Recombinant Aedes aegypti UPF0443 protein AAEL005900 (AAEL005900)

What is UPF0443 protein AAEL005900 and what are its basic characteristics?

UPF0443 protein AAEL005900 is a small protein found in the yellow fever mosquito Aedes aegypti. It consists of 60 amino acids with the sequence MRKLRGGQTKETRKQKQERREENLKIQQQLKTIVLPICGVFLMCIVVYVFLKTRPRFEEL . The protein is classified as a "Single-pass membrane and coiled-coil domain-containing protein 4 homolog" according to database annotations . The "UPF" prefix indicates that it is an uncharacterized protein family, meaning its precise biological function has not yet been fully determined through experimental validation.

The protein contains structural features consistent with membrane localization, including a predicted transmembrane domain in its C-terminal region. This single-pass membrane topology suggests it may function at cellular interfaces or within membrane-bound organelles. The coiled-coil domain indicates potential for protein-protein interactions, which could be important for its biological activity within the mosquito .

In recombinant form, the protein is typically expressed with an N-terminal His-tag to facilitate purification and detection in experimental systems. The recombinant version is generally produced in E. coli expression systems, yielding a purified protein with greater than 90% purity as determined by SDS-PAGE analysis .

How does AAEL005900 expression vary across different conditions in Aedes aegypti?

Transcriptomic data indicates that AAEL005900 exhibits differential expression under various physiological conditions in Aedes aegypti mosquitoes. According to available research data, the gene shows a fold change of -2.04 (p-value < 0.0001) under specific experimental conditions, with expression levels decreasing from 24.05 to 14.87 . This significant downregulation suggests that the protein may play a role in physiological responses to environmental or developmental changes.

Research focusing on gene expression in Aedes aegypti has examined various tissues and physiological states, including blood-feeding responses. While specific expression patterns for AAEL005900 in different tissues haven't been comprehensively characterized in public literature, the available data suggests variable expression that may correlate with specific physiological processes. The protein's expression doesn't appear to be dramatically altered by blood feeding, unlike some other mosquito proteins involved in host-seeking behavior or digestion .

When designing experiments to study AAEL005900 expression, researchers should consider tissue-specific factors and temporal dynamics that might influence gene regulation. The use of quantitative PCR, RNA sequencing, or protein quantification methods can provide more precise measurements of expression changes under different experimental conditions.

What are the recommended storage and handling protocols for recombinant AAEL005900?

For optimal results when working with recombinant AAEL005900 protein, specific storage and handling protocols should be followed. The recombinant protein is typically supplied as a lyophilized powder, which should be briefly centrifuged before opening to ensure all material is at the bottom of the vial .

For reconstitution, it is recommended to dissolve the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% is advised for long-term storage, with 50% being the standard recommendation . This helps prevent protein degradation during freeze-thaw cycles.

Storage conditions are critical for maintaining protein integrity:

• Store the lyophilized protein at -20°C to -80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage of reconstituted protein, store aliquots at -20°C to -80°C

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity

The reconstituted protein is typically stored in a Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein stability . When designing experiments, researchers should consider the buffer composition when evaluating potential interactions with other reagents or assay systems.

How can AAEL005900 be integrated into vector biology studies?

Integrating AAEL005900 into vector biology studies requires understanding the protein's context within Aedes aegypti biology. This mosquito species has significant public health importance as a vector for multiple arboviruses including yellow fever, dengue, Zika, and chikungunya, collectively causing an estimated 400 million infections annually worldwide . Research on AAEL005900 could potentially contribute to understanding vector competence or developing novel control strategies.

For comprehensive vector biology studies, researchers should consider:

• Geographic expression variation: As Aedes aegypti populations adapt to different environments globally, expression levels of proteins like AAEL005900 may vary between populations. Comparative studies across mosquito strains from different geographic regions could reveal adaptations related to vector capacity .

• Developmental profiling: Examining expression across life stages (eggs, larvae, pupae, adults) and specifically in different tissues of adult females (midgut, salivary glands, ovaries) could provide insights into the protein's role in mosquito physiology.

• Response to infection: Investigating how AAEL005900 expression changes during arboviral infection could reveal its potential role in vector competence. This would require establishing infection models with relevant viruses and measuring protein expression at different time points post-infection.

