Recombinant Anopheles gambiae Eukaryotic translation initiation factor 3 subunit B (eIF3-S9), partial

What is eIF3-S9 and what role does it play in Anopheles gambiae?

Eukaryotic translation initiation factor 3 subunit B (eIF3-S9) is a crucial component of the eIF3 complex in A. gambiae. The eIF3 complex is essential for translation initiation, functioning to stabilize the 43S pre-initiation complex and promote mRNA binding. In A. gambiae, eIF3-S9 is encoded by the gene AGAP012140-PA and forms part of the multi-protein complex that regulates protein synthesis . This protein plays a key role in mosquito development, immune response, and potentially in vector competence for Plasmodium parasites. While eIF3-S9 shares functional homology with mammalian counterparts, its specific interactions within the A. gambiae translational machinery may differ, making it an important subject for vector biology research.

How does eIF3-S9 relate to other eIF3 subunits in A. gambiae?

The eIF3 complex in A. gambiae consists of multiple subunits (designated A through J) that work together for efficient translation initiation. In A. gambiae, these subunits include:

• eIF3-S10 (subunit A, gene: AGAP002340-PA)

• eIF3-S9 (subunit B, gene: AGAP012140-PA)

• eIF3-S7 (subunit D, gene: AGAP002337-PA)

• eIF3-S6 (subunit E, gene: AGAP006944-PA)

• eIF3-S5 (subunit F, gene: AGAP002935-PA)

• eIF3-S4 (subunit G, gene: AGAP007668-PA)

• eIF3-S3 (subunit H, gene: AGAP009204-PA)

• eIF3-S2 (subunit I, gene: AGAP006607-PA)

• eIF3-S1 (subunit J, gene: AGAP006613-PB)

These subunits form a scaffold that bridges the ribosome and mRNA, with eIF3-S9 (subunit B) likely serving as a core structural component based on its conserved role across species.

What are the quality specifications for recombinant eIF3-S9?

Commercially available recombinant A. gambiae eIF3-S9 is typically produced with the following specifications:

Researchers should verify these specifications before using the protein in experimental workflows to ensure reliable results .

What are the optimal experimental conditions for working with recombinant eIF3-S9?

When working with recombinant A. gambiae eIF3-S9, researchers should maintain the following experimental conditions:

• Storage: Store the protein at -80°C in small aliquots to prevent repeated freeze-thaw cycles

• Buffer composition: Use buffers containing 10-20% glycerol with stabilizing agents

• Working temperature: Maintain at 4°C during handling; perform experiments at 25-28°C to mimic physiological conditions of A. gambiae

• pH range: Optimal activity is typically observed at pH 7.2-7.6

• Reducing agents: Include low concentrations (0.5-1 mM) of DTT or β-mercaptoethanol to maintain disulfide bonds

For in vitro translation assays, supplement reaction mixtures with other eIF3 subunits from A. gambiae, as the partial recombinant eIF3-S9 may require complex formation with other subunits for full functionality .

How can researchers validate the functionality of recombinant eIF3-S9?

Before incorporating recombinant eIF3-S9 into complex experiments, validate its functionality through:

• SDS-PAGE and Western blotting: Confirm molecular weight (typically 85-90 kDa) and immunoreactivity with anti-eIF3-S9 antibodies

• Binding assays: Test interaction with other eIF3 subunits and ribosomal components using co-immunoprecipitation or pull-down assays

• In vitro translation: Assess the protein's ability to enhance translation efficiency in reconstituted systems using A. gambiae cell extracts

• ATPase activity: Measure the protein's ATPase activity, which is essential for its role in translation initiation

• Thermal shift assays: Evaluate protein stability under various buffer conditions to optimize experimental parameters

Include appropriate positive controls (e.g., recombinant human eIF3-S9) and negative controls (e.g., heat-denatured protein) in validation experiments .

What techniques can be used to study eIF3-S9 interactions with other translation factors?

Researchers investigating eIF3-S9 interactions should consider these methodologies:

• Crosslinking and Immunoprecipitation (CLIP): Identify RNA binding sites and partners

• Proximity Labeling (BioID or APEX): Map the protein interaction network in mosquito cells

• Surface Plasmon Resonance (SPR): Determine binding kinetics with purified interaction partners

• Yeast Two-Hybrid screening: Identify novel protein interactions

• Cryo-EM structural analysis: Visualize eIF3-S9 within the larger translation initiation complex

• FRET/BRET assays: Monitor real-time interactions in cellular contexts

These approaches should be optimized for the specific properties of A. gambiae proteins and may require modification of standard protocols used in model organisms .

How is eIF3-S9 function potentially related to malaria parasite infection in Anopheles gambiae?

The relationship between eIF3-S9 and Plasmodium infection is complex and merits investigation:

• Translation regulation during infection: eIF3-S9 may regulate selective translation of immune-related transcripts during Plasmodium infection

• Stress response: Parasite invasion triggers cellular stress responses that alter translation patterns controlled by eIF3

• Potential polymorphisms: Similar to identified SNPs in immune signaling genes (like Toll5B), polymorphisms in eIF3-S9 might influence vector competence

• Midgut response: eIF3-S9 activity may be modified during midgut invasion by Plasmodium

• Parasite manipulation: Plasmodium may potentially alter host translation machinery to favor its development

Research approaches should include comparing eIF3-S9 expression and activity between susceptible and resistant A. gambiae strains, and analyzing potential associations between eIF3-S9 polymorphisms and infection outcomes .

What are the methodological considerations for studying eIF3-S9 in the context of vector competence?

