Recombinant Anopheles gambiae Cytosolic Fe-S cluster assembly factor NUBP1 homolog (AGAP011997)

What is the functional role of NUBP1 homolog in Anopheles gambiae?

NUBP1 homolog in Anopheles gambiae functions as a critical component of the cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery. Similar to other eukaryotic NUBP1 proteins, it is involved in the maturation of extramitochondrial Fe-S proteins, which are essential for various cellular processes. The protein belongs to the family of MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins .

The NUBP1 homolog in A. gambiae forms heterocomplexes with NUBP2, creating a scaffold for Fe-S cluster assembly. This heterotetramer mediates:

• De novo assembly of Fe-S clusters

• Transfer of assembled clusters to target apoproteins

• Maintenance of cellular iron homeostasis

Research indicates that NUBP1 is conserved across eukaryotes, suggesting an evolutionarily essential function. In mosquitoes, proper function of this protein may influence cellular processes that impact vector competence and development .

How can homologs of NUBP1 be identified across different species?

Identification of NUBP1 homologs across species requires a systematic bioinformatic approach. The following methodology is recommended based on NCBI guidelines:

Step-by-step approach to identifying NUBP1 homologs:

• Starting with the gene name:

  • Search the HomoloGene database with "NUBP1" and restrict by organism

  • For A. gambiae, use query format: NUBP1[gene name] AND "Anopheles gambiae"[orgn]

• If HomoloGene search is unsuccessful:

  • Search the Gene database with gene name "NUBP1"

  • From the desired record, follow the HomoloGene link in the right sidebar

  • If no HomoloGene link exists, locate a protein Reference Sequence and proceed to step 3

• Using protein accession numbers:

  • Search the Protein database with the accession number

  • Follow the "More about the gene" link to access homolog information

• Sequence-based homology search:

  • Use BLAST with the A. gambiae NUBP1 protein sequence

  • Select "protein blast" from the BLAST homepage

  • Enter the target organism in the "Organism" field

  • Examine resulting alignments for potential homologs

This methodology has successfully identified NUBP1 homologs across multiple species as demonstrated in the following table:

The high conservation of NUBP1 across species underscores its biological importance and provides valuable comparative models for functional studies .

What experimental controls should be included when working with recombinant NUBP1?

Proper experimental controls are essential for accurate interpretation of results when working with recombinant NUBP1. Based on established protocols in molecular biology research, the following controls should be implemented:

Essential controls for recombinant NUBP1 experiments:

• Expression system controls:

  • Empty vector control to account for vector-induced effects

  • Non-relevant protein expression (same tag system) to distinguish protein-specific from tag-related observations

  • Wild-type NUBP1 when studying mutations or variants

• Functional assays:

  • Enzymatic activity controls:

    • Heat-inactivated enzyme (negative control)

    • Known functional homolog from well-characterized species (positive control)

  • ATP hydrolysis assays:

    • No-substrate control

    • ATP regeneration system control

• Interaction studies:

  • NUBP2 co-expression studies (as NUBP1 and NUBP2 form heterocomplexes)

  • Negative controls lacking known interacting partners

  • Proteins with mutations in key binding residues

• RNAi experiments:

  • Non-targeting siRNA/shRNA controls

  • Positive knockdown controls targeting genes with known phenotypes

  • Rescue experiments with RNAi-resistant constructs

The importance of these controls is exemplified in studies of NUBP1 and NUBP2, where simultaneous knockdown experiments reveal synergistic effects that would be missed in single-gene studies. For instance, simultaneous double silencing of Nubp1 + KIFC5A was shown to restore the percentage of ciliated cells to control levels, highlighting complex functional interactions .

What experimental design is optimal for studying the function of NUBP1 in Anopheles gambiae?

An optimal experimental design for studying NUBP1 function in Anopheles gambiae requires careful consideration of variables, controls, and measurement techniques. Based on current research methodologies, the following comprehensive experimental approach is recommended:

Experimental Design Framework:

• Study objective definition:

  • Clearly state the research question (e.g., "What is the role of NUBP1 in mosquito development and vector competence?")

