Recombinant Pelophylax ridibundus Brevinin-2R

What is Brevinin-2R and what is its structural composition?

Brevinin-2R is a cationic antimicrobial peptide (AMP) belonging to the defensin family isolated from the skin secretions of Pelophylax ridibundus frogs. It has a unique amino acid sequence of KLKNFAKGVAQSLLNKASCKLSGQC, consisting of 25 amino acids, making it the shortest known defensin family member currently identified . The peptide contains a positively charged region with a centrally located hinge-forming region and a C-terminal hepta-membered ring stabilized by a disulfide bridge, which is characteristic of peptides isolated from Ranid frogs . Structurally, Brevinin-2R most closely resembles Brevinin-2Ee and Brevinin-2Ej from Rana esculenta . Like other antimicrobial peptides, it is cationic, relatively hydrophobic, and forms an amphipathic helix in membrane-mimetic environments .

How does Brevinin-2R differ from other brevinins in terms of activity?

Unlike most other brevinins, Brevinin-2R shows no significant hemolytic activity, making it particularly interesting for therapeutic applications . This unique property distinguishes it from other members of the brevinin family, which typically display considerable hemolytic effects. The non-hemolytic nature of Brevinin-2R has prompted researchers to investigate its potential anticancer properties across multiple cancer cell lines . While it shares the typical structural features of defensins, including the cationic nature and disulfide-bridged C-terminal region, its specific amino acid sequence confers this distinctive non-hemolytic property .

What cell lines have been used to evaluate Brevinin-2R's anticancer activity?

Brevinin-2R has been tested against multiple cancer cell lines, demonstrating preferential cytotoxicity toward malignant cells. Studies have evaluated its effects on Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219 and SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), and A549 (lung carcinoma) . Comparative studies with normal cells including peripheral blood mononuclear cells (PBMC), T cells, and human lung fibroblasts have shown that Brevinin-2R exhibits semi-selective cytotoxicity, preferentially killing cancer cells while having less effect on normal cells . This selectivity makes it a promising candidate for targeted cancer therapy development.

What experimental methods are commonly used to isolate and purify Brevinin-2R?

The isolation and purification of Brevinin-2R from frog skin secretions typically involves a multistep chromatographic process. Initially, crude peptide fractions are obtained from the skin secretions of Pelophylax ridibundus and subjected to a series of purification steps . The process generally includes:

• Collection of skin secretions from the frog

• Initial fractionation using reversed-phase chromatography

• Further purification of selected fractions using a Vydac semi-preparative C8 reversed-phase column

• Additional purification steps using a Macherey-Nagel semi-preparative C18 reversed-phase column

• Final confirmation of purity using analytical C8 reversed-phase chromatography

• Mass spectrometric analysis to determine the peptide sequence

This multi-step purification procedure ensures the isolation of pure Brevinin-2R for experimental studies.

What is the detailed mechanism of Brevinin-2R's selective anticancer activity?

Brevinin-2R kills cancer cells through a distinct lysosomal-mitochondrial death pathway that differs from conventional apoptotic mechanisms. Early indicators of Brevinin-2R-triggered death include decreased mitochondrial membrane potential (ΔΨm), reduced total cellular ATP levels, and increased reactive oxygen species (ROS) production . Importantly, caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G) are not observed, indicating a non-classical death pathway .

The mechanism involves several key steps:

• Interaction with both early and late endosomes

• Lysosomal membrane permeabilization

• Release of cathepsins (B and L) from lysosomes

• Mitochondrial damage and dysfunction

• Formation of autophagosomes

• Cell death resembling autophagy-like cell death

The selective nature of this cytotoxicity appears to be related to differences in membrane composition between cancer and normal cells, as well as differences in metabolic activities, though the precise targeting mechanism requires further investigation.

How do genetic modifications in target cells affect Brevinin-2R's efficacy?

Genetic modifications in target cells significantly impact Brevinin-2R's efficacy, providing important insights into its mechanism of action. Studies have shown that cells overexpressing the anti-apoptotic protein Bcl2 display substantial resistance to Brevinin-2R treatment . Specifically, Jurkat and MCF-7 cells overexpressing Bcl2 were largely protected from Brevinin-2R-induced cell death . Similarly, L929 and MCF-7 cells overexpressing a dominant-negative mutant of the pro-apoptotic protein BNIP3 (ΔTM-BNIP3) also exhibited significant resistance to Brevinin-2R treatment .

These findings suggest that despite not following a classical apoptotic pathway, Brevinin-2R-induced cell death involves the mitochondrial pathway regulated by Bcl2 family proteins. Researchers investigating Brevinin-2R should consider these genetic factors when designing experiments, as they may significantly impact experimental outcomes and interpretation of results.

