UMPS Human, Sf9

What is the biochemical function of UMPS and why is it significant in human metabolism?

UMPS (Uridine Monophosphate Synthetase) is a bifunctional enzyme that catalyzes the final two steps of the de novo pyrimidine biosynthetic pathway. In eukaryotes, UMPS uniquely links the orotate phosphoribosyltransferase and the orotidine-5'-monophosphate (OMP) decarboxylase activities into a single protein. This bifunctional arrangement is thought to stabilize the catalytic centers due to the low molar concentration of the protein in mammalian cells .

The enzyme plays a critical role in converting orotic acid to uridine monophosphate (UMP), which serves as a precursor for all pyrimidine nucleotides essential for DNA and RNA synthesis. Genetic mutations in UMPS are the underlying cause of hereditary orotic aciduria, a rare autosomal recessive disorder of pyrimidine metabolism .

What are the structural and biochemical characteristics of recombinant UMPS Human expressed in Sf9 cells?

UMPS Human Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 486 amino acids (positions 1-480 of the native sequence) with a molecular mass of 53kDa. The protein is expressed with a 6 amino acid His tag at the C-terminus to facilitate purification through affinity chromatography techniques .

Due to post-translational modifications, particularly glycosylation, the protein appears at approximately 50-70kDa when analyzed by SDS-PAGE. The expressed protein maintains both enzymatic domains necessary for its bifunctional activity. The protein solution (0.25mg/ml) is formulated in Phosphate Buffered Saline (pH 7.4) containing 30% glycerol, and typically achieves purity greater than 95.0% as determined by SDS-PAGE .

What are the optimal conditions for expression and purification of UMPS Human in the Sf9 expression system?

For optimal expression of UMPS Human in Sf9 cells, researchers should consider:

• Infection Parameters: Optimal multiplicity of infection (MOI) typically ranges from 1-10 for baculovirus infection, with protein expression monitored 48-72 hours post-infection.

• Cell Culture Conditions: Maintain Sf9 cells in appropriate insect cell media at 27°C, either as suspension or adherent cultures, with careful monitoring of cell viability throughout the expression period .

• Purification Strategy: Leverage the C-terminal His-tag for initial purification using nickel or cobalt affinity chromatography, followed by additional purification steps such as ion exchange or size exclusion chromatography to achieve high purity .

• Buffer Optimization: During purification, maintain pH stability (typically pH 7.4) and include stabilizing agents such as glycerol to prevent protein degradation and aggregation .

• Quality Control: Verify protein identity and purity through SDS-PAGE, Western blotting, and enzymatic activity assays for both functional domains.

What methodologies are recommended for studying UMPS mutations and their functional impacts?

Based on current research approaches, a comprehensive methodology for studying UMPS mutations includes:

• Genetic Analysis: Employ whole-exome sequencing (WES) for initial mutation identification, followed by Sanger sequencing with specific primers for confirmation in patient samples and family members .

• Bioinformatic Assessment: Use prediction tools like SIFT and PolyPhen-2 to assess the potential impact of mutations on protein function and structure .

• Metabolite Analysis: Implement gas chromatography-mass spectrometry (GC-MS) to analyze orotic acid levels in urine samples from affected individuals .

• Expression of Mutant Proteins: Generate constructs expressing identified mutations in Sf9 cells to assess their impact on protein expression, stability, and enzymatic activity.

• Functional Assays: Develop specific assays for both enzymatic activities to quantify how mutations affect catalytic function.

• Structural Biology: Use X-ray crystallography or molecular modeling to understand how mutations affect protein structure and domain interactions.

How can researchers differentiate between the two enzymatic activities of UMPS in experimental settings?

To differentiate and measure the two enzymatic activities of UMPS, researchers should implement domain-specific assays:

For orotate phosphoribosyltransferase (OPRT) activity:

• Measure the conversion of orotate to orotidine-5'-monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP).

• Track the reaction by spectrophotometric methods (decrease in absorbance at 295 nm) or by HPLC.

• Consider using radioisotope-labeled substrates ([14C]orotic acid) for increased sensitivity.

For orotidine-5'-phosphate decarboxylase (ODC) activity:

• Measure the conversion of orotidine-5'-monophosphate to uridine-5'-monophosphate.

