Recombinant Culex quinquefasciatus Adenosine monophosphate-protein transferase FICD homolog (CPIJ001789)

What is the taxonomic classification of Culex quinquefasciatus and why is it significant for FICD homolog research?

Culex quinquefasciatus (Say, 1823), commonly known as the southern house mosquito, belongs to the Culex pipiens species complex. Its taxonomic classification is as follows: Domain Eukaryota, Kingdom Animalia, Phylum Arthropoda, Class Insecta, Order Diptera, Family Culicidae, Genus Culex, Species C. quinquefasciatus . The significance of this mosquito for FICD homolog research lies in its role as a major disease vector for filariasis, avian malaria, and various arboviruses including West Nile virus, Zika virus, and St. Louis encephalitis . Understanding the function of proteins like FICD homolog in this species can provide insights into vector-pathogen interactions and potential targets for vector control strategies.

How does the FICD homolog in Culex quinquefasciatus compare structurally with FICD proteins in other insect species?

The Adenosine monophosphate-protein transferase FICD homolog in Culex quinquefasciatus shares several conserved domains with FICD proteins from other insect species such as Drosophila simulans (DsimGD23409) and Drosophila grimshawi (DgriGH10751) . All these proteins contain the characteristic FIC domain (Filamentation induced by cAMP) and retain the enzymatic function that typically includes AMPylation activity.

When conducting comparative structural analysis, researchers should focus on:

• Amino acid sequence alignment using tools like CLUSTAL W to identify conserved regions

• Domain prediction using databases such as PFAM or InterPro

• Tertiary structure modeling using homology modeling based on solved crystal structures

• Analysis of catalytic residues in the active site that are essential for AMPylation activity

A typical sequence identity matrix comparing FICD homologs across species would appear as follows:

Note: Percentage ranges are estimated based on typical conservation patterns among related species.

What expression systems are most effective for producing recombinant Culex quinquefasciatus FICD homolog?

The selection of an appropriate expression system for Culex quinquefasciatus FICD homolog depends on research objectives and downstream applications. Multiple expression platforms can be utilized with varying advantages:

Bacterial Expression (E. coli):
For basic structure-function studies, E. coli remains the most accessible and cost-effective system . Recommended strains include BL21(DE3) for general expression or Rosetta(DE3) for codon optimization of insect proteins. The protocol should incorporate:

• Cloning the CPIJ001789 gene into pET vectors (pET-28a or pET-SUMO for improved solubility)

• Induction with 0.1-0.5mM IPTG at lower temperatures (16-20°C) to enhance proper folding

• Lysis in buffer containing 50mM Tris-HCl (pH 8.0), 300mM NaCl, 10% glycerol, 1mM DTT, and protease inhibitors

Baculovirus-Insect Cell System:
For applications requiring post-translational modifications, Sf9 or High Five insect cells provide a more native environment for mosquito protein expression . This system typically yields:

• Improved protein solubility compared to bacterial systems

• Proper disulfide bond formation

• Appropriate glycosylation patterns when present

Yeast Expression (P. pastoris):
Offers a balance between bacterial simplicity and eukaryotic processing capability . The methanol-inducible AOX1 promoter system in P. pastoris provides:

• Controlled induction

• High cell density cultures

• Secretion options for simplified purification

What are the optimal purification strategies for maintaining the enzymatic activity of recombinant CPIJ001789?

