Recombinant Pseudoderopeltis foveolata Pyrokinin-5

What is Pyrokinin-5 and what organism does it originate from?

Pyrokinin-5 is a neuropeptide belonging to the pyrokinin family isolated from Pseudoderopeltis foveolata, a cockroach species . It is characterized by the common C-terminal pentapeptide sequence -FXPRL found in all pyrokinins, where X can be T, S, G, or V . Specifically, Pyrokinin-5 from P. foveolata contains the sequence GGGGSGETSGMWFGPRL, as indicated in recombinant protein specifications . Pyrokinins are produced primarily by neurosecretory cells located in the suboesophageal, abdominal, and thoracic ganglions of the ventral nerve cord and are subsequently secreted into the insect hemolymph .

What is the amino acid sequence and molecular characteristics of Recombinant Pseudoderopeltis foveolata Pyrokinin-5?

Recombinant Pseudoderopeltis foveolata Pyrokinin-5 has the following characteristics:

• Full sequence: GGGGSGETSGMWFGPRL

• Alternative sequence form: H-Ser-Ala-Ser-Ser-Gly-Glu-Ser-Ser-Gly-Met-Trp-Phe-Gly-Pro-Arg-Leu-NH2

• Molecular weight: 1654.75 Da

• Molecular formula: C71H107N21O23S

• Expression region: 1-17 amino acids

• C-terminal modification: The peptide typically has an amidated C-terminus (Leu-NH2)

• Purity: >85% as determined by SDS-PAGE for the recombinant product , with some synthetic versions available at higher purity levels (96.4%)

How should Recombinant Pseudoderopeltis foveolata Pyrokinin-5 be properly stored and reconstituted for experimental use?

For optimal stability and experimental reproducibility, Recombinant Pseudoderopeltis foveolata Pyrokinin-5 should be handled according to these guidelines:

Storage:

• Store lyophilized peptide at -20°C for regular storage

• For extended storage, conserve at -20°C or -80°C

• The shelf life of lyophilized form is approximately 12 months at -20°C/-80°C

• The shelf life of liquid form is approximately 6 months at -20°C/-80°C

Reconstitution:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) and aliquot for long-term storage (the default final concentration of glycerol is typically 50%)

Working guidelines:

• Repeated freezing and thawing is not recommended

• Working aliquots can be stored at 4°C for up to one week

What are the physiological functions of Pyrokinins in insect systems?

Pyrokinins serve multiple critical physiological functions in insects, making them important targets for both basic research and potential applied entomology applications:

• Myotropic activity: Stimulation of intestinal muscle contraction

• Pheromonotropic activity: Regulation of pheromone biosynthesis pathways through PBANs (Pheromone Biosynthesis Activating Neuropeptides), which are 33 amino acid peptides related to pyrokinins

• Regulation of secretion by Malpighian tubules, which are critical for insect excretion and osmoregulation

• Control of molting processes, essential for insect development

• Modulation of digestive enzyme release in the insect intestine

• Melanotropic roles, influencing pigmentation in certain insect species

The diverse regulatory functions of pyrokinins across multiple physiological systems make them valuable targets for understanding fundamental insect biology and potential pest management strategies.

What methodological approaches can be used to study receptor binding and functional activity of Pyrokinin-5?

Studying the receptor binding and functional activity of Pyrokinin-5 requires specialized methodological approaches:

Receptor characterization:

• RNA extraction and cDNA synthesis from target tissues:

  • Dissect relevant tissues (midgut, Malpighian tubules, reproductive organs) from study insects

  • Submerge tissues in RNA lysis buffer containing 1% 2-mercaptoethanol

  • Extract total RNA using appropriate RNA isolation kit (e.g., EZ-10 RNA Miniprep Kit)

  • Quantify RNA using spectrophotometric methods

  • Synthesize cDNA using 25 ng total RNA as template with appropriate reverse transcription reagents

• Structure-activity relationship studies:

  • Analysis of the C-terminal sequence -FSPRLa, which has been identified as the shortest peptide fragment responsible for activity

  • Substitution studies, such as replacing serine or arginine with D-isomer of phenylalanine to understand binding requirements

  • Development of cyclic analogs to improve stability against peptidases while maintaining activity

• Receptor antagonist development:

  • Synthesis of molecules that effectively block the binding site of the peptide in the receptor

  • Conformational modification through cyclization using side chains

These methodologies provide a comprehensive approach to understanding both the molecular interactions of Pyrokinin-5 with its receptors and the downstream physiological effects.

What challenges are associated with experimental use of synthetic Pyrokinin-5 and how can they be addressed?