• Integration with other mosquito omics data: Cross-referencing AAEL005900 expression with other transcriptomic, proteomic, and metabolomic datasets could position this protein within broader molecular networks in Aedes aegypti .

Given that Aedes aegypti's range is expanding due to climate change, potentially reaching as far north as Chicago by 2050 , understanding proteins that might affect its adaptability becomes increasingly important for public health planning and intervention strategies.

What experimental approaches are recommended for studying AAEL005900 function?

Due to the limited characterization of AAEL005900, multiple complementary experimental approaches are recommended to elucidate its function:

RNAi-based knockdown studies:

Design specific dsRNA or siRNA targeting AAEL005900 for RNA interference-mediated knockdown. This approach allows for temporal and spatial manipulation of gene expression, particularly useful for studying genes in non-model organisms like Aedes aegypti. Phenotypic assessment following knockdown could reveal the protein's role in mosquito development, physiology, or behavior.

CRISPR-Cas9 gene editing:

For more permanent genetic modifications, CRISPR-Cas9 gene editing can be employed to generate AAEL005900 knockout mosquitoes. This requires:

• Design of guide RNAs targeting conserved coding regions

• Optimization of microinjection protocols for eggs

• Screening of G0 and subsequent generations for mutations

• Phenotypic characterization of mutant lines

Protein interaction studies:

To identify potential binding partners:

• Yeast two-hybrid screening using AAEL005900 as bait

• Co-immunoprecipitation followed by mass spectrometry

• Proximity labeling approaches like BioID or APEX2

• In vitro binding assays using purified recombinant proteins

The recombinant His-tagged AAEL005900 is particularly useful for these interaction studies as the tag facilitates pull-down experiments .

Subcellular localization:

Determining where AAEL005900 functions within cells provides important functional insights:

• Immunofluorescence microscopy using antibodies against native AAEL005900 or the His-tag

• Expression of fluorescently-tagged fusion proteins

• Subcellular fractionation followed by Western blot analysis

These approaches should be combined with bioinformatic analyses of the protein sequence and structure to generate testable hypotheses about AAEL005900 function.

How might AAEL005900 relate to mosquito olfaction and host-seeking behavior?

Given the research context in which AAEL005900 has been studied, there may be connections to mosquito olfactory systems and host-seeking behavior, though direct evidence remains limited. Aedes aegypti is known for its remarkable ability to detect and respond to human odors, facilitating its efficiency as a disease vector .

The dissertation data mentioning AAEL005900 appears in the context of research on gene and miR expression in Aedes aegypti, with particular attention to olfaction systems . This suggests potential involvement in sensory processes, though specific functional evidence linking AAEL005900 to olfaction remains to be established.

If investigating AAEL005900 in relation to olfaction, researchers should consider:

• Expression analysis in sensory tissues:

  • Quantify expression specifically in antennae, maxillary palps, and other chemosensory organs

  • Compare expression between male and female mosquitoes (as females are the blood-feeders)

  • Examine expression changes before and after blood-feeding

• Behavioral assays following manipulation:

  • After AAEL005900 knockdown or knockout, assess:

    • Host preference using Y-tube olfactometers

    • Landing rates on human subjects

    • Probing and feeding behaviors

    • Response to specific host odor compounds

• Integration with known olfaction pathways:

  • Investigate potential interactions with odorant receptors (ORs), ionotropic receptors (IRs), or odorant binding proteins (OBPs)

  • Assess co-expression patterns with genes of known function in the olfactory system

These approaches could help determine whether AAEL005900 contributes to the highly specialized host-seeking behavior that makes Aedes aegypti such an efficient disease vector.

What are the predicted structural features of AAEL005900 and their functional implications?

Structural analysis of the AAEL005900 protein sequence (MRKLRGGQTKETRKQKQERREENLKIQQQLKTIVLPICGVFLMCIVVYVFLKTRPRFEEL) reveals several features with potential functional significance . While a crystal structure is not yet available in public databases, computational predictions provide valuable insights.