Purification of recombinant CPIJ001789 requires careful consideration of buffer conditions and chromatography techniques to preserve enzymatic activity. A multi-step purification approach is recommended:

Initial Capture:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Buffer composition: 50mM Tris-HCl pH 8.0, 300mM NaCl, 10% glycerol, 5-20mM imidazole for binding, 250-300mM imidazole for elution

• Addition of 1mM DTT or 5mM β-mercaptoethanol helps maintain reduced cysteines

Intermediate Purification:

• Ion Exchange Chromatography (IEX) using Q-Sepharose or SP-Sepharose depending on the protein's theoretical pI

• Buffer optimization: lower salt concentration (50mM NaCl) for binding, gradient to 500mM NaCl for elution

Polishing Step:

• Size Exclusion Chromatography (SEC) using Superdex 200 column

• Running buffer containing 25mM HEPES pH 7.5, 150mM NaCl, 5% glycerol, 1mM DTT

Activity preservation considerations:

• Include 1-5mM MgCl₂ in all buffers as FICD proteins typically require divalent cations for activity

• Add 10% glycerol to prevent aggregation and stabilize the protein

• Store purified protein at concentrations >1mg/ml to prevent surface adsorption

• Flash-freeze aliquots in liquid nitrogen for long-term storage at -80°C

How can researchers accurately assess the AMPylation activity of Culex quinquefasciatus FICD homolog?

AMPylation activity assessment for the Culex quinquefasciatus FICD homolog requires specialized assays that detect the transfer of AMP to target proteins. Several complementary approaches are recommended:

Radiometric Assay:
The gold standard for quantitative analysis involves using radiolabeled ATP (α-³²P-ATP):

• Incubate purified FICD homolog (0.5-1μM) with potential substrate proteins (2-5μM)

• Reaction buffer: 50mM HEPES pH 7.5, 150mM NaCl, 5mM MgCl₂, 0.1mM ATP, 1μCi α-³²P-ATP

• Incubate at 30°C for 30-60 minutes

• Resolve proteins by SDS-PAGE, dry the gel, and expose to phosphorscreen

• Quantify incorporated radioactivity using phosphorimager analysis

Fluorescence-based Assay:
A non-radioactive alternative using fluorescent ATP analogs:

• Utilize ATP-γ-AmNS (aminonaphthalene sulfonate) or similar fluorescent ATP analogs

• Monitor fluorescence changes (excitation: 330nm, emission: 460nm) upon AMP transfer

• Calculate enzyme kinetics parameters (Km, Vmax) using varying substrate concentrations

Mass Spectrometry Detection:
For identification of specific AMPylation sites:

• Perform in vitro AMPylation reaction with unlabeled ATP

• Digest products with trypsin or other suitable proteases

• Analyze by LC-MS/MS to detect the characteristic +329 Da mass shift

• Confirm AMPylation sites by targeted MS/MS fragmentation patterns

What experimental approaches can identify the physiological substrates of CPIJ001789 in Culex quinquefasciatus?

Identifying the physiological substrates of CPIJ001789 requires integration of multiple experimental approaches:

Affinity Purification-Mass Spectrometry (AP-MS):

• Express epitope-tagged CPIJ001789 (FLAG, HA, or biotin) in mosquito cells

• Perform crosslinking with formaldehyde or DSS to capture transient interactions

• Immunoprecipitate the protein complex using appropriate antibodies or affinity resins

• Identify co-purifying proteins by LC-MS/MS analysis

• Validate interactions using reciprocal pulldowns and co-immunoprecipitation

Proximity-dependent Biotin Identification (BioID):

• Generate a fusion protein of CPIJ001789 with BirA* biotin ligase

• Express in mosquito cells and supply biotin to the medium

• Harvest cells and purify biotinylated proteins using streptavidin beads

• Identify proteins by mass spectrometry

• Create interaction network maps based on identified proteins

In vitro AMPylation Screening:

• Prepare mosquito cell lysates or recombinant protein libraries

• Perform AMPylation reactions with purified CPIJ001789 and α-³²P-ATP

• Resolve by 2D gel electrophoresis

• Identify labeled proteins by mass spectrometry

• Confirm direct AMPylation using purified candidate proteins

Typical substrate identification results may be presented as follows:

How is CPIJ001789 expression related to insecticide resistance mechanisms in Culex quinquefasciatus?

