Recombinant Aedes aegypti Adenosine monophosphate-protein transferase FICD homolog (AAEL005383)

What is the AAEL005383 protein and what is its role in Aedes aegypti?

AAEL005383 is the gene identifier for a protein in Aedes aegypti that functions as an adenosine monophosphate-protein transferase FICD homolog. This protein belongs to the family of Fic domain-containing proteins, which typically catalyze post-translational modifications of target proteins through AMPylation (the addition of adenosine monophosphate). In Aedes aegypti, this protein likely plays a role in cellular stress responses and protein homeostasis similar to FICD homologs in other organisms. The gene appears in orthologous gene collections used in comparative genomic studies across arthropod species .

How does AAEL005383 compare to FICD proteins in other species?

AAEL005383 shares structural and functional similarities with FICD proteins found in other organisms, particularly the conserved Fic domain responsible for catalyzing AMPylation reactions. Comparative genomic analyses suggest orthology with similar proteins in other arthropods, including the tick species Ixodes scapularis (ISCW016921) . While the core enzymatic function is likely conserved, species-specific adaptations may exist that reflect the unique physiological requirements of Aedes aegypti. Sequence alignment and phylogenetic analyses would be necessary to determine the exact evolutionary relationships and functional divergence from FICD proteins in other insects and vertebrates.

Is there evidence for AAEL005383 involvement in dengue virus resistance?

Current research has not specifically identified AAEL005383 among the genes associated with dengue virus (DENV) resistance. An exome-wide association study in Aedes aegypti from Bakoumba, Gabon identified 263 genes significantly associated with resistance to both DENV-1 and DENV-3, with additional genes uniquely associated with resistance to specific dengue virus types . The shared significant genes were enriched in ATP binding activity and sulfur compound transmembrane transporter activity, while DENV-3 resistance-specific genes showed enrichment in zinc ion binding activity . Further studies would be needed to determine if AAEL005383 plays a role in viral resistance, particularly given that FICD proteins can regulate stress responses that might influence viral replication.

What are the key considerations for designing experiments to characterize AAEL005383 function?

When designing experiments to characterize AAEL005383 function, researchers should:

• Define clear variables: Establish independent variables (e.g., expression conditions, mutation sites) and dependent variables (e.g., enzymatic activity, substrate specificity) .

• Develop testable hypotheses: Formulate specific hypotheses about AAEL005383 function based on knowledge of FICD proteins in other systems .

• Include appropriate controls: Design positive controls (known FICD proteins), negative controls (enzymatically inactive mutants), and vehicle controls .

• Control extraneous variables: Minimize confounding factors such as temperature fluctuations, pH variations, and contamination .

• Plan for replication: Ensure sufficient biological and technical replicates to establish statistical significance .

• Select appropriate assay systems: Choose assay methods sensitive enough to detect AMPylation activity and substrate interactions.

How should researchers approach the recombinant expression of AAEL005383?

The recombinant expression of AAEL005383 should be approached systematically by:

• Vector selection: Choose an expression vector with appropriate promoters, tags, and selection markers based on the experimental goals.

• Expression system evaluation: Test multiple expression systems (bacterial, insect, mammalian) to identify optimal conditions. For mosquito proteins, bacterial systems like E. coli may present challenges due to codon usage differences and post-translational modifications .

• Optimization strategies: Consider codon optimization for the expression host, use of solubility tags (MBP, SUMO), and expression at lower temperatures to enhance protein solubility.

• Expression conditions: Systematically test induction conditions (temperature, inducer concentration, duration) to maximize yield while maintaining protein stability and activity.

• Monitoring for autocatalytic activity: Be aware that FICD proteins may exhibit autocatalytic behavior or autodigestive properties similar to what has been observed with Aedes aegypti serine proteases .

What purification strategies are most effective for recombinant AAEL005383?

Based on experience with similar Aedes aegypti proteins, the following purification approach is recommended:

• Initial clarification: After cell lysis, perform centrifugation at 10,000-15,000 × g for 30 minutes to remove cell debris.

• Affinity chromatography: Utilize appropriate affinity tags (His, GST, or MBP) for initial capture. For His-tagged constructs, immobilized metal affinity chromatography (IMAC) with Ni-NTA resin at pH 8.0 typically yields good results.

• Buffer optimization: Screen various buffer compositions to identify conditions that maintain protein stability and solubility. Consider including:

  • 25-50 mM Tris or HEPES buffer (pH 7.5-8.0)

  • 150-300 mM NaCl to reduce non-specific interactions

  • 5-10% glycerol as a stabilizing agent

  • 1-5 mM reducing agent (DTT or TCEP)

• Secondary purification: Apply size exclusion chromatography or ion exchange chromatography to achieve higher purity and remove aggregates.

• Stability monitoring: Track protein stability during purification using activity assays and thermal shift assays to ensure the protein remains functional.

What methods are appropriate for confirming the AMPylation activity of purified AAEL005383?

Several complementary approaches can be used to confirm and characterize the AMPylation activity:

• Radioactive assays: Utilize [α-32P]ATP to track the transfer of radiolabeled AMP to substrate proteins, followed by SDS-PAGE and autoradiography detection.

• Mass spectrometry: Employ high-resolution mass spectrometry to identify AMP modifications on target proteins, which appear as a mass increase of 329 Da corresponding to the addition of AMP.

• Antibody-based detection: Use anti-AMP-Thr and anti-AMP-Tyr antibodies to detect AMPylated substrates via Western blotting.

• Enzymatic coupled assays: Measure pyrophosphate (PPi) release during the AMPylation reaction using commercially available enzyme-coupled assays that produce a colorimetric or fluorescent readout.

