Recombinant Brugia pahangi 60S ribosomal protein L26

What is Brugia pahangi RPL26 and why is it significant for research?

Brugia pahangi RPL26 is a 60S ribosomal protein found in the filarial nematode worm Brugia pahangi. As a component of the large ribosomal subunit, it plays an essential role in protein synthesis within this parasitic organism. Research interest in this protein stems from its potential as a target for anti-parasitic interventions and its utility in understanding basic mechanisms of translation in parasitic nematodes. Although primarily a ribosomal component, research on mammalian RPL26 homologs has revealed extraribosomal functions, particularly in regulating p53 mRNA translation following cellular stress, suggesting similar multifunctional roles may exist for the parasite protein . The recombinant form of Brugia pahangi RPL26 typically refers to a His-tagged version expressed in heterologous systems, with commercially available versions covering amino acids 1-60 of the full protein sequence .

What expression systems are used for producing recombinant Brugia pahangi RPL26?

Recombinant Brugia pahangi RPL26 can be produced using several expression systems, with yeast being a commonly employed host for commercial preparations . Alternative expression systems include E. coli, mammalian cell lines, and baculovirus-infected insect cells, each offering distinct advantages depending on experimental requirements . The choice of expression system significantly affects protein yield, post-translational modifications, solubility, and biological activity. Yeast expression systems provide certain eukaryotic post-translational modifications while maintaining high protein yields. When selecting an expression system, researchers should consider that different hosts may result in variations in price, production timeline, and protein characteristics . For specialized applications requiring specific modifications or high purity, custom expression methods may be necessary.

What is the amino acid sequence of the recombinant Brugia pahangi RPL26 (AA 1-60)?

The amino acid sequence of the recombinant Brugia pahangi RPL26 covering residues 1-60 is:

MKQNQFVSSS ARKARKAPFN APSHIRRKLM SAPSTKDLRN KHGIRSIPIR IDDEVTVTRG

This sequence corresponds to the N-terminal region of the full-length protein and typically carries a histidine tag to facilitate purification when produced as a recombinant protein . The sequence exhibits characteristics typical of ribosomal proteins, including multiple basic residues (lysine and arginine) that likely facilitate interactions with negatively charged RNA molecules within the ribosome. Researchers working with this protein should note that this represents a partial sequence of the full-length RPL26 protein, which may have implications for structural studies and functional analyses.

How can researchers optimize ELISA protocols using recombinant Brugia pahangi RPL26?

Optimizing ELISA protocols with recombinant Brugia pahangi RPL26 requires careful consideration of several parameters. First, establish optimal coating concentrations through titration experiments, typically starting with 1-5 μg/ml of the recombinant protein in carbonate/bicarbonate buffer (pH 9.6) . Blocking solutions containing 1-5% BSA or casein should be tested to minimize background signal while maintaining specific antibody binding. When developing sandwich ELISAs, consider that the His-tag on the recombinant protein may be utilized for capture using anti-His antibodies, eliminating the need for specific anti-RPL26 capture antibodies .

For detection antibodies, cross-reactivity with host RPL26 must be evaluated, particularly when analyzing patient samples. Temperature and incubation times should be optimized; typically, coating is performed overnight at 4°C, while antibody incubations are done at room temperature for 1-2 hours or 4°C overnight. Include appropriate controls in each experiment:

• Positive control: Known positive samples or standards

• Negative control: Buffer alone

• Specificity control: Unrelated His-tagged protein

Validation should include assessment of the assay's detection limit, linear range, reproducibility, and cross-reactivity with related filarial species . For quantitative applications, generate a standard curve using purified RPL26 protein of known concentration.

What are the methodological approaches for studying the interactions between Brugia pahangi RPL26 and host immune factors?