What experimental methods can be used to investigate Brevinin-2R's interaction with cellular components?

Several experimental approaches can be employed to investigate Brevinin-2R's interactions with cellular components:

• FITC-labeling assay: FITC-labeled Brevinin-2R can be used to study binding to cell surfaces. Cells are incubated with FITC-labeled peptide, washed, and analyzed by flow cytometry to quantify binding. Nonspecific binding can be determined using FITC-labeled scrambled Brevinin-2R as a control .

• Immunofluorescence microscopy: Cells treated with Brevinin-2R can be fixed, permeabilized, and stained with antibodies against cellular components (e.g., endosomal markers like EEA-1 for early endosomes, mannose 6-phosphate for late endosomes, and LAMP-1 for lysosomes) to visualize co-localization .

• Mitochondrial function assays: Changes in mitochondrial membrane potential can be measured using specific dyes or probes. ATP levels and ROS production can also be quantified using appropriate assays .

• Lysosomal function assays: Lysosomal membrane permeabilization can be assessed using lysosomal-specific dyes or by monitoring the release of lysosomal enzymes into the cytosol .

• Immunoblotting: Expression of relevant proteins (e.g., Bcl2, Bcl-XL, Mcl-1, Bax, Bad) can be detected by immunoblotting to understand the molecular pathways involved .

These methods provide comprehensive insights into Brevinin-2R's cellular interactions and mechanism of action.

How does conjugation with fatty acids alter Brevinin-2R's biological activity?

The mechanism of action of the conjugated peptide involves:

• Cell membrane disruption

• Changes in electrical membrane potential

• Alterations in mitochondrial membrane potential

Interestingly, despite its increased in vitro cytotoxicity and hemolytic effects, L-Brevinin-2R did not show site-specific adverse reactions in animal models. In L. major-infected mice, treatment with L-Brevinin-2R resulted in decreased parasite load in lymph nodes adjacent to the infection site . This suggests that fatty acid conjugation may enhance the therapeutic potential of Brevinin-2R in vivo, possibly by improving its stability, tissue penetration, or target specificity.

What analytical techniques are most effective for characterizing recombinant Brevinin-2R?

Effective characterization of recombinant Brevinin-2R requires a combination of analytical techniques:

• Mass spectrometry: Essential for confirming the correct amino acid sequence and molecular weight of the recombinant peptide. MS can also identify any post-translational modifications or truncations .

• Reversed-phase HPLC: Critical for assessing peptide purity, with analytical C8 or C18 columns being particularly useful for Brevinin-2R characterization .

• Circular dichroism (CD) spectroscopy: Useful for analyzing the secondary structure of the peptide, particularly its tendency to form amphipathic helices in membrane-mimetic environments.

• Nuclear magnetic resonance (NMR) spectroscopy: Provides detailed structural information, including the three-dimensional arrangement of the peptide.

• Disulfide bond analysis: Important for confirming the correct formation of the C-terminal disulfide bridge that stabilizes the hepta-membered ring structure characteristic of Brevinin-2R .

• Functional assays: Including antimicrobial, cytotoxicity, and hemolytic assays to confirm that the recombinant peptide retains the biological activities of the native peptide.

• Phylogenetic analysis: Comparison with other brevinin family members to establish evolutionary relationships, as has been done with 25 Brevinin-2 amino acid sequences from six Rana species .

What experimental controls should be included when studying Brevinin-2R's anticancer effects?

When investigating Brevinin-2R's anticancer effects, several critical controls should be included:

• Scrambled peptide control: A peptide with the same amino acid composition but a randomized sequence (e.g., KFALGKVNAKLQSLNAKSLKQSGCC) should be used to demonstrate sequence-specific effects .

• Cell viability assays: Multiple assays (e.g., MTT assay, trypan blue exclusion) should be performed to ensure robust measurement of cell death .

• Normal cell controls: Primary cells such as peripheral blood mononuclear cells (PBMC), T cells, and human lung fibroblasts should be included to assess cancer cell selectivity .

• Genetic modification controls: Cells overexpressing anti-apoptotic proteins (e.g., Bcl2) or expressing dominant-negative mutants of pro-apoptotic proteins (e.g., ΔTM-BNIP3) provide valuable insights into the mechanism of action .

• Inhibitor controls: Various inhibitors should be tested, including lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L, to elucidate the death pathway .

• Time-course and dose-response studies: These are essential to understand the kinetics and concentration-dependence of Brevinin-2R's effects.

These controls ensure the specificity and mechanistic understanding of Brevinin-2R's anticancer activities.

What are the most effective expression systems for producing recombinant Brevinin-2R?