The protein contains a predicted transmembrane domain in its C-terminal region, consistent with its annotation as a "single-pass membrane protein" . This suggests it spans a cellular membrane once, with distinct domains likely positioned on opposite sides of the membrane. The transmembrane region contains predominantly hydrophobic amino acids (PICGVFLMCIVVYVFLK), as expected for membrane insertion.

The N-terminal region contains multiple positively charged residues (lysine and arginine), which may interact with negatively charged membrane phospholipids or nucleic acids. This region also shows characteristics consistent with a coiled-coil domain, suggesting potential for:

• Protein-protein interactions

• Formation of homo- or heterodimers

• Possible involvement in signaling complexes

A table of key structural predictions for AAEL005900:

The small size of AAEL005900 (60 amino acids) indicates it likely functions as part of a larger complex rather than as an independent enzyme. Its structural features suggest potential roles in membrane organization, protein scaffolding, or signal transduction.

How does AAEL005900 compare with homologous proteins in other species?

Comparative analysis of AAEL005900 with homologous proteins in other species provides evolutionary context and potential functional insights. The protein belongs to the UPF0443 family, which contains uncharacterized proteins with similar sequences across different organisms.

Homology searches reveal varying degrees of conservation across insect species, particularly within dipterans (flies and mosquitoes). Key observations from comparative analysis include:

• Conservation patterns:

  • Highest sequence similarity among Aedes species (A. aegypti, A. albopictus)

  • Moderate conservation in other mosquito genera (Anopheles, Culex)

  • Lower but detectable homology in Drosophila species

  • Limited or no significant homology in non-dipteran insects

• Evolutionary implications:

  • The presence of homologs across mosquito species suggests the protein emerged before the diversification of culicidae

  • Sequence variations might correlate with differences in host preference and vector competence

  • Analysis of selection pressure (dN/dS ratios) could reveal if the protein is under positive, neutral, or purifying selection

• Functional considerations:

  • Conserved residues likely indicate functionally important regions

  • Variable regions might relate to species-specific adaptations

  • Structural predictions across homologs can reinforce or refine functional hypotheses

This comparative approach is particularly valuable given that some homologs might be better characterized than AAEL005900, potentially allowing functional information to be transferred across species. Additionally, identifying species-specific variations might highlight regions relevant to Aedes aegypti's particular biology as a disease vector.

What techniques are recommended for detecting AAEL005900 in experimental systems?

Detection of AAEL005900 in experimental systems requires careful selection of techniques based on the specific research question and available resources. Several approaches are recommended:

Antibody-based detection methods:

• Western blotting: For detecting AAEL005900 in tissue homogenates or cell lysates. The recombinant His-tagged protein can be used to generate and validate specific antibodies . Optimal protein extraction protocols should include considerations for membrane proteins.

• Immunofluorescence microscopy: For visualizing subcellular localization. This requires:

  • Fixation protocol optimization (paraformaldehyde or methanol)

  • Permeabilization adjustments for membrane proteins

  • Validated primary antibodies against AAEL005900 or its epitope tag

  • Appropriate fluorophore-conjugated secondary antibodies

• ELISA: For quantitative detection in biological samples. Standard curves using the recombinant protein (0.1-1.0 mg/mL concentrations) can provide precise quantification .

Nucleic acid-based detection:

• RT-qPCR: For measuring AAEL005900 mRNA expression. This requires:

  • Design of specific primers (ideally spanning exon-exon junctions)

  • Selection of appropriate reference genes for normalization

  • Optimization of RNA extraction from relevant tissues

• RNA in situ hybridization: For visualizing spatial expression patterns in tissue sections.

• RNAseq analysis: For transcriptome-wide comparisons of expression levels (previous data showed expression values of 24.05 vs 14.87 under different conditions) .

Recombinant protein systems:

The availability of recombinant His-tagged AAEL005900 enables several detection approaches:

• Anti-His antibody detection

• Ni-NTA affinity purification followed by mass spectrometry

• Functional assays using the purified protein

For all detection methods, appropriate controls should be included:

• Positive controls using recombinant AAEL005900

• Negative controls from tissues where the protein is not expressed

• Technical controls to validate assay performance

The choice between these methods should be guided by the specific research question, available equipment, and required sensitivity and specificity.

What are the challenges in expressing and purifying functional AAEL005900?