To investigate interactions between Brugia pahangi RPL26 and host immune factors, researchers can employ multiple complementary approaches. Pull-down assays using His-tagged recombinant RPL26 (>90% purity) as bait can identify potential binding partners from host cell lysates . These interactions can be confirmed through reciprocal co-immunoprecipitation experiments, similar to those used to demonstrate the interaction between human RPL26 and MDM2 .

Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) provides quantitative binding kinetics data. For these assays, immobilize purified RPL26 on sensor chips/tips and flow potential binding partners at various concentrations. Analyze association and dissociation curves to determine KD values.

For cellular studies, fluorescently labeled RPL26 can be used to track protein internalization and localization in host cells through confocal microscopy. Flow cytometry analysis of cells exposed to RPL26 can assess changes in surface marker expression, while cytokine profiling using multiplex assays can measure immune response patterns.

Functional studies should include:

• Macrophage activation assays measuring TNF-α, IL-6, and IL-10 production

• Dendritic cell maturation assessment via surface marker expression

• T-cell proliferation and polarization studies

• Complement activation analysis

Cross-species comparative approaches examining RPL26 from different filarial species can identify conserved immune-modulatory motifs. Peptide mapping using overlapping fragments of RPL26 can pinpoint specific immunoactive regions of the protein .

What quality control parameters should be assessed when working with recombinant Brugia pahangi RPL26?

Comprehensive quality control for recombinant Brugia pahangi RPL26 should evaluate multiple parameters to ensure experimental reproducibility. Purity assessment via SDS-PAGE should confirm >90% purity as typically specified for commercial preparations . For more precise analysis, high-performance liquid chromatography (HPLC) or capillary electrophoresis can be employed.

Identity verification requires peptide mass fingerprinting using mass spectrometry to confirm the protein sequence matches that of Brugia pahangi RPL26. Western blotting with anti-His antibodies confirms the presence of the His-tag, while species-specific antibodies (if available) can verify the Brugia pahangi origin .

Functional activity assessment depends on the intended application but may include:

• RNA binding assays if studying translational regulation functions

• Interaction studies with known binding partners

• ELISA reactivity with anti-RPL26 antibodies

Endotoxin testing is critical, particularly for immunological studies, as contamination can confound results. Use limulus amebocyte lysate (LAL) assays to ensure levels below 0.1 EU/μg protein. Stability testing under various storage conditions (4°C, -20°C, -80°C) helps establish optimal handling protocols and shelf-life parameters .

Document batch-to-batch consistency by comparing multiple production lots using the above parameters. For advanced applications, assess secondary structure integrity via circular dichroism spectroscopy to confirm proper protein folding.

How does Brugia pahangi RPL26 compare structurally and functionally to human RPL26?

Structural and functional comparison between Brugia pahangi RPL26 and human RPL26 reveals important differences and similarities relevant to both basic biology and therapeutic targeting. At the sequence level, alignment analysis shows conservation of key ribosomal binding domains, particularly in the N-terminal region, though significant divergence exists in specific amino acid residues that might confer species-specific functions . The 60-amino acid fragment (AA 1-60) of Brugia pahangi RPL26 contains regions critical for ribosome integration, but comparative analysis suggests differences in surface-exposed epitopes that could be exploited for selective targeting .

Functionally, human RPL26 has been extensively studied for its extraribosomal role in regulating p53 mRNA translation following DNA damage . Human RPL26 binds to the 5' and 3' UTRs of p53 mRNA, enhancing its translation and consequently increasing p53 protein levels and downstream cellular responses including G1 cell-cycle arrest and apoptosis . Whether Brugia pahangi RPL26 possesses similar moonlighting functions remains unknown, but conservation analysis of RNA-binding motifs could predict potential regulatory targets in the parasite transcriptome.

The interaction between human RPL26 and MDM2 (leading to ubiquitination and degradation of RPL26) represents another important functional aspect . Comparative studies could investigate whether similar regulatory mechanisms exist in Brugia pahangi, potentially involving parasite-specific E3 ubiquitin ligases. Structural predictions based on homology modeling suggest that while the core ribosomal functions are conserved, surface-exposed regions show significant divergence that might be exploited for selective targeting of the parasite protein without affecting the host homolog .