• Monitor the reaction by tracking the decrease in absorbance at 285 nm or by measuring CO2 release.

• Implement coupled enzyme assays for continuous monitoring of activity.

Researchers must account for the interdependence of these domains in the native bifunctional enzyme when interpreting results from isolated domain assays.

How do UMPS mutations manifest clinically, and what are the research considerations when studying patient-derived variants?

UMPS deficiency results in hereditary orotic aciduria (HOA), with clinical manifestations that can include:

• Metabolic Abnormalities: Excessive orotic acid excretion in urine is the defining biochemical feature, detectable through GC-MS analysis .

• Hematological Effects: Megaloblastic anemia resistant to vitamin B12 and folate treatment is common.

• Neurological Manifestations: Recent research has identified cases with epilepsy and intellectual disability associated with UMPS mutations, suggesting broader neurological implications of pyrimidine metabolism disruption .

• Genotype-Phenotype Correlation: The recent identification of a novel missense mutation (c.517G>C, p.Val173Leu) in the UMPS gene demonstrated variable phenotypic expression within the same family, with the proband exhibiting severe manifestations (epilepsy and intellectual disability) while other carriers presented with mild orotic aciduria without clinical symptoms .

Research considerations when studying patient variants include:

• Distinguishing between homozygous mutations (typically causing clinical disease) and heterozygous carriers (often with biochemical abnormalities but minimal clinical manifestations)

• Accounting for potential modifier genes and environmental factors that may influence disease expression

• Establishing appropriate control groups including both healthy controls and asymptomatic carriers

What are the potential interactions between UMPS activity and cellular signaling pathways in Sf9 cells?

Studies of Sf9 cells have revealed complex interactions between cellular signaling and metabolic pathways that may impact UMPS function:

• Hormone Response Pathways: Research on hormone agonists in Sf9 cells has demonstrated distinct effects on cell proliferation and cell cycle progression, suggesting that endocrine signaling might interact with pyrimidine metabolism pathways .

• Cell Cycle Regulation: Different treatments can induce cell cycle arrest in different phases (G2/M or G1), indicating that metabolic enzymes like UMPS may have varying activities or regulation depending on cell cycle stage .

• Transcriptional Effects: Microarray experiments in Sf9 cells have shown that treatments can differentially affect gene expression, with distinct patterns observed for different compounds. This suggests that UMPS expression and activity might be regulated as part of broader transcriptional programs .

• Signal Transduction Pathways: The observation of different signaling pathways activated by different treatments in Sf9 cells indicates that UMPS function might be modulated through multiple regulatory mechanisms .

Researchers studying UMPS in Sf9 cells should consider these potential interactions, particularly when investigating how mutations or inhibitors might affect not only enzymatic activity but also broader cellular functions.

What strategies can address contradictions between in vitro enzyme assays and clinical phenotypes in UMPS research?

Researchers frequently encounter discrepancies between in vitro enzymatic measurements and clinical observations. To reconcile such contradictions:

• Tissue-Specific Effects: Consider that UMPS activity and regulation may vary across different tissues. While in vitro assays may show residual activity, tissue-specific factors might lead to more profound deficiencies in certain organs (particularly the central nervous system, as suggested by neurological symptoms in some patients) .

• Compensatory Mechanisms: Investigate alternate pyrimidine synthesis or salvage pathways that might partially compensate for UMPS deficiency in vivo but wouldn't be captured in isolated enzyme assays.

• Protein Stability Factors: Some mutations might primarily affect protein stability or half-life rather than catalytic activity. While the enzyme might show activity in short-term in vitro assays, decreased stability could lead to functional deficiency in vivo.

• Metabolic Context: The cellular environment, including substrate availability and product utilization, may significantly influence the functional impact of mutations. Comprehensive metabolomic profiling can reveal downstream effects not predicted by enzyme activity alone.

• Patient-Derived Models: Develop cellular models from patient samples (such as induced pluripotent stem cells) to better capture the complex genetic background and physiological context in which the mutant enzyme functions.

What are the key considerations for structural biology studies of UMPS requiring large-scale protein production?

