Recombinant Anopheles gambiae Cytosolic Fe-S cluster assembly factor NUBP2 homolog (AGAP010873)

What is the function of NUBP2 homolog in Anopheles gambiae?

NUBP2 functions as a component of the cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery, similar to its homolog NUBP1. Based on characterized homologs, NUBP2 is required for the maturation of extramitochondrial Fe-S proteins . NUBP proteins form heterotetramer complexes that serve as Fe-S scaffold assemblies, mediating the de novo assembly of Fe-S clusters and their transfer to target apoproteins . In eukaryotic cells, these proteins play critical roles in the cytosolic iron-sulfur cluster assembly (CIA) system, which exists in the cytosol and is responsible for adding Fe-S clusters to proteins made in the cytosol or nucleus . Research indicates that NUBP2 is enriched in centrosomes during mitosis, suggesting a potential role in centrosome biology .

How do NUBP1 and NUBP2 homologs interact in Anopheles?

NUBP1 and NUBP2 homologs interact directly with each other to form functional heterocomplexes. Studies in mammalian cells have demonstrated that these proteins interact not only with each other but also with motor proteins such as KIFC5A . Pull-down experiments have confirmed the direct physical interaction between these proteins, as demonstrated when recombinant His6-tagged proteins bound to Ni2+-NTA beads successfully recovered their interaction partners . The interaction between NUBP1 and NUBP2 is essential for their function in Fe-S cluster assembly, as they form a heterotetramer that serves as a scaffold for Fe-S cluster formation before transfer to recipient proteins .

What is the evolutionary conservation of NUBP2 across insect vectors?

NUBP2 belongs to the highly conserved Mrp/NBP35 ATP-binding protein family, specifically the NUBP1/NBP35 subfamily . While the search results don't provide specific information about conservation across insect vectors, the functional importance of Fe-S cluster assembly suggests high conservation. In Anopheles gambiae, studies of allele-specific expression have revealed that many genes, particularly Anopheles-specific genes, show imbalanced expression patterns that are regulated by factors on the same chromosome . This suggests that species-specific adaptations may influence the regulation of genes like NUBP2, even as the core functional domains remain conserved. Researchers interested in evolutionary aspects should conduct comparative genomic analyses across multiple insect vector species to fully characterize NUBP2 conservation.

What methods are most effective for producing recombinant Anopheles gambiae NUBP2?

Based on successful approaches with related proteins, the most effective expression systems for recombinant Anopheles gambiae NUBP2 include:

• Baculovirus-infected insect cells: This system has been successfully used for producing complex eukaryotic proteins with high purity (>90%) . For NUBP2, which likely requires proper folding and potential post-translational modifications, this system offers advantages over bacterial expression.

• E. coli expression systems: For initial characterization or when large quantities are needed, E. coli expression with appropriate tags (such as His6) can be effective. This approach was successfully employed for KIFC5A expression in interaction studies with NUBP2 .

• Mammalian expression systems: For functional studies requiring authentic post-translational modifications, transient expression in mammalian cells (such as HEK293) with appropriate epitope tags for detection and purification is recommended.

For optimal results, researchers should include appropriate affinity tags (His, GST, or FLAG) to facilitate purification, and consider expressing truncated versions if the full-length protein proves challenging. Expression and purification should be verified using techniques such as Western blotting with specific antibodies raised against unique peptide sequences in the NUBP2 C-terminus .

How can researchers investigate NUBP2's role in centrosome regulation in Anopheles?

To investigate NUBP2's role in centrosome regulation in Anopheles, researchers should employ a multi-faceted approach:

• RNAi-mediated silencing: Implement RNA interference targeting NUBP2 alone or in combination with NUBP1. When designing experiments, it's important to note that previous studies in mammalian cells showed that Nubp2 silencing alone did not produce a discernible phenotype, while Nubp1 silencing or double Nubp1/Nubp2 knockdown resulted in significant increases in centrosome numbers and multi-nucleated cells .

• Immunolocalization studies: Develop specific antibodies against Anopheles NUBP2 to track its subcellular localization throughout the cell cycle, particularly during mitosis. Studies in mammalian cells have shown that NUBP2 is enriched in centrosomes during mitosis .

• Co-immunoprecipitation experiments: Identify NUBP2-interacting proteins in Anopheles, focusing on potential interactions with centrosome components and motor proteins. This approach can reveal whether NUBP2 interacts with proteins similar to KIFC5A in mammals .

• Phenotypic analysis: Quantify centrosome numbers in interphase and mitotic cells, and assess the frequency of multi-nucleated cells following NUBP2 manipulation. Statistical analysis should be performed to determine significance, as demonstrated in the table below:

Statistical significance should be assessed using appropriate tests (p<0.05 considered significant) .