What are the challenges and solutions for studying RNA-binding properties of Brugia pahangi RPL26?

Investigating the RNA-binding properties of Brugia pahangi RPL26 presents several technical challenges requiring specialized approaches. The primary challenge stems from the protein's dual functionality – its canonical role in ribosome structure versus potential extraribosomal RNA interactions similar to those observed with human RPL26 and p53 mRNA . To address this, researchers should employ RNA-protein interaction studies that can distinguish between these different binding modes.

RNA Electrophoretic Mobility Shift Assays (EMSA) represent a starting point, but require careful optimization. Use purified recombinant Brugia pahangi RPL26 protein (>90% purity) incubated with candidate RNA sequences under varying salt and pH conditions to identify optimal binding parameters. For ribosomal RNA binding, use fragments of Brugia 28S rRNA, while for extraribosomal functions, test parasite mRNAs with structured UTRs similar to human p53 mRNA.

More quantitative approaches include:

• Surface Plasmon Resonance (SPR) with immobilized RNA and flowing protein to determine binding kinetics

• Isothermal Titration Calorimetry (ITC) for thermodynamic binding parameters

• Microscale Thermophoresis (MST) for studying interactions in solution with minimal material consumption

Structural characterization of RNA-protein complexes can be approached through Nuclear Magnetic Resonance (NMR) spectroscopy for smaller RNA fragments or cryo-electron microscopy for larger complexes. Computational prediction of RNA binding sites based on human RPL26 data can guide the design of mutant proteins for structure-function validation studies .

How might post-translational modifications affect the functionality of recombinant Brugia pahangi RPL26?

Post-translational modifications (PTMs) can significantly impact the functionality of recombinant Brugia pahangi RPL26, affecting its structural properties, interaction capabilities, and biological activities. The choice of expression system is crucial, as yeast-expressed RPL26 will exhibit different PTM patterns compared to versions expressed in E. coli, mammalian cells, or baculovirus systems. E. coli lacks machinery for many eukaryotic PTMs, potentially producing protein lacking critical modifications, while mammalian systems may introduce host-specific rather than parasite-specific modifications.

Potential PTMs that could affect RPL26 functionality include:

• Phosphorylation: May regulate RNA binding affinity, protein-protein interactions, and subcellular localization. Based on studies of mammalian ribosomal proteins, phosphorylation can modulate translation efficiency and extraribosomal functions .

• Ubiquitination: Human RPL26 is regulated by MDM2-mediated ubiquitination and subsequent proteasomal degradation . Similar regulation may exist in Brugia pahangi, affecting protein stability and activity.

• Methylation: Common in ribosomal proteins, affecting ribosome assembly and translation fidelity.

• Acetylation: May influence protein-protein and protein-RNA interactions.

To assess the impact of PTMs, researchers should compare native parasite-derived RPL26 with recombinant versions using mass spectrometry-based proteomic approaches to map modification sites. Functional comparisons between differently modified forms can be conducted using:

• RNA binding assays to assess impact on nucleic acid interactions

• Ribosome incorporation efficiency tests

• Protein stability assessments under various cellular conditions

• Interaction studies with potential parasite binding partners

Engineered mutants mimicking or preventing specific PTMs (phosphomimetic or phosphodeficient mutations) can help establish the functional significance of individual modifications .

What is the potential of Brugia pahangi RPL26 as a diagnostic biomarker for filariasis?

Immunogenicity studies are essential to determine if natural infections elicit detectable antibody responses against RPL26. Using recombinant protein with >90% purity , researchers should evaluate serum from confirmed filariasis patients versus healthy controls and individuals with other helminth infections to establish specificity and sensitivity parameters. Cross-reactivity with human RPL26 must be carefully assessed to prevent false positives.

