Function and Mechanism of Antiviral Wasp Venom Peptide Protopolybia-MP III and Its Derivatives against HSV-1.

Viruses are one of the leading causes of human disease, and many highly pathogenic viruses still have no specific treatment drugs. Therefore, producing new antiviral drugs is an urgent matter. In our study, we first found that the natural wasp venom peptide Protopolybia-MP III had a significant inhibitory effect on herpes simplex virus type 1 (HSV-1) replication in vitro by using quantitative real-time PCR (qPCR), Western blotting, and plaque-forming assays. Immunofluorescence analysis showed Protopolybia-MP III could enter cells, and it inhibited multiple stages of the HSV-1 life cycle, including the attachment, entry/fusion, and post-entry stages. Furthermore, ultracentrifugation and electron microscopy detected that Protopolybia-MP III significantly suppressed HSV-1 virion infectivity at different temperatures by destroying the integrity of the HSV-1 virion. Finally, by comparing the antiviral activity of Protopolybia-MP III and its mutants, a series of peptides with better anti-HSV-1 activity were identified. Overall, this work found the function and mechanism of the antiviral wasp venom peptide Protopolybia-MP III and its derivatives against HSV-1 and laid the foundation for the research and development of wasp venom-derived antiviral candidate peptide drugs.

Cationicity Enhancement on the Hydrophilic Face of Ctriporin Significantly Reduces Its Hemolytic Activity and Improves the Antimicrobial Activity against Antibiotic-Resistant ESKAPE Pathogens.

The ESKAPE pathogen-associated antimicrobial resistance is a global public health issue, and novel therapeutic strategies are urgently needed. The short cationic antimicrobial peptide (AMP) family represents an important subfamily of scorpion-derived AMPs, but high hemolysis and poor antimicrobial activity hinder their therapeutic application. Here, we recomposed the hydrophilic face of Ctriporin through lysine substitution. We observed non-linear correlations between the physiochemical properties of the peptides and their activities, and significant deviations regarding the changes of antimicrobial activities against different bacterial species, as well as hemolytic activity. Most importantly, we obtained two Ctriporin analogs, CM5 and CM6, these two have significantly reduced hemolytic activity and more potent antimicrobial activities against all tested antibiotic-resistant ESKAPE pathogens. Fluorescence experiments indicated they may perform the bactericidal function through a membrane-lytic action model. Our work sheds light on the potential of CM5 and CM6 in developing novel antimicrobials and gives clues for optimizing peptides from the short cationic AMP family.

Characterization of the Hemolytic Activity of Mastoparan Family Peptides from Wasp Venoms.

Biologically active peptides have attracted increasing attention in research on the development of new drugs. Mastoparans, a group of wasp venom linear cationic α-helical peptides, have a variety of biological effects, including mast cell degranulation, activation of protein G, and antimicrobial and anticancer activities. However, the potential hemolytic activity of cationic α-helical peptides greatly limits the clinical applications of mastoparans. Here, we systematically and comprehensively studied the hemolytic activity of mastoparans based on our wasp venom mastoparan family peptide library. The results showed that among 55 mastoparans, 18 had strong hemolytic activity (EC50 ≤ 100 µM), 14 had modest hemolytic activity (100 µM < EC50 ≤ 400 µM) and 23 had little hemolytic activity (EC50 > 400 µM), suggesting functional variation in the molecular diversity of mastoparan family peptides from wasp venom. Based on these data, structure-function relationships were further explored, and, hydrophobicity, but not net charge and amphiphilicity, was found to play a critical role in the hemolytic activity of mastoparans. Combining the reported antimicrobial activity with the present hemolytic activity data, we found that four mastoparan peptides, Parapolybia-MP, Mastoparan-like peptide 12b, Dominulin A and Dominulin B, have promise for applications because of their high antimicrobial activity (MIC ≤ 10 µM) and low hemolytic activity (EC50 ≥ 400 µM). Our research not only identified new leads for the antimicrobial application of mastoparans but also provided a large chemical space to support the molecular design and optimization of mastoparan family peptides with low hemolytic activity regardless of net charge or amphiphilicity.

Characterization of the Molecular Diversity and Degranulation Activity of Mastoparan Family Peptides from Wasp Venoms.