What is the relationship between NUBP2 and insecticide resistance in Anopheles gambiae?

The relationship between NUBP2 and insecticide resistance in Anopheles gambiae remains largely unexplored and represents an important research direction. Current evidence suggests several potential connections:

• Differential expression analysis: Studies examining allele-specific expression (ASE) in Anopheles gambiae have identified 115 genes showing consistently different messenger RNA levels between gene copies, suggesting cis-regulatory control . While NUBP2 was not specifically mentioned among these genes, its role in Fe-S cluster assembly could influence metabolic enzymes involved in detoxification pathways.

• Metabolic resistance mechanisms: As a component of the Fe-S cluster assembly machinery, NUBP2 potentially influences the function of multiple Fe-S proteins involved in metabolic processes. Metabolic resistance to insecticides often involves altered activity of detoxification enzymes, many of which require cofactors like Fe-S clusters for proper function .

• Research approach: To investigate potential connections, researchers should:

  • Compare NUBP2 expression levels between insecticide-resistant and susceptible strains

  • Perform genetic crosses between resistant and susceptible strains and analyze NUBP2 allele-specific expression

  • Examine the effects of NUBP2 knockdown on insecticide susceptibility

  • Identify Fe-S proteins involved in insecticide metabolism that might depend on NUBP2 function

This research direction could provide novel insights into insecticide resistance mechanisms, potentially identifying NUBP2 as a new target for vector control strategies.

How should researchers design experiments to study NUBP2's interaction with other CIA machinery components?

To effectively study NUBP2's interactions with other CIA machinery components in Anopheles gambiae, researchers should employ a systematic approach:

• Yeast two-hybrid screening: Identify potential interacting partners by using NUBP2 as bait against an Anopheles cDNA library. This provides an initial screen for interacting proteins, though results should be validated with additional methods.

• Co-immunoprecipitation assays: Perform both forward and reverse co-IP experiments using epitope-tagged NUBP2 expressed in Anopheles cells or tissues. For example:

  • Express His-tagged NUBP2 and analyze co-precipitating proteins by mass spectrometry

  • Use antibodies against known or suspected interacting partners to precipitate protein complexes and detect NUBP2

• Pull-down assays with recombinant proteins: Express and purify His-tagged NUBP2 from E. coli or insect cells, bind to Ni²⁺-NTA beads, and incubate with potential interacting partners or cell lysates. This approach has been successfully used to demonstrate direct interactions between NUBP2 and other proteins . The experimental design should include appropriate controls:

  • Negative control lacking His-tagged NUBP2

  • Control using unrelated tagged proteins to test for non-specific binding

• Bimolecular Fluorescence Complementation (BiFC): For in vivo validation of interactions, express NUBP2 and potential partners as fusion proteins with complementary fragments of a fluorescent protein in Anopheles cells.

• Crosslinking mass spectrometry: Apply protein crosslinking followed by mass spectrometry to identify not only interacting partners but also specific interaction interfaces.

The experimental approach should systematically characterize NUBP2's interactions with both known CIA components (based on homology to other organisms) and novel Anopheles-specific interactors.

What techniques are most effective for analyzing the function of NUBP2 in Fe-S cluster assembly?

Several complementary techniques can effectively analyze NUBP2's function in Fe-S cluster assembly:

• In vitro Fe-S cluster reconstitution assays:

  • Express and purify recombinant NUBP2 and NUBP1

  • Reconstitute Fe-S clusters under anaerobic conditions using iron sources (Fe²⁺) and sulfur sources (cysteine desulfurase)

  • Monitor cluster assembly spectroscopically (UV-visible absorption at 400-420 nm characteristic of Fe-S clusters)

  • Compare cluster assembly kinetics with and without NUBP2

• Target protein maturation assays:

  • Select Fe-S proteins known to require the CIA machinery

  • Express these proteins in systems where NUBP2 activity is modulated (knockdown, mutation, overexpression)

  • Assess Fe-S cluster incorporation by measuring enzymatic activity of target proteins

  • The core process of Fe-S cluster biosynthesis involves assembly onto scaffold proteins, recruitment of recipient proteins, and transfer of clusters

• Cellular fractionation and localization:

  • Use immunofluorescence with specific antibodies to track NUBP2 localization

  • Perform subcellular fractionation to isolate cytosolic components

  • Analyze co-localization with known CIA components

  • Studies have shown NUBP2 enrichment in centrosomes during mitosis, indicating specific localization patterns

• Genetic complementation studies:

  • Generate NUBP2-deficient cell lines or organisms

  • Reintroduce wild-type or mutant NUBP2 variants

  • Assess restoration of Fe-S protein activities

  • This approach can identify critical functional domains and residues

These techniques collectively enable a comprehensive analysis of NUBP2's functional role in the CIA machinery and its impact on cellular Fe-S protein maturation.