For antigen detection assays, researchers need to determine if RPL26 is detectable in patient specimens (blood, urine, or other fluids). This requires development of capture antibodies with high affinity for parasite RPL26 but minimal reactivity with host homologs. Sandwich ELISA configurations using the recombinant protein as a standard can establish detection limits and quantification ranges .

A multiplex approach combining RPL26 with other established filarial biomarkers could enhance diagnostic accuracy. Field-applicable formats such as lateral flow assays or microfluidic devices should be explored for point-of-care applications in endemic regions. Longitudinal studies are needed to determine if RPL26 levels correlate with parasite burden, disease progression, or treatment efficacy, potentially establishing its utility as a prognostic marker .

How can researchers investigate the immunomodulatory effects of Brugia pahangi RPL26 on host immunity?

Investigating the immunomodulatory effects of Brugia pahangi RPL26 requires a multi-faceted approach examining both innate and adaptive immune responses. Begin by exposing various immune cell populations (dendritic cells, macrophages, T-cells, B-cells) to purified recombinant RPL26 protein (>90% purity) and measure activation markers, cytokine production, and functional changes.

For dendritic cells, evaluate maturation status through flow cytometry analysis of surface markers (CD80, CD86, MHC-II) before and after RPL26 exposure. The response pattern may parallel the biphasic regulation observed with human RPL26 during LPS-induced dendritic cell maturation, where RPL26 translation is initially upregulated and later suppressed . Cytokine profiling should measure both pro-inflammatory (IL-12, TNF-α) and regulatory cytokines (IL-10, TGF-β).

Macrophage polarization studies can determine if RPL26 skews responses toward M1 (inflammatory) or M2 (regulatory) phenotypes, typical of many helminth-derived immunomodulatory molecules. For adaptive immunity, assess:

• T-cell differentiation in co-culture systems with RPL26-treated antigen-presenting cells

• Direct effects on T-cell subsets (Th1, Th2, Treg, Th17)

• B-cell activation, antibody production, and isotype switching

In vivo models using RPL26 administration in mice can evaluate systemic effects on immune responses to unrelated antigens or inflammatory challenges. Comparative studies with human RPL26 would determine if immunomodulatory effects are parasite-specific adaptations or conserved ribosomal protein functions .

Molecular mechanisms should be investigated through signaling pathway analysis, focusing on NF-κB, MAPK, and JAK/STAT pathways commonly targeted by parasite immunomodulators. RNA-seq of immune cells following RPL26 exposure can reveal broader transcriptional reprogramming events .

What structural and functional analyses would help identify potential drug targets within Brugia pahangi RPL26?

Comprehensive structural and functional analyses of Brugia pahangi RPL26 can reveal vulnerable sites for targeted drug development against filariasis. The initial approach should involve high-resolution structural determination of the full-length protein using X-ray crystallography or cryo-electron microscopy, building upon the known sequence data for the N-terminal fragment (AA 1-60) . Homology modeling based on solved structures of RPL26 from other species can provide preliminary structural insights while experimental structures are being developed.

Structure-function mapping studies should identify:

• RNA-binding domains essential for ribosomal incorporation

• Interfaces involved in interactions with other ribosomal proteins

• Regions mediating potential extraribosomal functions, similar to the p53 mRNA binding activity of human RPL26

• Surface-exposed epitopes unique to parasite RPL26 compared to human homologs

Functional assays to complement structural analysis include:

To identify druggable pockets, computational approaches like molecular dynamics simulations and binding site prediction algorithms should be applied to the structural data. Virtual screening of compound libraries targeting identified pockets can generate initial hit compounds for experimental validation. Fragment-based drug discovery approaches, starting with small chemical fragments that bind to specific protein sites, can be particularly effective for targeting protein-protein or protein-RNA interfaces .

Validation of potential inhibitors should assess:

• Binding affinity to parasite versus human RPL26

• Effects on ribosome assembly and function

• Antiparasitic activity in whole-organism assays

• Cytotoxicity in mammalian cells to establish therapeutic windows

How does Brugia pahangi RPL26 differ from RPL26 proteins in other nematode species?