  • Develop specific, testable hypotheses

• Variable identification:

  • Independent variable: NUBP1 expression levels (normal, reduced, increased)

  • Dependent variables: Phenotypic changes (development rate, fertility, vector competence)

  • Control variables: Temperature, humidity, feeding regimen, genetic background

• Experimental treatments:

  • RNAi-mediated silencing of NUBP1

  • Overexpression using transgenesis

  • CRISPR-Cas9 genome editing for knockout or mutation studies

• Mosquito rearing conditions:

  • Standard insectary conditions:

    • 12-hour light/dark cycle

    • 26°C ± 2°C

    • 70% relative humidity

    • 10% sucrose solution for adult feeding

• Sampling strategy:

  • Multiple biological replicates (n≥3)

  • Appropriate sample sizes for statistical power

  • Samples collected at various developmental stages

• Data collection procedures:

  • Molecular analyses: qRT-PCR, Western blotting, immunofluorescence

  • Phenotypic analyses: Microscopy for cellular phenotypes, development timing, mortality rates

  • Vector competence assays: Direct membrane feeding assays with Plasmodium

• Data analysis plan:

  • Statistical methods appropriate for data type

  • Control for multiple testing

  • Visualization techniques for complex datasets 15

Example of experimental timeline:

This experimental design incorporates robust controls and multiple assessment methods to comprehensively characterize NUBP1 function in A. gambiae .

How can RNAi be effectively used to study NUBP1 function in Anopheles gambiae?

RNA interference (RNAi) provides a powerful tool for investigating NUBP1 function in Anopheles gambiae. Based on successful studies in related organisms, the following methodological approach is recommended:

RNAi Methodology for NUBP1 Knockdown:

• Target sequence selection:

  • Identify unique regions of NUBP1 not shared with other genes

  • Target the coding region rather than UTRs

  • Design multiple siRNAs targeting different regions to validate phenotypes

  • Avoid regions with similarity to NUBP2 to prevent off-target effects

• dsRNA preparation:

  • Amplify target region using PCR with T7 promoter-containing primers

  • Generate dsRNA using in vitro transcription

  • Purify dsRNA and confirm quality by gel electrophoresis

  • Quantify concentration for consistent dosing

• Delivery methods:

  • Adult mosquitoes:

    • Microinjection into thorax (preferred for systemic effect)

    • Concentration: 2-3 μg/μL in injection buffer

    • Volume: 69 nL per mosquito

  • Larvae:

    • Soaking method for early instars

    • Feeding with dsRNA-containing food particles

• Validation of knockdown:

  • qRT-PCR to measure NUBP1 transcript levels

  • Western blot to confirm protein reduction

  • Timing: Assess 24-72 hours post-injection/treatment

• Phenotype assessment:

  • Cellular phenotypes: Focus on cilia formation in sensory neurons

  • Developmental effects: Monitor larval progression and pupal transformation

  • Fertility and vector competence metrics

The effectiveness of RNAi for studying NUBP1 is supported by research in C. elegans, where "RNAi-mediated silencing of nubp-1 causes the formation of morphologically aberrant and additional cilia in sensory neurons" . Similar approaches can be adapted for Anopheles gambiae, with appropriate modifications for species-specific delivery methods.

Expected phenotypes based on homologous systems:

• Abnormal cilia formation

• Altered development rates

• Potential defects in iron metabolism

• Changes in vector competence for Plasmodium

How does genetic diversity in wild Anopheles gambiae populations impact NUBP1 research?

Genetic diversity in wild Anopheles gambiae populations significantly impacts NUBP1 research, necessitating consideration of natural variation when designing experiments and interpreting results. The Anopheles gambiae 1000 Genomes Project (Ag1000G) has revealed extensive genetic diversity that directly affects research approaches:

Impact of genetic diversity on NUBP1 research:

• Sequence variation considerations:

  • Over 50 million SNPs have been identified across the A. gambiae genome

  • This variation may affect:

    • Primer binding sites for PCR and qPCR

    • RNAi efficiency due to sequence mismatches

    • Protein function due to amino acid substitutions

• Population structure implications:

  • A. gambiae populations show complex structure and gene flow patterns

  • Research using lab colonies must consider their genetic representativeness

  • Studies should identify the source population of specimens used

• Experimental design adjustments:

  • Sampling strategy:

    • Include specimens from multiple geographical locations

    • Consider both A. gambiae s.s. and A. coluzzii in comparative studies

    • Include wild-caught specimens alongside laboratory colonies

  • Genetic validation:

    • Sequence NUBP1 in study specimens to identify variants

    • Design primers/probes accounting for known polymorphisms

    • Validate RNA expression quantification methods against sequence variants

• Data interpretation considerations:

  • Phenotypic differences may result from genetic background rather than experimental manipulation

  • Population-specific effects may limit generalizability

  • Natural selection on NUBP1 or interacting genes could influence findings

The Ag1000G project has sampled mosquitoes from at least 13 countries across Africa, revealing significant genetic diversity even within local populations . This diversity is summarized in the following table:

Researchers must account for this genetic diversity when designing primers, probes, and reference sequences for NUBP1 studies, and should consider population genetic context when interpreting functional results .

What techniques are most effective for studying protein-protein interactions involving NUBP1 in Anopheles gambiae?