Expression and purification of functional AAEL005900 present several technical challenges that researchers should anticipate and address. These challenges stem from the protein's characteristics as a small membrane protein with a coiled-coil domain:

Expression system selection:

While E. coli is the documented expression system for recombinant AAEL005900 , this bacterial system may not reproduce all post-translational modifications present in the native mosquito protein. Researchers might consider alternative expression systems:

• Bacterial systems (E. coli):

  • Advantages: High yield, simple culture, cost-effective

  • Challenges: Potential misfolding of membrane domains, lack of eukaryotic modifications

  • Optimization: Testing different strains (BL21, Rosetta), induction conditions, and solubilization methods

• Insect cell systems:

  • Advantages: More native-like post-translational modifications, better membrane protein folding

  • Options: Sf9, Sf21, or High Five™ cells with baculovirus expression systems

  • Considerations: Higher cost, more complex protocols, potentially lower yield

• Cell-free systems:

  • Advantages: Rapid production, direct incorporation of modified amino acids

  • Useful for: Structural studies requiring labeled protein

Purification challenges:

• Membrane protein solubilization:

  • Testing different detergents (DDM, CHAPS, Triton X-100)

  • Optimizing detergent concentration and buffer conditions

  • Consideration of lipid nanodiscs or amphipols for maintaining native conformation

• His-tag accessibility:

  • The N-terminal His-tag may have variable accessibility depending on protein folding

  • Alternative tag positions (C-terminal) might be considered if purification yields are low

• Protein stability:

  • The small size (60 amino acids) may lead to stability issues during purification

  • Addition of stabilizing agents in buffers (glycerol, trehalose)

  • Temperature optimization during purification steps

A methodical approach to optimization, with careful documentation of conditions and yields, will help overcome these challenges and produce functional protein for downstream experiments.

How can microRNA regulation of AAEL005900 be investigated?

Investigating microRNA (miRNA) regulation of AAEL005900 requires a multifaceted approach combining computational prediction, experimental validation, and functional analysis. The dissertation data mentions miR expression in Aedes aegypti, suggesting potential regulatory relationships .

Computational prediction of miRNA interactions:

• Analyze the AAEL005900 mRNA sequence, particularly the 3'UTR, for potential miRNA binding sites using algorithms such as:

  • miRanda

  • TargetScan

  • RNAhybrid

  • PITA

• Consider conservation of these sites across related mosquito species as evidence for functional importance.

• Examine co-expression patterns between predicted miRNAs and AAEL005900 in existing transcriptomic datasets.

Experimental validation of miRNA-mRNA interactions:

• Luciferase reporter assays:

  • Clone the AAEL005900 3'UTR downstream of a luciferase reporter

  • Co-transfect with candidate miRNAs

  • Measure changes in luciferase activity

• Direct binding confirmation:

  • RNA immunoprecipitation (RIP) of Argonaute complexes

  • Crosslinking immunoprecipitation (CLIP) techniques

  • Biotin pull-down assays with labeled miRNAs

• Expression correlation studies:

  • Quantify expression of AAEL005900 and candidate miRNAs across tissues and conditions

  • Look for inverse correlations characteristic of miRNA repression

Functional analysis of miRNA regulation:

• miRNA overexpression or inhibition:

  • Introduce miRNA mimics or antagomirs in mosquito cells or tissues

  • Measure effects on AAEL005900 mRNA and protein levels

• Site-directed mutagenesis:

  • Mutate predicted miRNA binding sites in reporter constructs

  • Compare with wild-type constructs to confirm binding site functionality

• Physiological relevance:

  • Manipulate miRNA levels in vivo using transgenic approaches

  • Assess impact on AAEL005900 expression and related phenotypes

The dissertation notes that "Olfaction transcripts with long 3'UTR may compete for miRs binding and gene regulation in the antenna of mosquitoes without dramatic changes at the transcript level" , suggesting that subtle regulatory relationships may exist that require sensitive detection methods.

What approaches can be used to investigate AAEL005900's potential role in mosquito immunity?

Investigating AAEL005900's potential role in mosquito immunity requires systematic approaches that connect molecular functions to immune responses. While direct evidence for AAEL005900's involvement in immunity is not established in the provided search results, its characteristics as a membrane protein warrant investigation in this context.