The potential relationship between CPIJ001789 expression and insecticide resistance in Culex quinquefasciatus can be understood by examining mechanisms observed in related proteins. Studies on aldehyde oxidase in insecticide-resistant strains of C. quinquefasciatus have shown gene amplification and elevated enzyme activity , suggesting similar mechanisms may apply to other proteins involved in xenobiotic metabolism.

To investigate CPIJ001789's role in resistance, researchers should:

• Compare CPIJ001789 expression levels between susceptible and resistant mosquito strains using:

  • qRT-PCR for transcript quantification

  • Western blotting for protein levels

  • Activity assays for functional enzyme levels

• Analyze genomic copy number variations:

  • Quantitative PCR to determine if gene amplification occurs

  • Fluorescence in situ hybridization (FISH) to visualize amplicons

  • Whole genome sequencing to identify structural variations

• Investigate regulatory mechanisms:

  • Promoter sequence analysis for mutations affecting transcription

  • ChIP-seq to identify transcription factors regulating expression

  • Reporter assays to measure promoter activity differences

Research on aldehyde oxidase in C. quinquefasciatus found that the resistant strain contained an amplified AO gene on a 30-kb DNA amplicon alongside resistance-associated esterases, resulting in elevated AO activity across all life stages, with highest activity in 3rd instar larvae . This provides a model for studying CPIJ001789, as AMPylation may similarly modulate proteins involved in detoxification pathways.

What methodologies are most effective for determining if CPIJ001789 directly modulates insecticide metabolism or clearance?

To establish whether CPIJ001789 directly affects insecticide metabolism or clearance, a multi-faceted approach is necessary:

In vitro Insecticide Modification Assays:

• Incubate purified CPIJ001789 with various insecticide classes (pyrethroids, organophosphates, carbamates)

• Use HPLC-MS to detect potential chemical modifications

• Quantify reaction rates and substrate specificity

• Compare activity against insecticides with activity of known detoxification enzymes

RNAi-mediated Knockdown Studies:

• Develop dsRNA targeting CPIJ001789

• Deliver to mosquitoes via microinjection or ingestion

• Confirm knockdown via qRT-PCR and Western blotting

• Assess insecticide susceptibility using WHO tube bioassays or CDC bottle bioassays

• Calculate LC50 values and compare to controls

CRISPR-Cas9 Gene Editing:

• Design guide RNAs targeting CPIJ001789

• Introduce CRISPR-Cas9 components into embryos

• Screen for successful mutants

• Characterize phenotypes related to insecticide tolerance

• Perform complementation studies with wild-type gene

Metabolomics Analysis:

• Expose wild-type and CPIJ001789-knockdown mosquitoes to sublethal insecticide doses

• Extract and analyze metabolites using LC-MS/MS

• Identify differences in insecticide metabolite profiles

• Map metabolic pathways affected by CPIJ001789 activity

Previous studies on AO in C. quinquefasciatus demonstrated partial inhibition of elevated AO activity by various insecticides, suggesting a role in insecticide resistance . Similar experimental designs could reveal whether CPIJ001789's AMPylation activity modifies proteins in detoxification pathways, potentially altering their function or stability in response to insecticide exposure.

What crystallization conditions are optimal for obtaining diffracting crystals of Culex quinquefasciatus FICD homolog?

Obtaining high-quality protein crystals of Culex quinquefasciatus FICD homolog for X-ray crystallography requires systematic screening and optimization. Based on crystallization studies of related proteins, the following approach is recommended:

Initial Preparation:

• Ensure protein purity >95% as assessed by SDS-PAGE and SEC-MALS

• Concentrate protein to 8-15 mg/ml in crystallization buffer (20mM HEPES pH 7.5, 150mM NaCl, 5% glycerol, 1mM DTT)

• Remove aggregates by centrifugation at 16,000×g for 10 minutes at 4°C

• Filter through 0.22μm filter immediately before setting up crystallization trials

Primary Screening:

• Employ commercial sparse matrix screens (Hampton Research Crystal Screen 1 & 2, Molecular Dimensions PACT premier)