Researchers working with synthetic Pyrokinin-5 face several important experimental challenges:

• TFA (Trifluoroacetic acid) contamination:

  • TFA is used during peptide synthesis and often remains as residual salt in the final product

  • TFA can cause unpredictable fluctuations in experimental data

  • At nanomolar (nM) concentrations, TFA can influence cell experiments:

    • It can inhibit cell growth at concentrations as low as 10 nM

    • It can promote cell growth at higher concentrations (0.5-7.0 mM)

  • TFA can act as an allosteric regulator on glycine receptors (GlyR)

  • In vivo, TFA can trifluoroacetylate amino groups in proteins and phospholipids, potentially inducing unwanted antibody responses

Mitigation strategies:

• Request TFA removal during peptide synthesis

• Use alternative counterions like acetate or hydrochloride

• Validate experimental outcomes with appropriate controls to account for TFA effects

• Consider dialysis or reverse-phase chromatography to remove TFA before sensitive experiments

• Stability and degradation concerns:

  • The hydrophilic nature of pyrokinins makes it difficult for them to penetrate through hydrophobic insect cuticle

  • Rapid degradation occurs in the insect digestive tract by intestinal peptidases

Mitigation strategies:

• Development of synthetic analogs with modified chemical properties

• Use of cyclic forms that are less susceptible to degradation by peptidases

• For example, a cyclic PBAN analogue: cyclo[ASN1]Leuma-PK showed 70% activity compared to natural PBAN at 100 nM concentration, with improved resistance to peptidase degradation

How does Pyrokinin-5 compare structurally and functionally to other members of the Pyrokinin family?

Pyrokinins represent a diverse family of neuropeptides with varying structures but conserved functional domains:

Structural characteristics:

• All pyrokinins share the conserved C-terminal pentapeptide motif -FXPRL, where X can be T, S, G, or V

• Sequence variations:

  • Simple pyrokinins (PKs): Short peptides with few amino acids

  • Complex pyrokinins like PBAN, diapause hormone, or melanization and reddish coloration hormones (MRCH): Longer peptides with tens of amino acids

  • Pyrokinin-5 from P. foveolata: 17 amino acids with the sequence GGGGSGETSGMWFGPRL

  • Pyrokinin-6 from P. foveolata is also documented, showing the diversity within a single species

• Table: Comparison of Pyrokinin Family Members

Functional relationships:

• The shortest peptide fragment responsible for the activity of the molecule is the C-terminal sequence -FSPRLa

• PBANs (Pheromone Biosynthesis Activating Neuropeptides) are functionally specialized pyrokinins that regulate pheromone biosynthesis pathways - substances responsible for attracting the opposite sex

• Structure-activity studies have shown that modifications to the C-terminal region significantly affect binding and activity

Understanding these structural relationships helps researchers design experiments to probe specific aspects of pyrokinin function and develop targeted interventions for research or potential pest management applications.

What potential applications exist for Recombinant Pseudoderopeltis foveolata Pyrokinin-5 in insect control research?

Pyrokinins, including Pyrokinin-5, show promising potential as alternative bioinsecticides due to their specific action on insect physiology:

Research applications:

• Development of PBAN antagonists:

  • Studies of pyrokinin receptor structure and identification of domains responsible for ligand interaction

  • Construction of molecules that block the binding site, arresting reactions initiated by peptide hormones

  • Conformational changes of peptides through cyclization using side chains

• Advantages as bioinsecticides:

  • Specificity to insects, reducing off-target effects on non-target organisms

  • The pyrokinin-based approach targets specific physiological systems in insects

  • Potential for lower environmental persistence compared to conventional insecticides

• Challenges to overcome:

  • The hydrophilic nature of pyrokinins makes cuticular penetration difficult

  • Rapid degradation by insect peptidases limits effectiveness

  • Delivery mechanisms need development for field applications

• Synthetic analogs research:

  • Addition of various chemical groups to change physical and chemical properties

  • Development of bicyclic analogs with improved stability

  • For example, the cyclic form cyclo[ASN1]Leuma-PK showed 70% activity compared to natural PBAN at 100 nM concentration, while its linear counterpart showed no activity at concentrations below 100 nM

This research direction addresses growing concerns about conventional insecticides, which are associated with human poisonings (250,000-370,000 cases globally per year), environmental persistence, bioaccumulation, and ecosystem disruption .

What are the recommended protocols for in vitro studies using Recombinant Pseudoderopeltis foveolata Pyrokinin-5?

