycogen synthesis or aberrant glycosylation

• Metabolic engineering platforms:

  • Rationale: UGP2 controls UDP-glucose availability for multiple biosynthetic pathways

  • Approach: Engineer UGP2 variants with altered substrate specificity or regulatory properties

  • Challenge: Balancing enhanced activity with metabolic homeostasis

  • Potential application: Production of glycosylated biopharmaceuticals or valuable glycan-based products

• Biomarker development:

  • Rationale: UGP2 expression is regulated by YAP, a transcription factor altered in many cancers

  • Approach: Develop assays for UGP2 activity or glycosylation profiles as disease indicators

  • Challenge: Establishing specificity for particular disease states

  • Potential application: Early detection or monitoring of cancers with altered YAP-UGP2 axis

• Synthetic biology applications:

  • Rationale: UGP2 connects primary metabolism to specialized glycan production

  • Approach: Incorporate engineered UGP2 variants into synthetic pathways for novel glycan synthesis

  • Challenge: Coordinating UGP2 activity with downstream glycosyltransferases

  • Potential application: Production of customized glycans for research and therapeutic applications

These applications leverage the fundamental understanding of UGP2's roles in both glycogen synthesis and protein N-glycosylation to address unmet needs in biotechnology and medicine.

What key questions remain unresolved about UGP2 structure, function, and regulation?

Despite significant progress, several important questions about UGP2 remain unanswered:

• Structural mechanisms:

  • How do substrate binding events trigger conformational changes during catalysis?

  • What structural features determine the oligomeric state of UGP2 and how does this affect function?

  • Are there allosteric regulatory sites beyond the active site that modulate activity?

• Metabolic regulation:

  • How is UGP2 activity partitioned between glycogen synthesis and protein glycosylation pathways?

  • What post-translational modifications regulate UGP2 activity in response to metabolic signals?

  • How does UGP2 integrate into broader glucose sensing and utilization networks?

• Species-specific differences:

  • How do structural and functional properties of Cricetulus griseus UGP2 differ from human homologs?

  • Are there species-specific regulatory mechanisms that have evolved to meet different metabolic demands?

  • Can these differences be exploited for selective targeting in biotechnology applications?

• Disease relevance:

  • Beyond its identified role in supporting cancer cell survival , how does UGP2 dysfunction contribute to metabolic disorders?

  • Are there natural variants that affect UGP2 function with clinical consequences?

  • What compensatory mechanisms exist when UGP2 activity is compromised?

Addressing these questions will require integrating structural biology, enzymology, cell biology, and systems approaches to fully understand UGP2's multifaceted roles in cellular metabolism and its potential as a therapeutic target.