When investigating eIF3-S9's role in vector competence, researchers should:

• Use genetically diverse A. gambiae populations to capture natural variation

• Implement RNAi-mediated knockdown of eIF3-S9 to assess effects on Plasmodium infection rates

• Employ CRISPR-Cas9 genome editing to create eIF3-S9 mutants for functional studies

• Develop tissue-specific and time-specific expression systems to understand temporal dynamics

• Integrate proteomics approaches to identify post-translational modifications occurring during infection

• Use polysome profiling to identify transcripts differentially translated during infection

These approaches should follow standardized protocols for A. gambiae infection studies as outlined in the Methods in Anopheles Research manual, with appropriate controls for mosquito age, feeding conditions, and environmental parameters .

How can researchers investigate potential polymorphisms in eIF3-S9 and their functional consequences?

To study eIF3-S9 polymorphisms:

• Perform targeted sequencing of eIF3-S9 across diverse A. gambiae populations with varying vector competence

• Use association mapping approaches similar to those that identified SNPs in Toll5B and insulin-like peptide 3 precursor genes

• Analyze non-synonymous changes that may affect protein structure and function

• Test for departures from Hardy-Weinberg equilibrium as indicators of selection pressure

• Functionally characterize identified variants through:

  • Recombinant expression of variant proteins

  • Comparative binding assays with translation components

  • In vitro translation efficiency measurements

  • Structural analysis of protein variants

Research should consider molecular form-dependent patterns, as observed with other immune-related genes in A. gambiae .

What genomic and bioinformatic resources are available for eIF3-S9 research in Anopheles gambiae?

Researchers have access to several resources for eIF3-S9 studies:

These resources allow for comprehensive analysis of eIF3-S9 sequence, structure, and function across different mosquito populations .

What protocols are recommended for expression and purification of recombinant eIF3-S9?

For optimal expression and purification of recombinant eIF3-S9:

• Expression system selection:

  • E. coli: Use BL21(DE3) with pET vector systems for high yield

  • Baculovirus: Prefer for more complex proteins requiring post-translational modifications

  • Mammalian: Consider for functional studies requiring authentic folding

• Purification strategy:

  • Affinity chromatography using His-tag or GST-tag

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing

• Quality control:

  • SDS-PAGE to confirm >85% purity

  • Western blot for identity confirmation

  • Mass spectrometry for accurate molecular weight determination

  • Activity assays to confirm functionality

• Storage recommendations:

  • Store in small aliquots at -80°C

  • Include 10-20% glycerol in storage buffer

  • Avoid repeated freeze-thaw cycles

Researchers should optimize these protocols based on specific experimental requirements and downstream applications .

How can researchers integrate eIF3-S9 studies with broader investigations of Anopheles gambiae biology?

To integrate eIF3-S9 research with broader A. gambiae biology:

• Developmental studies: Investigate eIF3-S9 expression across life stages using techniques from the Methods in Anopheles Research manual

• Insecticide resistance: Study potential translational regulation of insecticide resistance genes by eIF3-S9

• Reproductive biology: Examine eIF3-S9 roles in gametogenesis and early embryonic development

• Environmental adaptation: Investigate how environmental factors modulate eIF3-S9 activity

• Population genetics: Incorporate eIF3-S9 polymorphism analysis in population genomics studies

• Field applications: Develop molecular tools to monitor eIF3-S9 variants in natural populations

Researchers should employ standardized protocols for mosquito rearing, molecular analysis, and field collection to ensure comparability with existing research in vector biology .

What are the potential applications of eIF3-S9 research in vector control strategies?

Emerging applications of eIF3-S9 research include:

• Novel insecticide target development: If eIF3-S9 has mosquito-specific features, it could represent a selective target for new control agents

• Genetic modification approaches: eIF3-S9 could be targeted in gene drive systems to reduce vector competence

• Resistance monitoring: eIF3-S9 variants might influence resistance to existing control methods

• Biomarker development: eIF3-S9 expression patterns could serve as biomarkers for physiological states relevant to vectorial capacity

• Translational inhibitors: Compounds that specifically disrupt A. gambiae eIF3-S9 function could offer new vector control tools

These applications require detailed understanding of eIF3-S9 structure, function, and variability across Anopheles populations .

How does eIF3-S9 research contribute to our understanding of mosquito-parasite interactions?

eIF3-S9 research provides insights into mosquito-parasite interactions through:

• Selective translation regulation: eIF3-S9 may control translation of specific mRNAs during infection

• Immune response modulation: Translation initiation factors can influence immune signaling pathways similar to those containing SNPs associated with Plasmodium infection

• Metabolic adaptation: eIF3-S9 may regulate translation of metabolic enzymes that create favorable or unfavorable environments for parasite development

• Cellular stress response: Parasite infection induces stress responses that affect translation initiation

• Evolutionary implications: Comparing eIF3-S9 across vector and non-vector species may reveal adaptations relevant to vectorial capacity

These research directions complement existing studies on immune signaling genes associated with natural Plasmodium infection in A. gambiae .

What are the key considerations for designing comprehensive eIF3-S9 research projects?

When designing comprehensive eIF3-S9 research:

• Integrate multiple approaches: Combine molecular, biochemical, and genomic techniques for robust results

• Consider physiological context: Study eIF3-S9 in relevant tissues (midgut, fat body, salivary glands) and developmental stages

• Account for environmental factors: Include temperature, microbiome, and other variables that influence vector biology

• Incorporate population diversity: Sample across different A. gambiae molecular forms and geographical regions

• Validate with functional assays: Move beyond correlative studies to establish causative relationships

• Standardize methodologies: Follow established protocols from resources like the Methods in Anopheles Research manual

• Collaborate across disciplines: Engage experts in translation, vector biology, and parasite-host interactions