• Use sitting drop vapor diffusion method with 1:1 ratio of protein:reservoir (0.5μl each)

• Incubate at both 4°C and 18°C

• Observe regularly for crystal formation over 2-4 weeks

Optimization Strategies:

• Fine-tune promising conditions by varying:

  • pH in 0.2 unit increments

  • Precipitant concentration in 2% increments

  • Protein concentration between 5-20 mg/ml

• Implement additive screening with Hampton Research Additive Screen

• Try seeding techniques from initial microcrystals

• Explore alternative crystallization methods (hanging drop, microbatch under oil)

Co-crystallization with Ligands:

• Include ATP or non-hydrolyzable analogs (AMPPNP, ATPγS) at 2-5mM

• Add 5mM MgCl₂ to stabilize nucleotide binding

• Pre-incubate protein with ligands for 30 minutes on ice before setting up trials

Based on successful crystallization of related FICD proteins, the following conditions often yield promising results:

How can molecular dynamics simulations provide insights into the catalytic mechanism of CPIJ001789?

Molecular dynamics (MD) simulations offer powerful insights into the atomic-level movements and interactions that govern CPIJ001789's catalytic mechanism. A comprehensive MD study should include:

System Preparation:

• Build an accurate homology model of CPIJ001789 using templates from related FICD proteins

• Include substrate ATP, cofactor Mg²⁺, and potential protein substrates

• Embed in explicit solvent (TIP3P water model) with physiological ion concentration

• Apply AMBER or CHARMM force fields optimized for protein-nucleotide interactions

• Perform energy minimization to resolve steric clashes

Simulation Protocol:

• Equilibration: gradually heat system to 300K over 1ns, maintaining constant pressure

• Production runs: minimum 100ns for basic dynamics, 1μs or longer for rare events

• Replicate simulations (at least 3) with different starting velocities

• Enhanced sampling techniques for exploring high-energy transitions:

  • Umbrella sampling for free energy profiles

  • Metadynamics for mapping reaction pathways

  • Accelerated MD for overcoming energy barriers

Analysis Focus Areas:

• Active site dynamics:

  • Monitor distances between catalytic residues and ATP

  • Track water molecule positions for potential nucleophilic attack

  • Analyze Mg²⁺ coordination geometry

• Conformational transitions:

  • Principal Component Analysis (PCA) to identify major motions

  • RMSD and RMSF calculations to quantify structural flexibility

  • Identify allosteric networks using dynamic cross-correlation matrices

• Substrate recognition:

  • Calculate binding free energies using MM/PBSA or MM/GBSA

  • Identify key residue interactions through hydrogen bond analysis

  • Map electrostatic complementarity between enzyme and substrate

Key findings from MD simulations typically reveal:

• Conformational changes associated with substrate binding

• Water-mediated interactions essential for catalysis

• Proton transfer pathways during the reaction

• Rate-limiting steps in the catalytic cycle

These insights can guide mutagenesis experiments to validate mechanistic hypotheses and inform protein engineering efforts to modify enzyme activity.

How has the FICD homolog evolved across mosquito vectors, and what are the implications for vector control strategies?

The evolutionary analysis of FICD homologs across mosquito vectors provides critical insights into protein conservation and potential vector control targets. A comprehensive evolutionary study should include:

Sequence Collection and Alignment:

• Extract FICD homolog sequences from:

  • Culex quinquefasciatus (southern house mosquito)

  • Aedes aegypti (yellow fever mosquito)

  • Anopheles gambiae (malaria mosquito)

  • Other relevant disease vectors

• Include outgroups from non-vector insects (Drosophila) and mammals

• Perform multiple sequence alignment using MUSCLE or MAFFT with iterative refinement

• Manually inspect alignments, especially in catalytic regions

Phylogenetic Analysis:

• Construct phylogenetic trees using:

  • Maximum Likelihood (RAxML or IQ-TREE)

  • Bayesian Inference (MrBayes)

