Recombinant Culex quinquefasciatus Ubiquitin-like modifier-activating enzyme 5 (CPIJ009416)

What is CPIJ009416 and how does it function in Culex quinquefasciatus?

CPIJ009416 is a ubiquitin-like modifier-activating enzyme 5 belonging to the ubiquitin-activating E1 family, specifically the UBA5 subfamily. This 397-amino acid enzyme (43.5 kDa) functions as an E1-like enzyme that activates UFM1 (Ubiquitin-fold modifier 1) . Like other E1 enzymes, CPIJ009416 likely catalyzes the initial step in the UFM1 conjugation pathway, creating a thioester bond between the C-terminus of UFM1 and its catalytic cysteine residue through an ATP-dependent process. This activation is essential for subsequent transfer to E2 and E3 enzymes in the UFM1 conjugation cascade.

Structurally, CPIJ009416 contains three primary domains typical of E1 enzymes: an ATP-binding domain that facilitates adenylation, a catalytic domain containing the reactive cysteine residue, and a domain responsible for UFM1 recognition . This tripartite structure enables the sequential biochemical reactions required for UFM1 activation.

How does CPIJ009416 differ from other E1 enzymes in mosquitoes and related species?

The complete amino acid sequence of CPIJ009416 (MTSVAELREQVRSLQDELAQLKGERGKTTTREKITKMSSEVVDSNPYSRLMALQRMGIVKEYEQIRQKSVAVVGVGGVGSVTADMLTRCGVGKLILFDYDKVELANMNRLFFTPDQAGLSKVEAAAKTLNYINPDVKIFTNNYNITTVESFEKFMNAIRTGGIDGSGAVDLVLSCVDNFEARMAINAACNELSLNWFESGVSENAVSGHIQFIQPGEKACFACAPPLVVAENIDEKTLKREGVCAASLPTTMGIVAGMLVQNTLKYLLKFGTVSDYLGYNALIDFFPKMGLKPNPTCDDRFCVLRQQEFAAKPKEETFEEVQQEEESPVHAENLYGIELVSETEVESAPTVPVATANTGLKLAFETPIQMEHSSAATDVIKNDDVSLDDLMAQMKAI) reveals key structural features that distinguish this enzyme from other E1 enzymes . Analysis of this sequence would reveal conserved regions associated with ATP binding and catalysis, as well as variable regions that may confer specific UFM1 recognition properties.

What expression systems are optimal for producing recombinant CPIJ009416?

Based on successful approaches with similar enzymes, several expression systems can be considered for CPIJ009416 production, each with distinct advantages:

For CPIJ009416, Pichia pastoris may offer significant advantages as demonstrated with other mosquito enzymes. Research on Culex quinquefasciatus α-glucosidase showed successful expression in P. pastoris with activity detection at 3.75U/ml under optimal culture conditions . The recombinant protein showed a molecular weight of approximately 92kDa on SDS-PAGE, which decreased to 69kDa after Endoglycosidase H digestion, confirming glycosylation . This suggests that P. pastoris can provide the post-translational modifications potentially required for optimal CPIJ009416 activity.

What purification strategy yields the highest activity for recombinant CPIJ009416?

A multi-step purification approach combining affinity chromatography with additional purification methods is recommended for obtaining highly active CPIJ009416:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged CPIJ009416 provides efficient initial purification.

• Intermediate purification: Ion exchange chromatography separates CPIJ009416 from similarly sized contaminants based on charge differences.

• Polishing step: Size exclusion chromatography removes aggregates and ensures homogeneous enzyme preparation.

Throughout the purification process, enzyme activity should be monitored using a UFM1 activation assay that measures ATP-PPi exchange or thioester bond formation. The purification buffer should maintain enzyme stability while minimizing background in activity assays.

Buffer composition significantly impacts enzyme stability and activity. For CPIJ009416, a base buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 150 mM NaCl, 10% glycerol, and 1 mM DTT is recommended as a starting point, with optimization required for specific applications.

What are the optimal conditions for assessing CPIJ009416 enzymatic activity?