For structural biology applications requiring substantial quantities of pure UMPS:

• Expression System Optimization:

  • Evaluate different promoters and signal sequences in the baculovirus construct

  • Optimize cell density at infection (typically 1-2 × 10^6 cells/mL)

  • Determine optimal harvest time post-infection (typically 48-72 hours)

  • Consider the addition of protease inhibitors during harvesting

• Purification Strategy Enhancement:

  • Develop a multi-step purification protocol beginning with His-tag affinity chromatography

  • Implement ion exchange chromatography to separate charge variants

  • Use size exclusion chromatography as a final polishing step to ensure homogeneity

  • Consider tag removal if the His-tag interferes with crystallization

• Protein Quality Assessment:

  • Verify both enzymatic activities to confirm proper folding

  • Analyze protein by dynamic light scattering to assess monodispersity

  • Conduct thermal shift assays to identify stabilizing buffer conditions

  • Evaluate glycosylation status and its potential impact on crystallization

• Crystallization Considerations:

  • Screen both full-length protein and individual domains

  • Test the effect of substrate analogs or inhibitors on crystal formation

  • Explore the impact of reducing surface entropy through targeted mutations

  • Consider limited proteolysis to identify stable domains for crystallization

• Alternative Structural Approaches:

  • Implement cryo-electron microscopy for structure determination without crystallization

  • Explore small-angle X-ray scattering (SAXS) for solution structure analysis

  • Consider hydrogen-deuterium exchange mass spectrometry for dynamics studies

How can advanced computational methods enhance the understanding of novel UMPS mutations?

For novel mutations like the recently identified c.517G>C variant, advanced computational approaches offer valuable insights:

• Molecular Dynamics Simulations:

  • Simulate the behavior of wild-type and mutant proteins over nanosecond to microsecond timescales

  • Analyze changes in protein flexibility, domain interactions, and catalytic site geometry

  • Identify potential allosteric effects that might not be apparent from static structural analysis

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Model the enzymatic reaction mechanism at the quantum level

  • Determine how mutations might affect transition states and energy barriers

  • Predict changes in catalytic efficiency with atomic-level precision

• Machine Learning Approaches:

  • Develop predictive models based on existing mutation-phenotype correlations

  • Implement neural networks trained on protein structural data to predict functional impacts

  • Use natural language processing to extract relevant information from scientific literature

• Network Analysis:

  • Model the pyrimidine synthesis pathway as an integrated network

  • Predict how UMPS mutations might affect flux through the pathway

  • Identify potential compensatory mechanisms or vulnerabilities

• Comparative Genomics:

  • Analyze UMPS sequence conservation across species

  • Identify co-evolving residues that might functionally interact with the mutated position

  • Examine whether homologous mutations exist in other species and their phenotypic consequences

These computational approaches, when integrated with experimental data, provide a comprehensive framework for understanding the molecular basis of UMPS-related disorders and developing potential therapeutic strategies.

What emerging technologies might advance UMPS research in the coming years?

Several cutting-edge technologies hold promise for advancing UMPS research:

• CRISPR/Cas9 Gene Editing:

  • Creation of precise UMPS mutations in cellular and animal models

  • Development of isogenic cell lines differing only in UMPS genotype

  • High-throughput screening of UMPS variants using CRISPR libraries

• Single-Cell Omics:

  • Analysis of cell-to-cell variation in UMPS expression and activity

  • Identification of subpopulations with differential response to UMPS deficiency

  • Integration of transcriptomic, proteomic, and metabolomic data at single-cell resolution

• Organoid Models:

  • Development of three-dimensional tissue models expressing UMPS variants

  • Study of tissue-specific effects of UMPS deficiency

  • Testing of therapeutic approaches in physiologically relevant systems

• In Situ Structural Biology:

  • Cryo-electron tomography to visualize UMPS in its cellular context

  • Integration of structural data with functional assays in living cells

  • Analysis of UMPS interactions with other cellular components

• Precision Medicine Approaches:

  • Development of patient-specific treatment strategies based on specific UMPS mutations

  • Pharmacogenomic studies to predict response to uridine supplementation

  • Exploration of gene therapy approaches for severe UMPS deficiency