How can researchers accurately assess the impact of NUBP2 mutations on protein function?

To accurately assess the impact of NUBP2 mutations on protein function, researchers should implement a multi-level analysis approach:

• Structural prediction and conservation analysis:

  • Identify conserved domains and motifs in NUBP2 using sequence alignment across species

  • Generate structural models based on homologs with known structures

  • Target mutations to highly conserved residues, known functional domains, or residues predicted to be involved in protein-protein interactions

  • NUBP2 belongs to the Mrp/NBP35 ATP-binding protein family , which provides guidance on critical functional domains

• In vitro biochemical characterization:

  • Express and purify wild-type and mutant NUBP2 proteins

  • Assess basic biochemical properties including protein stability, oligomerization, and ATP binding/hydrolysis

  • Compare Fe-S cluster binding capacity using spectroscopic methods

  • Evaluate interaction with NUBP1 and other partners using pull-down assays or surface plasmon resonance

• Cellular complementation assays:

  • Generate NUBP2-deficient cells through CRISPR/Cas9 or RNAi approaches

  • Reintroduce wild-type or mutant NUBP2 variants

  • Quantify restoration of:

    • Fe-S protein activities

    • Cellular phenotypes (centrosome numbers, nuclear morphology)

    • Growth or viability

• Iron homeostasis assessment:

  • Measure cellular iron levels in cells expressing mutant NUBP2

  • Assess expression of iron-responsive genes

  • Evaluate mitochondrial function, as disruption of Fe-S cluster assembly can impact iron homeostasis

• Specific functional readouts:

  • Analyze centrosomal phenotypes, including centrosome numbers in interphase and mitosis

  • Quantify multi-nucleated cells to assess cytokinesis defects

  • These phenotypes were significantly affected in Nubp1/Nubp2 studies in mammalian systems

This systematic approach provides a comprehensive evaluation of how specific mutations affect NUBP2's diverse functions, from biochemical activities to cellular phenotypes.

How should researchers interpret discrepancies between in vitro and in vivo NUBP2 functional studies?

When researchers encounter discrepancies between in vitro and in vivo NUBP2 functional studies, they should consider several factors for proper interpretation:

• Protein context and interaction network:

  • In vivo, NUBP2 functions within a complex network of interacting proteins

  • Studies in mammalian systems show NUBP2 interacts with both NUBP1 and motor proteins like KIFC5A

  • In vitro studies may lack these interaction partners, potentially altering NUBP2 function

  • Consider supplementing in vitro systems with known interaction partners to better mimic cellular conditions

• Functional redundancy:

  • In vivo silencing experiments have shown that Nubp2 knockdown alone may not produce observable phenotypes, while Nubp1 silencing does

  • This suggests possible compensatory mechanisms or functional redundancy

  • When interpreting discrepancies, consider whether other proteins might compensate for NUBP2 function in vivo

• Post-translational modifications:

  • In vivo, NUBP2 may undergo PTMs that are absent in recombinant systems

  • Recombinant protein expression systems differ in their ability to perform eukaryotic PTMs

  • Expression systems like baculovirus-infected insect cells provide better PTM profiles than bacterial systems

  • Consider analyzing the PTM status of native versus recombinant NUBP2

• Statistical analysis approach:

  • When analyzing phenotypic data, apply appropriate statistical tests as demonstrated in studies of Nubp1/Nubp2 silencing

  • For example, when analyzing centrosome numbers or multi-nucleated cells, significance testing (p-values) should be used to determine whether observed differences are statistically meaningful

  • The table below illustrates how to organize and compare such data:

This structured approach helps researchers systematically evaluate and explain discrepancies between different experimental systems.

What bioinformatic approaches are most valuable for identifying potential NUBP2 interaction partners in Anopheles?

For identifying potential NUBP2 interaction partners in Anopheles gambiae, researchers should employ a multi-layered bioinformatic strategy:

• Homology-based prediction:

  • Identify known interaction partners of NUBP2 homologs in well-studied organisms

  • The CIA machinery components are generally conserved across eukaryotes

  • Search for Anopheles orthologs of these interaction partners using reciprocal BLAST

  • Prioritize interactions that are conserved across multiple species

• Protein domain and motif analysis:

  • Identify conserved domains in NUBP2 that mediate protein-protein interactions

  • NUBP2 belongs to the Mrp/NBP35 ATP-binding protein family , which provides information about potential interaction interfaces

  • Search for proteins containing complementary interaction domains

  • Analyze protein sequences for short linear motifs that could mediate interactions