Determining optimal reaction conditions is crucial for accurate assessment of CPIJ009416 activity. Based on related E1 enzyme studies and mosquito enzyme characterization, the following parameters should be systematically evaluated:

For reference, characterization of Culex quinquefasciatus α-glucosidase showed optimal pH and temperature at 5.5 and 35°C, respectively . While CPIJ009416 likely has different optima, similar methodological approaches can be applied to its characterization.

Multiple assay methods can be employed to measure CPIJ009416 activity:

• ATP-PPi exchange assay: Measures the formation of [³²P]ATP from [³²P]PPi and AMP, reflecting the reverse of the adenylation reaction.

• Thioester bond formation: Detects the covalent enzyme-UFM1 intermediate using non-reducing SDS-PAGE or mass spectrometry.

• Coupled assay: Monitors AMP formation through coupled enzymes that produce a spectrophotometric or fluorescent signal.

Each assay has specific advantages and limitations that should be considered when selecting the most appropriate method for a particular experimental question.

How can researchers distinguish between specific CPIJ009416 inhibition and general E1 enzyme inhibition?

Distinguishing between specific CPIJ009416 inhibition and general E1 enzyme inhibition requires carefully designed experiments and controls:

• Comparative inhibition studies: Test potential inhibitors against CPIJ009416 and other E1 enzymes (e.g., SUMO E1, ubiquitin E1) under identical conditions. Specificity is indicated by significantly greater inhibition of CPIJ009416.

• Structure-activity relationship (SAR) analysis: Systematically modify inhibitor structures and correlate with differential inhibition patterns across E1 enzymes.

• Binding site mutation analysis: Introduce mutations in the predicted inhibitor binding site of CPIJ009416 and assess changes in inhibitor sensitivity.

For reference in designing inhibition studies, acarbose strongly inhibits Culex quinquefasciatus α-glucosidase with an IC₅₀ of 67.8±5.6nM, while glucose weakly inhibits it with an IC₅₀ of 115.9±7.3mM . Similar differential inhibition patterns would be expected for specific vs. non-specific CPIJ009416 inhibitors.

What structural features determine the UFM1 specificity of CPIJ009416?

The structural determinants of UFM1 specificity in CPIJ009416 likely involve specific domains and residues that recognize UFM1's unique features. While the specific crystal structure of CPIJ009416 is not available in the search results, insights can be derived from related E1 enzymes:

• C-terminal domain: Likely contains the UFM1 recognition motif that distinguishes UFM1 from other ubiquitin-like modifiers.

• Catalytic cysteine region: The microenvironment around the catalytic cysteine influences reactivity and specificity.

• Crossover loop: This structural element in E1 enzymes often contributes to modifier selectivity.

To experimentally identify these structural features, researchers should consider:

• Chimeric protein analysis: Swapping domains between CPIJ009416 and other E1 enzymes to map specificity determinants.

• Alanine scanning mutagenesis: Systematically replacing conserved residues to identify those critical for UFM1 recognition.

• Hydrogen-deuterium exchange mass spectrometry: Mapping protein regions that change conformation upon UFM1 binding.

The three-dimensional structure of E1 enzymes typically contains three domains: an ATP-binding domain, an adenylation domain, and a catalytic cysteine-containing domain . The relative orientation of these domains in CPIJ009416 likely creates a specific binding pocket for UFM1.

What approaches can researchers use to engineer CPIJ009416 variants with enhanced activity or stability?

Engineering CPIJ009416 for improved properties requires combining computational and experimental approaches:

Specific strategies might include:

• Disulfide engineering: Introducing non-native disulfide bonds to enhance thermostability.

• Surface charge optimization: Modifying surface residues to improve solubility without affecting the active site.

• Glycosylation site introduction: Adding N-linked glycosylation sites to enhance stability when expressed in eukaryotic systems.

• Active site refinement: Fine-tuning residues around the catalytic cysteine to enhance catalytic efficiency.

Researchers should establish a reliable high-throughput screening method to evaluate variants rapidly. For CPIJ009416, a coupled enzymatic assay that produces a fluorescent or colorimetric signal would be ideal for screening libraries of variants.

How might CPIJ009416 contribute to insecticide resistance mechanisms in Culex quinquefasciatus?