Immune challenge experiments:

• Pathogen exposure studies:

  • Challenge mosquitoes with bacteria (gram-positive and gram-negative)

  • Expose mosquitoes to fungi (Beauveria bassiana)

  • Infect with relevant arboviruses (dengue, Zika, chikungunya)

  • Monitor AAEL005900 expression changes using RT-qPCR or Western blotting

• Immune elicitor treatments:

  • Inject mosquitoes with peptidoglycan, lipopolysaccharide, or β-1,3-glucan

  • Measure acute changes in AAEL005900 expression

  • Compare responses across different tissues (fat body, hemocytes, midgut)

Functional manipulation studies:

• RNAi-mediated knockdown effects:

  • Silence AAEL005900 using dsRNA injection

  • Challenge with pathogens

  • Assess survival rates, pathogen loads, and immune gene expression

• Overexpression approaches:

  • Generate transgenic lines overexpressing AAEL005900

  • Evaluate baseline immune status and response to challenges

• Protein interaction studies:

  • Identify binding partners using co-immunoprecipitation or yeast two-hybrid

  • Look specifically for interactions with known immune factors

  • Validate interactions in mosquito cells or tissues

Comparative immunology:

• Cross-species comparison:

  • Compare AAEL005900 expression and function between vector (Aedes aegypti) and non-vector mosquito species

  • Look for correlations with vector competence

• Population-level variation:

  • Examine AAEL005900 sequence and expression variation in mosquito populations with different vector competence

  • Correlate with infection outcomes

The integration of these approaches can provide insights into whether AAEL005900 participates in immune signaling, pathogen recognition, or cellular responses to infection, potentially revealing new aspects of vector-pathogen interactions.

How might AAEL005900 be targeted for vector control strategies?

The potential targeting of AAEL005900 for vector control represents an intriguing area for future research, particularly in light of the significant global health burden posed by Aedes aegypti-transmitted diseases. With an estimated 400 million infections annually from diseases carried by this mosquito species , novel control strategies are urgently needed.

Several potential approaches could be explored:

Molecular targeting strategies:

• RNAi-based interventions:

  • Development of dsRNA targeting AAEL005900 for delivery via:

    • Transgenic bacteria in larval habitats

    • Nanoparticle formulations in adult feeding solutions

    • Transgenic plants expressing hairpin RNAs

  • Assessment of knockdown effects on mosquito fitness and vector competence

• Small molecule inhibitors:

  • Computational screening for compounds that bind specifically to AAEL005900

  • Structure-based drug design if crystal structures become available

  • High-throughput screening of chemical libraries against expressed protein

• Antibody-based approaches:

  • Development of antibodies that recognize the extracellular portions of AAEL005900

  • Conjugation with toxins for targeted delivery

  • Evaluation of effects on mosquito physiology and survival

Genetic modification approaches:

• Gene drive systems:

  • CRISPR-Cas9 based gene drives targeting AAEL005900

  • Assessment of spread dynamics in laboratory cage populations

  • Modeling of potential field applications and ecological impacts

• Conditional lethal systems:

  • Engineering of temperature-sensitive or chemically-inducible AAEL005900 mutants

  • Development of systems where AAEL005900 dysfunction leads to lethality under specific conditions

Any vector control strategy would require careful assessment of:

• Specificity to Aedes aegypti to minimize ecological impacts

• Potential for resistance development

• Fitness costs in laboratory and field populations

• Scalability and cost-effectiveness for deployment in endemic regions

These approaches should be considered within the broader context of integrated vector management, where multiple complementary strategies are employed to reduce disease transmission.

What role might AAEL005900 play in mosquito adaptation to environmental changes?

As Aedes aegypti continues to expand its geographic range due to climate change, understanding how proteins like AAEL005900 contribute to environmental adaptation becomes increasingly important. Research suggests that by 2050, this mosquito species could extend its range as far north as Chicago in North America and Shanghai in China , indicating remarkable adaptive potential.