Comparative analysis of Brugia pahangi RPL26 with homologs from other nematode species reveals both conservation patterns and species-specific variations that may reflect functional adaptations. At the sequence level, the N-terminal region (AA 1-60) of Brugia pahangi RPL26 shows high conservation among filarial nematodes (Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus), with identity typically >90%. This conservation reflects the fundamental role of this region in ribosome structure and function.

When compared to free-living nematodes like Caenorhabditis elegans, moderate sequence divergence becomes apparent, particularly in surface-exposed regions, while core functional domains remain conserved. This pattern suggests potential adaptation to parasitic lifestyle in filarial worms. Analysis of codon usage patterns across nematode RPL26 genes can provide insights into translational efficiency optimization within different host environments.

Functional differences may exist in:

• RNA binding specificity and affinity

• Interactions with other ribosomal components

• Potential extraribosomal functions (similar to the p53 regulation seen in humans)

• Post-translational modification patterns

• Immunogenicity and host immune interaction profiles

These comparative insights help identify both broadly applicable anti-nematode strategies and species-specific targeting approaches.

What methods are best for comparing the RNA binding properties of RPL26 across different filarial species?

To effectively compare RNA binding properties of RPL26 across different filarial species, researchers should employ a multi-method approach that provides both qualitative and quantitative insights. Begin by expressing recombinant RPL26 proteins from multiple filarial species (Brugia pahangi, Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus) using identical expression systems and purification protocols to ensure comparable starting materials . Yeast expression systems provide advantages for eukaryotic proteins, though E. coli systems may yield higher quantities for initial comparative studies.

RNA Electrophoretic Mobility Shift Assays (EMSAs) offer a straightforward approach for qualitative comparison, using identical RNA substrates with RPL26 proteins from different species. Begin with conserved ribosomal RNA sequences, then expand to potential regulatory target mRNAs. Quantitative methods should include:

• Surface Plasmon Resonance (SPR): Immobilize identical RNA sequences and flow different RPL26 proteins to determine comparative kinetic parameters (kon, koff, KD).

• Fluorescence Anisotropy: Label RNA probes with fluorophores and measure binding through changes in rotational diffusion, allowing direct comparison of binding affinities in solution.

• Isothermal Titration Calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, ΔG) that can reveal mechanistic differences in binding interactions across species.

For high-throughput comparison of RNA binding preferences, RNA Bind-n-Seq or similar approaches can identify preferred binding motifs for each species' RPL26, potentially revealing functional specializations. Structural analysis of RNA-protein complexes using NMR or X-ray crystallography can identify atomic-level differences in binding mechanisms .

Cross-linking and immunoprecipitation followed by sequencing (CLIP-seq) using native proteins from different parasites can reveal the actual RNA targets in vivo, though this requires availability of species-specific antibodies or expression of tagged proteins in the respective parasites.

How can researchers leverage comparative genomics to understand the evolution of RPL26 function in parasitic nematodes?

Leveraging comparative genomics to understand RPL26 evolution in parasitic nematodes requires integration of sequence, structural, and functional data across diverse species. Begin with comprehensive phylogenetic analysis using RPL26 sequences from parasitic nematodes (including Brugia pahangi ), free-living nematodes, and outgroups to establish evolutionary relationships and identify clade-specific patterns. Use maximum likelihood and Bayesian methods to construct robust phylogenetic trees, with special attention to branch length variations that may indicate rate heterogeneity.

Perform selection pressure analysis using dN/dS ratios to identify sites under positive, negative, or relaxed selection. Sites under positive selection may indicate adaptation to parasitism or host evasion functions, while conserved sites likely represent core ribosomal functions. Sliding window analysis can reveal domains with different evolutionary trajectories.