While the search results don't specifically discuss expression systems for recombinant Brevinin-2R, based on research with similar antimicrobial peptides, several expression systems could be considered:

• Bacterial expression systems: E. coli-based systems are commonly used for antimicrobial peptides, though care must be taken to prevent toxicity to the host cells. Expression as fusion proteins with partners like thioredoxin, SUMO, or glutathione S-transferase can mitigate toxicity and improve solubility.

• Yeast expression systems: Pichia pastoris or Saccharomyces cerevisiae may offer advantages for correct disulfide bond formation, which is critical for Brevinin-2R's C-terminal hepta-membered ring structure .

• Cell-free expression systems: These can circumvent toxicity issues and may be suitable for producing Brevinin-2R without the complications of cellular expression.

• Chemically synthesized peptides: Given Brevinin-2R's relatively small size (25 amino acids) , chemical synthesis may be a practical alternative to recombinant expression, allowing for precise control over the sequence and potential modifications.

The selection of an expression system should consider factors such as yield, correct folding, disulfide bond formation, ease of purification, and cost-effectiveness.

How can the therapeutic window of Brevinin-2R be optimized for potential clinical applications?

Optimizing Brevinin-2R's therapeutic window for potential clinical applications requires addressing several key aspects:

• Structural modifications: Specific amino acid substitutions could enhance selectivity for cancer cells while reducing toxicity to normal cells. The non-hemolytic nature of Brevinin-2R already provides an advantage over other brevinin family members .

• Conjugation strategies: As demonstrated with lauric acid conjugation, attaching specific molecules to Brevinin-2R can enhance its activity . Other conjugation partners, including cell-targeting ligands or PEGylation, might improve pharmacokinetics and reduce off-target effects.

• Delivery systems: Encapsulation in liposomes, nanoparticles, or other drug delivery vehicles could enhance tumor targeting while minimizing systemic exposure.

• Combination therapies: Combining Brevinin-2R with conventional cancer therapies might allow for lower doses, reducing toxicity while maintaining efficacy. Since Brevinin-2R activates a distinct lysosomal-mitochondrial death pathway , it might synergize with therapies targeting other cell death mechanisms.

• Dosing schedules: Optimized administration protocols could maximize anticancer effects while minimizing toxicity to normal tissues.

Research in these areas could help translate Brevinin-2R's promising selective cytotoxicity into clinically viable therapeutic applications.

How does Brevinin-2R compare with other antimicrobial peptides in terms of structure and function?

Brevinin-2R shares features with other antimicrobial peptides but also possesses distinctive characteristics:

Brevinin-2R stands out for its unique combination of non-hemolytic properties and selective cytotoxicity toward cancer cells, which distinguishes it from many other antimicrobial peptides that often exhibit significant hemolytic activity .

What structural features of Brevinin-2R contribute to its selective cytotoxicity against cancer cells?

Several structural features of Brevinin-2R likely contribute to its selective cytotoxicity against cancer cells:

• Cationic nature: Brevinin-2R's positive charge may facilitate interaction with negatively charged components of cancer cell membranes, which often have altered membrane composition compared to normal cells .

• Amphipathic helical structure: In membrane environments, Brevinin-2R likely forms an amphipathic helix with hydrophobic and hydrophilic faces, enabling membrane interaction and potential disruption .

• C-terminal disulfide bridge: The hepta-membered ring stabilized by a disulfide bridge at the C-terminus is a characteristic feature that may contribute to its specific interactions with cellular components .

• Sequence specificity: The unique amino acid sequence of Brevinin-2R (KLKNFAKGVAQSLLNKASCKLSGQC) confers specific properties that distinguish it from other brevinins . The centrally located hinge-forming region may allow for conformational flexibility when interacting with different cellular targets.

• Non-hemolytic property: Unlike most other brevinins, Brevinin-2R lacks significant hemolytic activity , suggesting specific structural features that prevent disruption of red blood cell membranes while still enabling interaction with cancer cell membranes.

Understanding these structure-function relationships could guide the design of optimized peptides with enhanced anticancer activity and selectivity.

How do the anticancer mechanisms of Brevinin-2R differ from conventional chemotherapeutic agents?

Brevinin-2R's anticancer mechanisms differ substantially from conventional chemotherapeutic agents in several key aspects:

• Cell death pathway: Brevinin-2R activates a distinct lysosomal-mitochondrial death pathway , whereas many conventional chemotherapeutics primarily induce apoptosis through DNA damage or disruption of microtubule assembly.

• Caspase independence: Unlike many chemotherapeutics that activate caspase-dependent apoptosis, Brevinin-2R-induced cell death is not associated with caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G) .

• Lysosomal involvement: Brevinin-2R interacts with both early and late endosomes and induces lysosomal membrane permeabilization, with cathepsin B and L playing critical roles in the death process . This lysosomal targeting is uncommon among conventional chemotherapeutics.