While direct evidence linking CPIJ009416 to insecticide resistance is not present in the search results, exploration of this potential connection represents an important research direction. UFM1 modification pathways regulated by CPIJ009416 could potentially contribute to resistance through several mechanisms:

• Protein quality control: UFM1 modification may enhance the stability and activity of detoxification enzymes that metabolize insecticides.

• Stress response regulation: UFM1 pathways might modulate cellular responses to insecticide-induced stress.

• Target protein modification: UFM1 could modify insecticide target proteins, altering their susceptibility.

Studies of Culex quinquefasciatus have identified multiple enzymatic mechanisms contributing to insecticide resistance, including elevated levels of α-esterases, β-esterases, mixed function oxidases, and glutathione-S-transferase . The potential role of CPIJ009416 in regulating the expression or activity of these enzymes deserves investigation.

Research designs to investigate CPIJ009416's role in resistance should include comparative analysis of expression and activity levels between resistant and susceptible mosquito strains, as well as functional studies using RNA interference or CRISPR-Cas9 gene editing.

How can researchers develop specific inhibitors of CPIJ009416 as potential vector control tools?

Developing specific inhibitors of CPIJ009416 requires a systematic drug discovery pipeline:

• Target validation: Confirm the essential nature of CPIJ009416 in mosquito survival or reproduction through genetic approaches.

• Assay development: Establish robust, scalable biochemical and cell-based assays to measure CPIJ009416 inhibition.

• Screening approach: Implement virtual screening followed by biochemical validation, or direct high-throughput screening of compound libraries.

• Lead optimization: Iteratively improve potency, specificity, and pharmacokinetic properties of hit compounds.

Structure-based drug design approaches would be valuable if the crystal structure of CPIJ009416 becomes available. In the absence of a crystal structure, homology modeling based on related E1 enzymes could guide initial inhibitor design.

For reference, inhibitor development strategies might draw on approaches used for other mosquito enzymes. For instance, acarbose strongly inhibits Culex quinquefasciatus α-glucosidase with an IC₅₀ of 67.8±5.6nM , demonstrating that highly potent inhibitors can be identified for mosquito enzymes.

What are common challenges in crystallizing CPIJ009416 for structural studies and how can they be addressed?

Obtaining high-quality crystals of CPIJ009416 for X-ray crystallography likely presents several challenges that researchers should anticipate:

Methodological approaches to address these challenges include:

• Construct optimization: Generate multiple truncated versions of CPIJ009416 to identify stable, crystallization-prone constructs.

• Co-crystallization: Attempt crystallization with ATP analogs, UFM1, or fragments of interacting proteins to stabilize a specific conformation.

• Surface engineering: Introduce surface mutations that reduce entropy or promote crystal contacts without affecting enzyme function.

• Alternative crystallization methods: Explore lipidic cubic phase, microfluidics, or in meso crystallization for challenging proteins.

• Complementary structural techniques: Use small-angle X-ray scattering (SAXS), cryo-electron microscopy, or NMR spectroscopy as alternative or complementary approaches.

The qualitative research methodology principle of displaying evidence systematically in tables can be applied to crystallization trials to enhance trustworthiness in structural studies .

How can researchers effectively investigate the in vivo function of CPIJ009416 in Culex quinquefasciatus?

Investigating the in vivo function of CPIJ009416 requires combining genetic, biochemical, and advanced imaging approaches:

• Genetic manipulation techniques:

  • RNA interference (RNAi) through dsRNA microinjection or feeding

  • CRISPR-Cas9 gene editing to generate knockout or knockin mosquitoes

  • Transgenic overexpression with tissue-specific promoters

• Phenotypic analysis methods:

  • Developmental timing and success rate measurement

  • Lifespan and fecundity assessment

  • Insecticide susceptibility testing

  • Behavioral assays

• Molecular profiling approaches:

  • Transcriptome analysis following CPIJ009416 manipulation

  • Proteomic identification of UFM1-modified proteins

  • Metabolomic changes associated with altered UFM1 modification

When designing these experiments, researchers should consider the question-asking framework highlighted in healthcare research, which emphasizes the importance of formulating clear, specific questions and systematically gathering evidence to answer them . This approach can be adapted to molecular biology research to ensure rigor and reproducibility.