Synteny analysis comparing the genomic context of RPL26 genes across species can provide insights into regulatory evolution and potential gene duplication events. Analysis of upstream regulatory regions may reveal acquisition of parasite-specific expression patterns.

Molecular clock analyses calibrated with fossil data can estimate when functional divergence occurred in relation to the evolution of parasitism. Ancestral sequence reconstruction methods can predict the RPL26 sequence at key evolutionary nodes, allowing "resurrection" of ancestral proteins for functional testing.

For structure-function insights, map sequence variations onto predicted or experimentally determined structures to visualize how evolutionary changes might impact:

• RNA binding interfaces

• Protein-protein interaction sites

• Potential extraribosomal functions, like those observed with human RPL26 and p53 regulation

Cross-species complementation experiments, where RPL26 from one species is expressed in another, can test functional conservation and specialization. This approach can be particularly informative when comparing parasitic and free-living nematodes.

What are the challenges in expressing full-length Brugia pahangi RPL26 and strategies to overcome them?

Expressing full-length Brugia pahangi RPL26 presents several technical challenges not encountered with the truncated version (AA 1-60) typically available commercially . The primary challenge is protein solubility, as full-length ribosomal proteins often aggregate when expressed outside their native ribosomal context due to exposed hydrophobic surfaces normally buried within the ribosome structure. To address this, researchers should explore multiple expression strategies systematically.

For expression system selection, while E. coli offers high yield and simplicity, solubility issues are common. Using specialized E. coli strains (Rosetta, Arctic Express) can help with rare codon usage and folding. Yeast systems have proven successful for the N-terminal fragment and may work for the full-length protein with optimization. Insect cell and mammalian expression systems provide more sophisticated folding machinery but at higher cost and complexity.

Fusion tags can dramatically improve solubility:

• MBP (Maltose Binding Protein): Highly soluble, can be used with a cleavable linker

• SUMO: Enhances solubility and can be precisely removed with SUMO protease

• Thioredoxin: Smaller than MBP but effective for solubility enhancement

• GST: Offers solubility benefits and affinity purification options

Expression conditions require careful optimization, including:

• Temperature reduction (16-20°C) during induction

• Lower inducer concentrations for slower expression

• Rich vs. minimal media comparison

• Co-expression with chaperones (GroEL/ES, DnaK/J)

For purification, consider:

• Initial capture under denaturing conditions followed by refolding

• On-column refolding during affinity purification

• Size exclusion chromatography in buffers mimicking ribosomal environment

RNA co-expression or addition during purification may stabilize the protein by satisfying RNA-binding surfaces. Finally, protein engineering approaches such as surface entropy reduction or targeted mutation of aggregation-prone regions may improve solubility while maintaining functionality .

How can researchers optimize antibody production against Brugia pahangi RPL26 while minimizing cross-reactivity with host RPL26?

Developing antibodies against Brugia pahangi RPL26 with minimal cross-reactivity to host (human/animal) RPL26 requires strategic epitope selection and rigorous screening protocols. Begin with comprehensive sequence alignment of Brugia pahangi RPL26 against mammalian homologs to identify regions of low sequence conservation. While the N-terminal 60 amino acids available in recombinant form contain some conserved regions, focus on stretches showing <40% identity to host proteins.

For peptide antibody approaches:

• Select 2-3 divergent peptide sequences (15-20 amino acids) unique to parasite RPL26

• Ensure peptides are surface-exposed based on structural predictions

• Conjugate peptides to carrier proteins (KLH or BSA) for immunization

• Immunize multiple rabbits to increase chances of success

• Include proper adjuvants to enhance immunogenicity

For recombinant protein immunization:

• Use the available His-tagged recombinant fragment (AA 1-60) if it contains sufficient parasite-specific regions

• Consider expressing larger unique regions or full-length protein despite technical challenges

• Implement a subtraction strategy: pre-absorb sera with mammalian RPL26 to remove cross-reactive antibodies

Comprehensive screening is critical:

• ELISA against both parasite and host RPL26

• Western blots on parasite extract and host cell lysates

• Immunofluorescence on fixed parasite samples and host cells

• Immunoprecipitation to confirm specificity for native protein

For monoclonal antibody development, screen hybridoma supernatants against both parasite and host proteins simultaneously to identify clones with high specificity ratios. Consider phage display approaches with counter-selection against host RPL26 to enrich for parasite-specific binders.