Studying protein-protein interactions (PPIs) involving NUBP1 in Anopheles gambiae requires specialized techniques adapted to arthropod systems. Based on successful approaches with homologous proteins and mosquito studies, the following methodology is recommended:

Effective PPI techniques for NUBP1 research:

• Co-immunoprecipitation (Co-IP):

  • Methodology:

    • Generate antibodies against A. gambiae NUBP1 or use epitope tags

    • Prepare mosquito tissue lysates under non-denaturing conditions

    • Immunoprecipitate NUBP1 and identify interacting partners by mass spectrometry

  • Advantages: Detects interactions in near-native conditions

  • Limitations: Requires high-quality antibodies or successful expression of tagged proteins

• Yeast two-hybrid (Y2H) screening:

  • Methodology:

    • Clone NUBP1 into bait vector

    • Screen against A. gambiae cDNA library

    • Validate positive interactions with secondary assays

  • Advantages: High-throughput identification of binary interactions

  • Limitations: High false-positive rate; may miss interactions dependent on post-translational modifications

• Bimolecular Fluorescence Complementation (BiFC):

  • Methodology:

    • Fuse NUBP1 and candidate partners to complementary fragments of fluorescent protein

    • Express in mosquito cell lines or tissues

    • Visualize interactions through fluorescence microscopy

  • Advantages: Visualizes interactions in living cells with spatial information

  • Limitations: Potential artificial stabilization of weak interactions

• Proximity-dependent biotin identification (BioID):

  • Methodology:

    • Fuse NUBP1 to a promiscuous biotin ligase

    • Express in mosquito cells or tissues

    • Identify biotinylated proximity partners by pulldown and mass spectrometry

  • Advantages: Identifies transient interactions and nearby proteins in native cellular context

  • Limitations: Can identify proteins in proximity but not necessarily direct interactors

Based on studies with mammalian NUBP1, key interaction partners to investigate include:

Research has uncovered "novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which... [are] enriched at the basal body" . Similar interactions are likely conserved in A. gambiae and represent promising research targets.

How can data tables be effectively designed for presenting NUBP1 research findings?

Effective data table design is crucial for clearly communicating NUBP1 research findings. Following established scientific guidelines for data presentation ensures clarity and facilitates interpretation:

Data table design principles for NUBP1 research:

• Table structure fundamentals:

  • Include a clear, descriptive title that states the purpose of the experiment

  • Format: "The effect of ____ (independent variable) on ______ (dependent variable)"

  • Place independent variables in the left column

  • Place dependent variables and trial data in subsequent columns

  • Include a derived or calculated column (e.g., mean values) on the far right 15

• Variable organization:

  • For NUBP1 expression studies:

    • List experimental treatments (e.g., RNAi conditions, expression systems) in rows

    • Present measured outcomes in columns with appropriate units

    • Include multiple trials and statistical measures

• Statistical representation:

  • Include standard deviation or standard error values

  • Provide sample sizes (n values)

  • For Division C level research, maintain consistent significant figures

  • Include sample calculations below the table

• Example data table for NUBP1 knockdown experiments:

Sample calculation: Average Expression (%) = (Trial 1 + Trial 2 + Trial 3) / 3
For Control siRNA: (100.0 + 98.5 + 101.2) / 3 = 99.9%

• Advanced table design for complex experiments:

  • Use hierarchical row/column headers for multifactorial experiments

  • Consider heat map formatting for large datasets

  • Include footnotes explaining methodology or unusual observations

  • For gene expression analyses across species, include accession numbers

Remember that "in most cases, the independent variable (that which you purposefully change) is in the left column, the dependent variable (that which you measure) with the different trials is in the next columns, and the derived or calculated column (often average) is on the far right" .

What methodologies can be used to study NUBP1's role in vector competence for Plasmodium transmission?

Investigating NUBP1's potential role in vector competence requires specialized methodologies that bridge molecular biology and parasite transmission studies. The following comprehensive approach can effectively elucidate NUBP1's function in Plasmodium transmission:

Methodological framework for studying NUBP1 in vector competence:

• Gene expression manipulation:

  • RNAi-mediated knockdown of NUBP1 in adult female mosquitoes

  • CRISPR-Cas9 genome editing to generate NUBP1 mutants

  • Conditional expression systems to control timing of manipulation

• Direct membrane feeding assay (DMFA) protocol:

  • Preparation:

    • Maintain NUBP1-manipulated and control mosquito groups under identical conditions

    • Obtain Plasmodium-infected blood (P. falciparum gametocyte cultures or patient isolates)

    • Set up membrane feeders at 37°C

  • Feeding procedure:

    • For serum replacement studies:

      • Replace endemic serum with naive serum in parallel groups

      • Feed mosquitoes for 15-20 minutes

    • Use 3-5 day old female mosquitoes

    • Maintain fed mosquitoes at 26°C and 70-80% humidity

  • Infection assessment:

    • Dissect mosquito midguts 7-10 days post-feeding

    • Count oocysts using microscopy

    • Examine salivary glands for sporozoites at day 14-16

• Data collection and analysis:

  • Key metrics to measure:

    • Infection prevalence (% of mosquitoes infected)

    • Infection intensity (oocyst/sporozoite numbers per mosquito)

    • Transmission potential (sporozoite load in salivary glands)

  • Experimental setup example:

This experimental design is based on successful approaches used in similar studies, where "the proportion of mosquitoes infected via direct membrane feeding assay with either P. malariae monoinfections (16% [19 of 121]) or coinfections (28% [31 of 112]) was higher after serum replacement than in parallel groups without serum replacement" .

• Molecular mechanisms investigation:

  • Transcriptome analysis of NUBP1-depleted mosquitoes before and after infection

  • Immunolocalization of NUBP1 during Plasmodium development

  • Characterization of cellular changes (especially cilia-related) that might influence parasite development

This comprehensive methodology links molecular function to vector competence, providing insights into how NUBP1 might influence malaria transmission dynamics .

How can heterologous expression systems be optimized for producing functional recombinant A. gambiae NUBP1?

Optimizing heterologous expression systems for producing functional recombinant A. gambiae NUBP1 requires careful consideration of expression hosts, vectors, and purification strategies. Based on successful approaches with similar proteins, the following methodology is recommended:

Optimization strategy for recombinant A. gambiae NUBP1 expression:

• Expression system selection:

  • E. coli:

    • Advantages: Quick, high yield, inexpensive

    • Limitations: Potential improper folding, lack of post-translational modifications

    • Best systems: BL21(DE3), Rosetta for rare codon optimization

    • Recommended for structural studies and antibody production

  • Yeast (S. cerevisiae, P. pastoris):

    • Advantages: Eukaryotic folding machinery, moderate cost

    • Limitations: Lower yields than E. coli

    • Best for: Functional studies requiring proper folding

    • Particularly suitable as S. cerevisiae has NBP35 homolog

  • Baculovirus/insect cells:

    • Advantages: Insect-derived system, proper folding, post-translational modifications

    • Limitations: Higher cost, longer production time

    • Optimal for: Functional studies requiring native-like protein

    • Most biologically relevant for mosquito proteins

  • Mammalian cells:

    • Advantages: Advanced folding machinery, full post-translational modifications

    • Limitations: Highest cost, complex protocols, lower yield

    • Consider for: Complex interaction studies with mammalian proteins

• Expression vector optimization:

  • Codon optimization:

    • Adjust codons for expression host (critical for AT-rich A. gambiae genes)

    • Remove rare codons and RNA secondary structures

  • Fusion tags selection:

    • N-terminal: His6, GST, MBP (improves solubility)

    • C-terminal: FLAG, c-Myc (for detection)

    • Consider TEV or PreScission protease cleavage sites for tag removal

  • Promoter selection:

    • E. coli: T7 for high expression

    • Yeast: GAL1, AOX1 for inducible expression

    • Insect cells: polyhedrin, p10 promoters

    • Mammalian: CMV, EF1α promoters

• Expression conditions optimization:

  • Temperature:

    • Lower temperatures (16-25°C) often improve folding

    • Test expression at 37°C, 30°C, 25°C, and 18°C

  • Induction parameters:

    • IPTG concentration: 0.1-1.0 mM for E. coli

    • Induction OD: Test early (OD600 0.4-0.6) vs. late (OD600 0.8-1.0)

    • Duration: 3-6h vs. overnight expression

  • Media supplements:

    • Iron and sulfur sources for Fe-S protein

    • Metal chelators if needed

    • Chaperone co-expression for improved folding

• Purification strategy:

  • Initial capture:

    • IMAC (Ni-NTA) for His-tagged protein

    • Glutathione affinity for GST-fusion

  • Secondary purification:

    • Ion exchange chromatography

    • Size exclusion chromatography

  • Special considerations:

    • Anaerobic purification for Fe-S cluster preservation

    • Buffer optimization with reducing agents (DTT, β-ME)

    • Glycerol (10-20%) to improve stability

The choice of expression system significantly impacts yield and quality, as evidenced by commercial pricing: E. coli-expressed NUBP1 homolog costs approximately $930 for 0.02 mg, while mammalian cell-expressed protein is priced at $1,615 for the same amount, reflecting the increased complexity and biological relevance .