• Co-expression network analysis:

  • Utilize RNA-seq data from Anopheles gambiae to build co-expression networks

  • Genes with expression patterns similar to NUBP2 are candidates for functional association

  • Studies of allele-specific expression in Anopheles have identified genes regulated by cis-regulatory factors , which can inform co-expression analysis

• Protein-protein interaction prediction algorithms:

  • Apply machine learning-based PPI prediction tools trained on known interactions

  • Integrate multiple features including sequence, structure, and evolutionary conservation

  • Assign confidence scores to predicted interactions based on multiple lines of evidence

• Functional association prediction:

  • Use tools like STRING or FunCoup to predict functional associations

  • Integrate information from genomic context, experimental data, and text mining

  • Focus on proteins involved in Fe-S cluster assembly, centrosome regulation, and related processes

• Experimental validation planning:

  • Rank predicted interactions based on confidence scores and biological relevance

  • Design validation experiments prioritizing high-confidence predictions

  • Include both known (as positive controls) and novel interaction candidates

This comprehensive bioinformatic approach provides a strong foundation for subsequent experimental validation of NUBP2 interaction partners.

What are the key challenges in developing recombinant NUBP2 for functional studies in vector biology?

Developing recombinant NUBP2 for functional studies in vector biology faces several critical challenges that researchers must address:

• Protein expression and solubility:

  • Fe-S cluster assembly proteins often require specific conditions for proper folding

  • Expression systems must be carefully selected and optimized

  • Baculovirus-infected insect cells offer advantages for complex eukaryotic proteins

  • Bacterial expression systems may require optimization of growth conditions, including temperature and induction parameters

• Functional conservation versus species-specific adaptations:

  • While the core functions of NUBP2 are likely conserved across species, vector-specific adaptations may exist

  • Studies in Anopheles have shown that Anopheles-specific genes are more likely to show allele-specific expression

  • Researchers must distinguish conserved functions from species-specific adaptations

  • Comparative studies with NUBP2 from multiple vector species would be valuable

• Physiological relevance of in vitro studies:

  • Recombinant protein studies may not fully recapitulate the complex cellular environment

  • The function of NUBP2 depends on interactions with multiple partners including NUBP1

  • In vivo validation of findings from recombinant protein studies is essential

• Technical challenges in studying Fe-S proteins:

  • Fe-S clusters are oxygen-sensitive, requiring specialized handling

  • Spectroscopic analysis of Fe-S cluster formation requires specific expertise

  • Quantification of Fe-S cluster transfer to target proteins presents methodological challenges

• Bridging molecular function and vector biology:

  • Connecting NUBP2 function to vector-specific traits requires interdisciplinary approaches

  • Potential links to insecticide resistance mechanisms need to be explored systematically

  • Developing genetic manipulation tools specifically for vectors remains challenging

Addressing these challenges requires integrated approaches combining molecular biology, biochemistry, cell biology, and vector biology expertise.

How might future research on NUBP2 contribute to novel vector control strategies?

Future research on NUBP2 holds significant potential for contributing to novel vector control strategies through several promising avenues:

• Targeting essential cellular processes:

  • NUBP2's role in Fe-S cluster assembly represents a fundamental cellular process

  • Studies in mammalian systems have shown that disruption of the NUBP1/NUBP2 system affects critical cellular functions including centrosome duplication and cytokinesis

  • Identifying vector-specific vulnerabilities in this pathway could provide novel targets for vector control

• Connection to insecticide resistance mechanisms:

  • Fe-S proteins are involved in various metabolic pathways, including those potentially involved in insecticide detoxification

  • Studies in Anopheles have identified genes showing allele-specific expression that may confer metabolic resistance

  • If NUBP2 function influences the activity of detoxification enzymes, it could become a target for overcoming resistance

• Genetic approaches to vector control:

  • CRISPR-based gene drive systems targeting NUBP2 or its regulatory elements could be developed

  • Understanding cis-regulatory modules predicted through machine learning approaches could enable precise genetic manipulation

  • Careful characterization of NUBP2's role in reproduction and development is needed to assess the feasibility of such approaches

• Comparative studies across vector species:

  • Identifying conserved versus species-specific aspects of NUBP2 function across disease vectors

  • This knowledge could lead to broad-spectrum control strategies applicable to multiple vector species

  • Alternatively, species-specific features could enable highly targeted approaches

• Screening platforms for novel compounds:

  • Developing assay systems using recombinant NUBP2 for high-throughput screening

  • Compounds that specifically disrupt NUBP2 function or its interactions could become leads for new insecticides

  • The requirement for properly functioning Fe-S cluster assembly for cellular viability makes this pathway an attractive target