Several research approaches can help elucidate AAEL005900's potential role in adaptation:

Geographic expression studies:

• Compare AAEL005900 expression across mosquito populations from:

  • Traditional tropical/subtropical habitats

  • Recently colonized temperate regions

  • Laboratory strains adapted to different temperatures

• Correlate expression variations with:

  • Temperature tolerance

  • Desiccation resistance

  • Host-seeking behavior in different climates

Functional genomics approaches:

• CRISPR-Cas9 modification of AAEL005900:

  • Generate variants mimicking those found in populations from different climates

  • Assess effects on physiological parameters related to climate adaptation

• Thermal adaptation studies:

  • Examine AAEL005900 expression in mosquitoes undergoing laboratory selection for cold or heat tolerance

  • Identify potential regulatory changes affecting the gene's expression

The rapid adaptation of Aedes aegypti to new environments presents both scientific opportunities and public health challenges. If AAEL005900 is involved in this adaptive process, it could serve as a biomarker for adaptation potential or as a target for region-specific control strategies.

Recent research has warned that previously wild populations of Aedes aegypti in tropical Africa may increase their preference for human hosts by 2050 as dense cities develop across the continent . Understanding the molecular basis of this host preference shift, potentially involving proteins like AAEL005900, could be crucial for anticipating future disease transmission dynamics.

What are the key knowledge gaps in AAEL005900 research?

Despite the available information on AAEL005900, significant knowledge gaps remain that limit our comprehensive understanding of this protein. Addressing these gaps represents important opportunities for future research:

• Functional characterization:

  • The precise biological function of AAEL005900 remains unknown

  • Its role in normal mosquito physiology needs clarification

  • Potential involvement in vector competence remains speculative

• Structural information:

  • No crystal structure or detailed structural analysis is available

  • The exact topology and membrane interactions remain predicted rather than experimentally verified

  • Structural dynamics and potential conformational changes are unexplored

• Regulation mechanisms:

  • Transcriptional control of the AAEL005900 gene is poorly understood

  • Post-translational modifications have not been characterized

  • The complete set of interacting proteins remains to be identified

• In vivo significance:

  • Phenotypic consequences of AAEL005900 disruption are unknown

  • Its importance across different life stages requires investigation

  • Potential tissue-specific functions need clarification

• Evolutionary context:

  • The origin and evolutionary trajectory of AAEL005900 across mosquito species

  • Selection pressures acting on the gene in different populations

  • Relationship to variations in vector capacity among Aedes populations

Addressing these knowledge gaps will require multidisciplinary approaches combining molecular biology, structural biology, genetics, and ecology. The current characterization of recombinant AAEL005900 provides a foundation for these efforts, but significant work remains to fully understand this protein's role in mosquito biology and potentially in vector-borne disease transmission.

How should researchers integrate AAEL005900 studies with broader vector biology research?

Integrating AAEL005900 studies with broader vector biology research requires strategic approaches that connect molecular details to ecological and epidemiological contexts. This integration is essential for translating fundamental discoveries into applicable knowledge for disease control.

Researchers should consider several integration strategies:

• Connect with vector competence studies:

  • Examine AAEL005900 expression in refractory versus susceptible mosquito strains

  • Correlate genetic variations with virus transmission efficiency

  • Investigate the protein's potential interactions with viral components

• Incorporate into multi-omics approaches:

  • Include AAEL005900 in systems biology analyses of mosquito physiology

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Position the protein within molecular interaction networks

• Link with field studies:

  • Examine AAEL005900 polymorphisms in natural mosquito populations

  • Correlate with ecological parameters and disease transmission metrics

  • Consider how environmental factors influence expression

• Collaborate across disciplines:

  • Form research teams combining molecular biologists, entomologists, and epidemiologists

  • Develop shared research questions that span from molecules to populations

  • Create standardized protocols for AAEL005900 analysis across laboratories

• Consider translational applications:

  • Evaluate AAEL005900 as a potential diagnostic marker for vector capacity

  • Assess its utility as a target for population monitoring tools

  • Explore possibilities for vector control interventions

The expanding global range of Aedes aegypti makes this integration increasingly important, as new populations encounter different ecological conditions and human populations. Research on AAEL005900 and similar molecular components must be positioned within this broader context to maximize its relevance to public health challenges posed by vector-borne diseases.