• Autophagy-like features: Autophagosomes have been detected upon Brevinin-2R treatment, suggesting an autophagy-like cell death mechanism , which differs from the primarily apoptotic mechanisms of most chemotherapeutics.

• Selective targeting: Brevinin-2R exhibits preferential cytotoxicity toward malignant cells compared to normal cells , potentially offering improved selectivity over many conventional chemotherapeutics that target all rapidly dividing cells.

These distinct mechanisms suggest that Brevinin-2R may overcome resistance to conventional chemotherapeutics and could be effective in combination therapies targeting multiple cell death pathways.

What are the most promising applications of recombinant Brevinin-2R in cancer research?

Recombinant Brevinin-2R shows several promising applications in cancer research:

• Novel therapeutic development: Brevinin-2R's selective cytotoxicity toward cancer cells and unique death mechanism make it a promising lead molecule for developing new anticancer therapeutics . Its non-hemolytic nature gives it an advantage over other similar peptides .

• Combination therapy research: Investigating synergistic effects between Brevinin-2R and conventional chemotherapeutics could lead to more effective treatment regimens with reduced side effects. Since Brevinin-2R activates a distinct lysosomal-mitochondrial death pathway , it may complement therapies targeting other cell death mechanisms.

• Overcoming treatment resistance: Brevinin-2R's mechanism of action differs from conventional chemotherapeutics, potentially making it effective against drug-resistant cancers. Studies on genetic modifications affecting sensitivity (e.g., Bcl2 overexpression) provide insights into potential resistance mechanisms.

• Drug delivery systems: Conjugated or modified versions of Brevinin-2R could serve as targeting moieties in drug delivery systems, as demonstrated by the altered activity profile of lauric acid-conjugated Brevinin-2R .

• Biomarker discovery: Understanding which genetic or molecular features determine sensitivity to Brevinin-2R could lead to the identification of new biomarkers for cancer stratification and personalized treatment approaches.

What methodological challenges need to be addressed in studying Brevinin-2R's interactions with complex cellular systems?

Several methodological challenges must be addressed when studying Brevinin-2R's interactions with complex cellular systems:

• Maintaining peptide stability: Antimicrobial peptides like Brevinin-2R can be susceptible to proteolytic degradation in biological systems. Developing stable formulations or protective delivery systems is crucial for consistent experimental results.

• Tracking intracellular localization: Visualizing Brevinin-2R's interactions with subcellular compartments, particularly endosomes and lysosomes, requires sophisticated imaging techniques. FITC-labeled Brevinin-2R has been used , but additional approaches may be needed for real-time tracking in living cells.

• Distinguishing direct and indirect effects: Determining whether observed cellular responses are direct results of Brevinin-2R or secondary consequences requires careful experimental design, including appropriate controls like scrambled peptides .

• Quantifying membrane interactions: Brevinin-2R's interactions with cell membranes are likely critical to its mechanism, but quantifying these interactions in complex cellular systems presents technical challenges.

• Translating in vitro findings to in vivo models: As seen with lauric acid-conjugated Brevinin-2R, in vitro cytotoxicity doesn't always predict in vivo effects . Developing appropriate animal models that recapitulate human disease is essential for translational research.

• Standardizing recombinant production: Ensuring consistent quality and activity of recombinantly produced Brevinin-2R across different studies requires standardized expression and purification protocols.

Addressing these challenges will facilitate more robust and reproducible research on Brevinin-2R's potential therapeutic applications.

How might genetic engineering approaches enhance the therapeutic potential of Brevinin-2R?

Genetic engineering approaches offer several strategies to enhance Brevinin-2R's therapeutic potential:

• Amino acid substitutions: Systematic mutation of specific residues could enhance cancer cell selectivity, increase stability, or reduce potential immunogenicity. Given Brevinin-2R's small size (25 amino acids) , comprehensive alanine scanning or site-directed mutagenesis is feasible.

• Domain fusion: Creating fusion proteins that combine Brevinin-2R with cancer-targeting domains (e.g., antibody fragments, peptide ligands) could improve tumor specificity and reduce off-target effects.

• Multimerization: Designing multimeric versions of Brevinin-2R might enhance potency through avidity effects while potentially maintaining selectivity.

• Incorporation of unnatural amino acids: Using expanded genetic code technologies to incorporate non-standard amino acids could introduce novel properties not achievable with the 20 natural amino acids.

• Expression optimization: Codon optimization for specific expression systems could improve recombinant production yields and quality, facilitating larger-scale studies and potential therapeutic development.

• Conditional activation systems: Engineering Brevinin-2R variants that become active only under specific conditions found in the tumor microenvironment (e.g., acidic pH, specific proteases) could further enhance selectivity.