• Thermal shift assays: Monitor changes in protein thermal stability upon binding of substrates and cofactors to gain insight into binding interactions.

How can CRISPR-Cas9 gene editing be applied to study AAEL005383 function in vivo?

CRISPR-Cas9 gene editing offers powerful approaches to investigate AAEL005383 function:

• Knockout generation: Design guide RNAs targeting the AAEL005383 gene to create complete knockout mosquitoes. Guidelines for gRNA design include:

  • Target early exons to ensure complete loss of function

  • Select sequences with minimal off-target effects

  • Design multiple gRNAs to increase editing efficiency

• Knockin strategies: Introduce specific mutations to test the importance of key residues in the Fic domain or to add reporter tags for in vivo tracking.

• Conditional expression systems: Implement tissue-specific or inducible promoters to control AAEL005383 expression in specific contexts.

• Phenotypic analysis: Evaluate the effect of AAEL005383 disruption on:

  • Mosquito development and viability

  • Response to cellular stressors

  • Vector competence for arboviruses

  • Reproductive capacity

• Rescue experiments: Reintroduce wild-type or mutant versions of AAEL005383 in knockout backgrounds to confirm specificity of observed phenotypes.

How might AAEL005383 interact with dengue virus proteins during infection?

Understanding potential interactions between AAEL005383 and dengue virus proteins requires several investigative approaches:

• Protein-protein interaction studies: Use co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screening to identify direct interactions between AAEL005383 and viral proteins.

• AMPylation target identification: Employ proteomic approaches to identify viral proteins that might be AMPylated by AAEL005383, focusing on:

  • Non-structural proteins involved in viral replication

  • Structural proteins during virion assembly

  • Host factors recruited during viral replication

• Functional consequences assessment: Determine how AMPylation affects viral protein function through:

  • Enzymatic activity assays for viral enzymes

  • Viral replication assays in cell culture

  • Structural studies of modified viral proteins

• Temporal dynamics: Analyze the timing of AAEL005383 expression and activity relative to the viral life cycle, particularly whether AMPylation represents a host defense mechanism or viral subversion strategy.

How does AAEL005383 compare to its orthologs in other mosquito vectors?

AAEL005383 belongs to a family of conserved genes found across mosquito species. Comparative analysis reveals:

*Note: Exact values would require comprehensive sequence analysis which is beyond the scope of this document.

The comparative analysis of these orthologs could provide insights into:

• Conservation of catalytic residues suggesting functional importance

• Species-specific adaptations that might relate to different host preferences or vector competence

• Evolutionary patterns that could reveal selective pressures related to pathogen interactions

What insights can be gained from studying AAEL005383 expression patterns during different life stages and physiological conditions?

Understanding AAEL005383 expression dynamics can reveal functional importance:

• Developmental expression profiling: Quantitative PCR or RNA-seq analysis across embryonic, larval, pupal, and adult stages can identify critical developmental windows where AAEL005383 function may be essential.

• Blood-meal responsive expression: Unlike some Aedes aegypti serine proteases that show differential expression before and after blood meals , it remains to be determined whether AAEL005383 expression changes in response to blood feeding. This could provide clues about its role in digestion, reproduction, or immune responses.

• Stress-responsive regulation: As FICD proteins often function in stress response pathways, examining AAEL005383 expression under various stressors (heat shock, oxidative stress, viral infection) could reveal its role in cellular homeostasis.

• Tissue-specific expression: Determining which tissues express AAEL005383 at highest levels (midgut, salivary glands, ovaries, neural tissue) would provide functional insights.

What structural features of AAEL005383 are critical for its enzymatic function?

The functional architecture of AAEL005383 likely includes several conserved features typical of FICD proteins:

• Fic domain conservation: The canonical Fic domain typically contains an HPFx(D/E)GN(G/K)R motif that forms the catalytic core. Any variations in this motif in AAEL005383 could suggest altered substrate specificity or catalytic mechanism.

• ATP-binding pocket: Residues that coordinate ATP binding are likely conserved and critical for function. Mutations in these residues would be expected to abolish enzymatic activity.

• Substrate recognition elements: Regions outside the core catalytic domain likely contribute to substrate recognition and specificity. These may be less conserved across species and could represent adaptations to mosquito-specific substrates.

• Regulatory elements: Many FICD proteins contain auto-inhibitory elements that regulate enzymatic activity. Identifying such features in AAEL005383 would be important for understanding its regulation.

• Oligomerization interfaces: If AAEL005383 functions as a dimer or higher-order oligomer, the interfaces mediating these interactions would be structurally important.

What approaches can be used to identify potential inhibitors of AAEL005383?

Development of AAEL005383 inhibitors could proceed through several complementary strategies:

• Structure-based design: If structural data becomes available (through X-ray crystallography or cryo-EM), virtual screening of compound libraries against the ATP-binding pocket or substrate-binding sites could identify potential inhibitors.

• High-throughput screening: Develop a robust enzymatic assay suitable for screening compound libraries. This could involve:

  • Fluorescence-based detection of AMPylation using modified substrates

  • ATP consumption or pyrophosphate production measurement

  • Thermal shift assays to detect compounds that bind to AAEL005383

• Fragment-based approaches: Screen libraries of small molecular fragments that can bind to different regions of AAEL005383, then link promising fragments to create more potent inhibitors.

• Natural product screening: Test extracts from plants known to have mosquitocidal properties for inhibitory activity against AAEL005383.

• Rational design based on substrate mimics: Design competitive inhibitors that mimic natural substrates or ATP but cannot be utilized in the AMPylation reaction.