When designing in vitro experiments with Recombinant Pseudoderopeltis foveolata Pyrokinin-5, researchers should consider the following protocol recommendations:

Sample preparation:

• Reconstitution of lyophilized peptide:

  • Briefly centrifuge the vial prior to opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% for storage stability

  • Aliquot to minimize freeze-thaw cycles

• Working solution preparation:

  • For receptor binding studies, prepare dilutions in appropriate assay buffer

  • For cell-based assays, consider potential TFA effects and adjust accordingly

  • Store working aliquots at 4°C for up to one week

Quality control measures:

• Verify peptide integrity by:

  • HPLC analysis to confirm purity (target >85% for recombinant, >95% for synthetic)

  • Mass spectrometry to verify molecular weight

  • SDS-PAGE for recombinant protein verification

• Positive and negative controls:

  • Include known active pyrokinins as positive controls

  • Use scrambled or inactive peptide analogs as negative controls

  • Consider including TFA-only controls to account for potential TFA effects

These protocols help ensure experimental reproducibility and reliable results when working with Recombinant Pseudoderopeltis foveolata Pyrokinin-5 in vitro.

How can researchers optimize tissue-specific expression studies for Pyrokinin-5 receptors?

For researchers investigating tissue-specific expression of Pyrokinin-5 receptors, the following methodological approach is recommended:

Tissue collection and preparation:

• Immobilize experimental insects (e.g., mosquitoes) with brief exposure to CO2

• Submerge specimens in nuclease-free Dulbecco's phosphate-buffered saline (DPBS)

• Carefully dissect relevant tissues:

  • Midgut

  • Malpighian tubules

  • Pyloric valve (midgut-hindgut junction)

  • Ileum

  • Rectum

  • Reproductive organs (including ovaries, common and lateral oviducts, spermathecae)

RNA extraction:

• Transfer dissected tissues into RNA lysis buffer containing 1% 2-mercaptoethanol

• For whole-body samples, submerge 7-8 specimens in RNA lysis buffer and homogenize

• Extract total RNA using an appropriate kit (e.g., EZ-10 RNA Miniprep Kit)

• Quantify purified RNA using a microvolume spectrophotometer

• Synthesize cDNA using 25 ng total RNA as template

Expression analysis:

• Design primers specific to pyrokinin receptor sequences

• Perform qRT-PCR to quantify tissue-specific expression levels

• Use appropriate housekeeping genes as internal controls

• Analyze data using the comparative CT method (2^-ΔΔCT)

This comprehensive approach allows for detailed mapping of pyrokinin receptor expression across different tissues, providing insights into potential physiological roles and target sites for further functional studies.

What are the current research gaps in Pyrokinin-5 studies?

Despite significant advances in understanding Pyrokinin-5 and related neuropeptides, several important research gaps remain:

• Receptor diversity and cross-reactivity:

  • Limited understanding of how different pyrokinin family members interact with various receptor subtypes

  • Need for comprehensive characterization of receptor-ligand specificity

  • Investigation of potential cross-reactivity with other neuropeptide systems

• Signaling pathways:

  • Incomplete knowledge of downstream signaling cascades activated by Pyrokinin-5

  • Limited understanding of intracellular mechanisms mediating diverse physiological effects

  • Need for systems biology approaches to map complex regulatory networks

• Evolutionary aspects:

  • Further exploration of pyrokinin diversity across insect taxa

  • Comparative studies to understand evolutionary conservation and divergence

  • Investigation of potential analogous systems in non-insect arthropods

• Applied aspects:

  • Development of effective delivery systems for pyrokinin-based bioinsecticides

  • Long-term safety and environmental impact studies

  • Resistance management strategies for potential pyrokinin-based pest control

Addressing these research gaps would significantly advance both fundamental understanding of insect neuroendocrinology and potential applications in pest management.

What interdisciplinary approaches might advance Pyrokinin-5 research?

Advancing Pyrokinin-5 research will likely require interdisciplinary approaches combining:

• Structural biology and computational modeling:

  • Advanced crystallography or cryo-EM studies of pyrokinin-receptor complexes

  • Molecular dynamics simulations to understand binding mechanisms

  • In silico design of optimized pyrokinin analogs or antagonists

• Chemical biology:

  • Development of photoactivatable or fluorescent pyrokinin analogs

  • Click chemistry approaches for tracking pyrokinin distribution in vivo

  • Peptidomimetic design to overcome stability and delivery limitations

• Genomics and transcriptomics:

  • Comparative analysis across insect species to identify conserved regulatory elements

  • Single-cell transcriptomics to map cellular responses to pyrokinin signaling

  • CRISPR-Cas9 gene editing to probe receptor function in vivo

• Ecotoxicology and environmental science:

  • Assessment of ecological impacts of pyrokinin-based interventions

  • Development of targeted delivery systems with minimal off-target effects

  • Integration with integrated pest management (IPM) approaches