Table 1: Comparison of Antibody Development Approaches for Brugia pahangi RPL26

What are the best approaches for studying potential interactions between Brugia pahangi RPL26 and host cell proteins?

Investigating interactions between Brugia pahangi RPL26 and host cell proteins requires methodical approaches that balance sensitivity with specificity. Begin with in vitro pull-down assays using His-tagged recombinant RPL26 (>90% purity) as bait and host cell lysates (macrophages, dendritic cells, or other relevant cell types) as prey. Eluted proteins can be identified through mass spectrometry, with stringent filtering to eliminate common contaminants and false positives. Control experiments using unrelated His-tagged proteins help distinguish specific from non-specific interactions.

For validation of identified interactions, reciprocal co-immunoprecipitation provides strong evidence. This approach mirrors techniques used to confirm human RPL26 interactions with MDM2 , but requires antibodies specific to Brugia pahangi RPL26 to avoid precipitating host RPL26 complexes. Proximity ligation assays (PLA) in parasite-infected or RPL26-treated cells can visualize interactions in situ with subcellular resolution.

To quantify binding parameters of validated interactions, surface plasmon resonance (SPR) or bio-layer interferometry (BLI) using purified components determines association/dissociation kinetics and binding affinities. Isothermal titration calorimetry (ITC) provides complementary thermodynamic parameters.

For functional characterization:

• Expression of interaction domains as peptide mimetics to disrupt specific interactions

• Site-directed mutagenesis of key residues in RPL26 to map interaction interfaces

• Cellular assays measuring functional outcomes (signaling activation, gene expression) when interactions are present versus disrupted

Chemical crosslinking coupled with mass spectrometry (CXMS) can map interaction interfaces at amino acid resolution. For high-throughput screening, yeast two-hybrid or mammalian two-hybrid screens using RPL26 as bait against host cDNA libraries can identify additional interaction partners .

A comparative approach examining interactions of RPL26 from both parasitic and free-living nematodes can reveal parasite-specific adaptations that may represent targets for intervention.

How might Brugia pahangi RPL26 be involved in regulating parasite stress responses similar to human RPL26's role in p53 regulation?

Investigation into Brugia pahangi RPL26's potential role in stress response regulation could reveal critical insights into parasite biology. Human RPL26 has a well-established extraribosomal function in binding to p53 mRNA following DNA damage, enhancing its translation and consequently increasing p53 protein levels and downstream stress responses . While nematodes lack p53 orthologs, they possess other stress-responsive pathways that might be regulated by RPL26 through similar mechanisms.

To explore this possibility, researchers should first identify parasite transcripts containing structured 5' and 3' UTRs reminiscent of human p53 mRNA, as these could be potential RPL26 binding targets. Computational approaches analyzing the parasite transcriptome for RNA structures similar to those bound by human RPL26 provide initial candidates. RNA immunoprecipitation (RIP) using antibodies against native or tagged RPL26 followed by sequencing can identify actual RNA targets in vivo .

Functional studies should examine RPL26 relocalization under various stress conditions (heat shock, oxidative stress, anthelmintic exposure) using immunofluorescence microscopy. If RPL26 exhibits stress-induced redistribution from the nucleolus to the nucleoplasm or cytoplasm, this would suggest potential regulatory roles similar to mammalian ribosomal proteins. Polysome profiling comparing normal and stress conditions can determine if specific mRNAs show RPL26-dependent translation enhancement during stress .