  • Neighbor-Joining as a complementary approach

• Apply appropriate substitution models (LG+G+F, WAG+I+G)

• Assess node support through bootstrap replication (1000 replicates)

• Root trees with appropriate outgroups

Selection Pressure Analysis:

• Calculate dN/dS ratios across sequence alignments

• Identify sites under positive or purifying selection using PAML or HyPhy

• Map selection patterns onto protein structure

• Correlate evolutionary rates with functional domains

Implications for Vector Control:

• Identify highly conserved regions that may serve as broad-spectrum targets

• Evaluate species-specific variations that could affect inhibitor specificity

• Assess potential for resistance development based on genetic diversity

• Consider impact of targeting FICD function on vector fitness

Evolutionary patterns typical of FICD homologs include:

• High conservation of catalytic motifs across vector species

• Variable regulatory domains that may affect tissue-specific expression

• Evidence of occasional positive selection in regions interacting with pathogens

• Conservation of structural features despite sequence divergence

What bioinformatic approaches can predict the regulatory network controlling CPIJ001789 expression in different developmental stages and physiological conditions?

Predicting the regulatory network of CPIJ001789 requires integration of multiple bioinformatic approaches that span genomic, transcriptomic, and network analysis:

Promoter Analysis:

• Extract 2-3kb upstream region of CPIJ001789 from Culex quinquefasciatus genome

• Identify conserved transcription factor binding sites using:

  • JASPAR database with TFBS Scan

  • MEME Suite for de novo motif discovery

  • PhyloGibbs for phylogenetically conserved elements

• Compare with promoters of co-expressed genes for common regulatory elements

• Map enhancer elements using chromatin accessibility data (when available)

Expression Correlation Networks:

• Analyze RNA-seq data across developmental stages and tissues

• Build co-expression networks using WGCNA or similar tools

• Identify gene modules containing CPIJ001789

• Perform Gene Ontology enrichment of co-expressed genes

• Infer key transcription factors using master regulator analysis

Comparative Genomics Approach:

• Extract orthologous FICD promoters from related mosquito species

• Perform phylogenetic footprinting to identify conserved regulatory elements

• Map syntenic regions to identify potential distant regulatory elements

• Compare with expression patterns in related species

Integration with Physiological Data:

• Correlate expression with insecticide exposure responses

• Analyze patterns during immune challenges

• Examine regulation during blood feeding and reproduction

• Identify circadian expression patterns

A typical output of regulatory network analysis may include:

These predicted relationships can guide experimental validation through reporter assays, ChIP-qPCR, and functional studies of identified transcription factors.

How can CRISPR-Cas9 gene editing of CPIJ001789 be optimized for functional analysis in Culex quinquefasciatus?

Optimizing CRISPR-Cas9 gene editing of CPIJ001789 in Culex quinquefasciatus requires careful consideration of several technical aspects:

Guide RNA Design:

• Select target sites within early exons to ensure complete loss-of-function

• Use tools like CHOPCHOP or CRISPOR for guide design, considering:

  • On-target efficiency scores (>0.6 preferred)

  • Minimal off-target potential (<2 off-targets with >3 mismatches)

  • GC content between 40-60%

• Design multiple sgRNAs (at least 3-4) targeting different regions

• Include sgRNA targeting a visible marker gene (e.g., white, kmo) as control

Delivery Methods:

• Embryo microinjection:

  • Collect eggs within 1 hour of oviposition

  • Align along double-sided tape

  • Inject at posterior end with minimal volume (0.1-0.2nl)

  • Cas9 concentration: 300-500ng/μl

  • sgRNA concentration: 100-150ng/μl each

• RNP complex preparation:

  • Pre-assemble Cas9 protein with sgRNA (10:1 molar ratio)

  • Incubate at 37°C for 10 minutes before injection

  • Include tracrRNA if using crRNA system

Mutation Detection Strategies:

• Initial screening:

  • T7 Endonuclease I assay of PCR amplicons

  • Heteroduplex mobility assay

  • Direct Sanger sequencing with TIDE analysis

• Comprehensive characterization:

  • Next-generation sequencing of target region

  • Long-range PCR to detect larger indels

  • RT-PCR to assess transcript alterations

Generating Stable Lines:

• Cross G0 adults to wild-type

• Pool eggs from multiple crosses

• Screen F1 individuals by genotyping

• Establish homozygous lines through controlled crosses

• Confirm phenotype stability across generations

Functional Validation Approaches:

• Molecular characterization:

  • Verify protein loss by Western blotting

  • Assess transcript changes via qRT-PCR

  • Measure AMPylation activity in mutant tissues

• Phenotypic analysis:

  • Development rate and viability

  • Insecticide susceptibility

  • Reproductive capacity

  • Vector competence for pathogens

Success rates for CRISPR-Cas9 editing in Culex mosquitoes typically range from 5-30% of G0 individuals carrying mutations, with efficiency varying based on target site accessibility and technical parameters.

What implications does the AMPylation activity of CPIJ001789 have for designing novel mosquito control strategies?

The AMPylation activity of CPIJ001789 presents unique opportunities for developing targeted mosquito control strategies:

Small Molecule Inhibitor Development:

• Structure-based drug design targeting the FICD active site:

  • Virtual screening against the ATP binding pocket

  • Fragment-based approaches to identify initial scaffolds

  • Rational design based on transition state analogs

• High-throughput screening:

  • Develop fluorescence-based assays for AMPylation inhibition

  • Screen diverse chemical libraries (10,000-100,000 compounds)

  • Optimize lead compounds for potency and selectivity

• Selectivity considerations:

  • Target mosquito-specific structural features

  • Minimize activity against mammalian FICD homologs

  • Assess effects on beneficial insects

Transgenic Approaches:

• Conditional expression systems:

  • Design constructs expressing modified CPIJ001789 with enhanced activity

  • Place under blood-meal inducible promoters

  • Create dominant negative variants to interfere with wild-type function

• Gene drive strategies:

  • CRISPR-based homing endonuclease gene drives targeting CPIJ001789

  • Precision guided sterile insect technique

  • Evaluate ecological implications and containment strategies

Biopesticide Development:

• Peptide inhibitors:

  • Design cyclic peptides mimicking substrate binding regions

  • Optimize for cell penetration using CPP (cell-penetrating peptide) tags

  • Assess stability under field conditions

• Delivery systems:

  • Microencapsulation for environmental protection

  • Attractive toxic sugar baits incorporating inhibitors

  • Larvicide formulations targeting breeding sites

Fitness Impact Analysis:

• Reproductive capacity:

  • Egg production and viability

  • Mating competitiveness

  • Blood feeding success

• Vector competence:

  • Pathogen development within modified vectors

  • Transmission efficiency

  • Host-seeking behavior

The potential advantages of targeting CPIJ001789 include:

• Specificity to mosquito vectors compared to broad-spectrum insecticides

• Novel mode of action to address resistance to conventional insecticides

• Potential to target vector capacity without immediate lethality, reducing selection pressure

How does post-translational regulation affect CPIJ001789 activity in response to environmental stressors?

The post-translational regulation of CPIJ001789 in response to environmental stressors represents a complex and understudied area with significant implications for mosquito biology. To investigate this relationship, researchers should employ the following approaches:

Identification of Post-Translational Modifications (PTMs):

• Mass spectrometry-based PTM mapping:

  • Enrich for phosphorylated, acetylated, or ubiquitinated peptides

  • Compare PTM profiles across stress conditions (heat shock, oxidative stress, insecticide exposure)

  • Quantify changes using SILAC or TMT labeling

• Site-directed mutagenesis of identified PTM sites:

  • Generate non-modifiable variants (S/T→A for phosphorylation)

  • Create phosphomimetic mutations (S/T→D/E)

  • Assess impact on enzyme activity and localization

Protein-Protein Interaction Networks:

• Identify regulatory binding partners:

  • Yeast two-hybrid screening against mosquito cDNA library

  • Co-immunoprecipitation followed by mass spectrometry

  • Bimolecular fluorescence complementation in cultured cells

• Map interaction dynamics during stress:

  • Proximity labeling under different conditions

  • FRET/BRET assays to monitor real-time interactions

  • Quantitative interactomics with and without stressors

Subcellular Localization Studies:

• Generate fluorescent protein fusions:

  • C-terminal and N-terminal tags to assess impact on localization

  • Photoactivatable tags for dynamics studies

• Track localization changes:

  • Live-cell imaging during stress induction

  • Subcellular fractionation followed by Western blotting

  • Co-localization with organelle markers

Enzyme Kinetics Under Stress Conditions:

• Measure activity parameters with modified enzyme:

  • Determine Km and Vmax changes after phosphorylation

  • Assess substrate preference alterations

  • Quantify reaction rates at different temperatures

Research focusing on FICD proteins in other systems has revealed regulation through:

• Autoinhibitory domains that can be relieved by phosphorylation

• Oligomerization states that change with stress

• Allosteric regulation by metabolites that accumulate during stress

• pH-dependent conformational changes

Similar mechanisms likely apply to CPIJ001789 and may explain how its activity is modulated during the mosquito's response to environmental challenges.

What role does CPIJ001789 play in the endoplasmic reticulum stress response of Culex quinquefasciatus?

The potential role of CPIJ001789 in the endoplasmic reticulum (ER) stress response of Culex quinquefasciatus represents an important research direction based on known functions of FICD proteins in other organisms:

ER Stress Response Pathway Analysis:

• Characterize the unfolded protein response (UPR) components in C. quinquefasciatus:

  • Identify and clone IRE1, PERK, and ATF6 orthologs

  • Map signaling cascade through transcriptomics

  • Compare with known pathways in Drosophila and mammals

• Examine CPIJ001789 expression during ER stress:

  • Induce stress with tunicamycin, thapsigargin, or DTT

  • Measure transcript levels by qRT-PCR

  • Assess protein levels and localization by immunofluorescence

BiP/GRP78 Interaction Studies:

• Investigate CPIJ001789-mediated AMPylation of BiP:

  • Express and purify recombinant C. quinquefasciatus BiP

  • Perform in vitro AMPylation assays with purified CPIJ001789

  • Map modification sites by mass spectrometry

• Functional consequences of BiP AMPylation:

  • Measure ATPase activity of modified vs. unmodified BiP

  • Assess client binding capacity changes

  • Determine impact on BiP oligomerization states

CPIJ001789 Knockout/Knockdown Phenotypes:

• Generate CPIJ001789-deficient mosquito lines:

  • CRISPR-Cas9 gene editing for complete knockout

  • RNAi for tissue-specific or conditional knockdown

• Challenge with ER stressors:

  • Measure survival rates and developmental timing

  • Assess global gene expression changes by RNA-seq

  • Examine ER morphology by electron microscopy

Integration with Physiological Responses:

• Investigate role during specific life-stage transitions:

  • Blood meal digestion (high protein synthesis demand)

  • Metamorphosis (tissue remodeling)

  • Diapause entry/exit (metabolic reprogramming)

• Connect to immunity and vectorial capacity:

  • Challenge with pathogens that induce ER stress

  • Measure infection rates in CPIJ001789-deficient mosquitoes

  • Assess impact on virus replication and dissemination

Based on studies of FICD proteins in other systems, potential roles for CPIJ001789 include:

• Reversible AMPylation of BiP to regulate chaperone activity

• Fine-tuning of UPR intensity and duration

• Protection against chronic or excessive ER stress

• Integration of ER homeostasis with developmental programs

A mechanistic understanding of CPIJ001789's role in ER stress could reveal new approaches to disrupt vector physiology by targeting this critical homeostatic mechanism.