RNAi or CRISPR-based knockdown/knockout of RPL26 (if technically feasible in Brugia) followed by stress exposure would reveal its necessity for stress survival. Alternatively, transgenic overexpression of RPL26 might confer enhanced stress resistance if it positively regulates stress response pathways.

Comparison of potential regulatory mechanisms across filarial species and more distant nematodes could reveal evolutionary conservation or divergence of this potential moonlighting function .

What potential exists for developing RNA aptamers targeting Brugia pahangi RPL26 for therapeutic applications?

Developing RNA aptamers targeting Brugia pahangi RPL26 represents an innovative therapeutic approach with several potential advantages over conventional drugs. RNA aptamers—short, structured oligonucleotides that bind specifically to target proteins—could disrupt critical RPL26 functions while achieving parasite selectivity through targeted design. The development process would begin with Systematic Evolution of Ligands by Exponential Enrichment (SELEX) using purified recombinant Brugia pahangi RPL26 protein (>90% purity) as the target.

A counter-selection strategy incorporating human RPL26 during SELEX would eliminate aptamer candidates that cross-react with the host protein, enhancing parasite specificity. Multiple SELEX rounds with increasing stringency can yield aptamers with nanomolar or better affinity. High-throughput sequencing of enriched aptamer pools followed by clustering analysis can identify families of related aptamers for further characterization.

Functional screening should evaluate aptamers for their ability to:

• Disrupt RPL26 incorporation into ribosomes

• Interfere with potential extraribosomal functions

• Inhibit parasite growth in culture

• Demonstrate minimal toxicity to mammalian cells

Structure determination of aptamer-RPL26 complexes using crystallography or cryo-electron microscopy would guide rational optimization. For therapeutic development, aptamers would require chemical modifications to enhance stability in biological fluids and tissues:

• 2'-O-methyl or 2'-fluoro substitutions to resist nuclease degradation

• Phosphorothioate linkages at strategic positions

• 3' end capping to prevent exonuclease attack

• Conjugation to carriers for improved pharmacokinetics

Delivery strategies for filarial infections might include encapsulation in lipid nanoparticles or conjugation to cell-penetrating peptides. In vivo testing in animal models of filariasis would assess efficacy, toxicity, immunogenicity, and pharmacokinetics .

How can functional genomics approaches be applied to study the roles of RPL26 in Brugia pahangi life cycle stages?

Applying functional genomics to investigate RPL26 roles across Brugia pahangi life cycle stages requires innovative approaches adapted to this challenging parasite system. Begin with comprehensive transcriptomic profiling (RNA-seq) of all accessible life stages (microfilariae, L3 larvae, adult males and females) to establish RPL26 expression patterns relative to other ribosomal proteins and the global transcriptome. Ribosome profiling (Ribo-seq) can determine if RPL26 incorporation into ribosomes varies across stages, potentially indicating specialized translational landscapes .

For direct functional analysis, RNAi approaches offer the most accessible intervention in filarial nematodes. Design multiple siRNAs targeting RPL26 mRNA and deliver via soaking or electroporation to various parasite stages. Monitor effects on:

If stable transfection systems become available, CRISPR/Cas9 editing could generate hypomorphic RPL26 alleles or tagged versions for pulldown experiments. Conditional knockdown systems utilizing tetracycline-responsive promoters would allow temporal control of RPL26 depletion to identify stage-specific requirements .

For proteomics approaches, quantitative mass spectrometry comparing the interactome of RPL26 across life cycle stages can reveal stage-specific protein interactions. Parallel reaction monitoring (PRM) can quantify RPL26 protein levels with high precision across stages and subcellular fractions.

For systems-level analysis, integrate transcriptomic, proteomic, and phenotypic data from RPL26 perturbation experiments to build gene regulatory networks. This can reveal how RPL26 functions within broader molecular networks during parasite development and host adaptation .

Cross-species complementation experiments, introducing Brugia pahangi RPL26 into C. elegans RPL26 mutants, can provide insights into functional conservation and specialization between free-living and parasitic nematodes.