Wasp stings have become an increasingly serious public health problem because of their high incidence and mortality rates in various countries and regions. Mastoparan family peptides are the most abundant natural peptides in hornet venoms and solitary wasp venom. However, there is a lack of systematic and comprehensive studies on mastoparan family peptides from wasp venoms. In our study, for the first time, we evaluated the molecular diversity of 55 wasp mastoparan family peptides from wasp venoms and divided them into four major subfamilies. Then, we established a wasp peptide library containing all 55 known mastoparan family peptides by chemical synthesis and C-terminal amidation modification, and we systematically evaluated their degranulation activities in two mast cell lines, namely the RBL-2H3 and P815 cell lines. The results showed that among the 55 mastoparans, 35 mastoparans could significantly induce mast cell degranulation, 7 mastoparans had modest mast cell degranulation activity, and 13 mastoparans had little mast cell degranulation activity, suggesting functional variation in mastoparan family peptides from wasp venoms. Structure-function relationship studies found that the composition of amino acids in the hydrophobic face and amidation in the C-terminal region are critical for the degranulation activity of mastoparan family peptides from wasp venoms. Our research will lay a theoretical foundation for studying the mechanism underlying the degranulation activity of wasp mastoparans and provide new evidence to support the molecular design and molecular optimization of natural mastoparan peptides from wasp venoms in the future.

Functional Characterization of a New Degradation Peptide BmTX4-P1 from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpion is an important traditional Chinese medical material that has been widely used to treat various diseases in China for over one thousand years. Our recent work showed that thermally processed Buthus martensii Karsch scorpions contain many degraded peptides; however, the pharmacological activities of these peptides remain to be studied. Here, a new degraded peptide, BmTX4-P1, was identified from processed Buthus martensii Karsch scorpions. Compared with the venom-derived wild-type toxin peptide BmTX4, BmTX4-P1 missed some amino acids at the N-terminal and C-terminal regions, while containing six conserved cysteine residues, which could be used to form disulfide bond-stabilized α-helical and ß-sheet motifs. Two methods (chemical synthesis and recombinant expression) were used to obtain the BmTX4-P1 peptide, named sBmTX4-P1 and rBmTX4-P1. Electrophysiological experimental results showed that sBmTX4-P1 and rBmTX4-P1 exhibited similar activities to inhibit the currents of hKv1.2 and hKv1.3 channels. In addition, the experimental electrophysiological results of recombinant mutant peptides of BmTX4-P1 indicated that the two residues of BmTX4-P1 (Lys22 and Tyr31) were the key residues for its potassium channel inhibitory activity. In addition to identifying a new degraded peptide, BmTX4-P1, from traditional Chinese scorpion medicinal material with high inhibitory activities against the hKv1.2 and hKv1.3 channels, this study also provided a useful method to obtain the detailed degraded peptides from processed Buthus martensii Karsch scorpions. Thus, the study laid a solid foundation for further research on the medicinal function of these degraded peptides.

Gene expression of TRPMLs and its regulation by pathogen stimulation.

The transient receptor potential mucolipin (TRPML) subfamily in mammalian has three members, namely TRPML1, TRPML2, and TRPML3, who play key roles in regulating intracellular Ca2+ homeostasis, endosomal pH, membrane trafficking and autophagy. Previous studies had shown that three TRPMLs are closely related to the occurrence of pathogen invasion and immune regulation in some immune tissues or cells, but the relationship between TRPMLs expression and pathogen invasion in lung tissue or cell remains elusive. Here, we investigated the expression distribution of three TRPML channels in mouse different tissues by qRT-PCR, and then found that all three TRPMLs were highly expressed in the mouse lung tissue, as well as mouse spleen and kidney tissues. The expression of TRPML1 or TRPML3 in all three mouse tissues had a significant down-regulation after the treatment of Salmonella or LPS, but TRPML2 expression showed a remarkable increase. Consistently, TRPML1 or TRPML3 but not TRPML2 in A549 cells also displayed a decreased expression induced by LPS stimulation, which shared a similar regulation pattern in the mouse lung tissue. Furthermore, the treatment of the TRPML1 or TRPML3 specific activator induced a dose-dependent up-regulation of inflammatory factors IL-1ß, IL-6 and TNFα, suggesting that TRPML1 and TRPML3 are likely to play an important role in immune and inflammatory regulation. Together, our study identified the gene expression of TRPMLs induced by pathogen stimulation in vivo and in vitro, which may provide novel targets for innate immunity or pathogen regulation.

BmK86-P1, a New Degradation Peptide with Desirable Thermostability and Kv1.2 Channel-Specific Activity from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpions are a traditional Chinese medical material for treating various diseases. However, their pharmacological foundation remains unclear. Here, a new degraded peptide of scorpion toxin was identified in Chinese scorpion medicinal material by proteomics. It was named BmK86-P1 and has six conserved cysteine residues. Homology modeling and circular dichroism spectra experiments revealed that BmK86-P1 not only contained representative disulfide bond-stabilized α-helical and ß-sheet motifs but also showed remarkable stability at test temperatures from 20-95 °C. Electrophysiology experiments indicated that BmK86-P1 was a highly potent and selective inhibitor of the hKv1.2 channel with IC50 values of 28.5 ± 6.3 nM. Structural and functional dissection revealed that two residues of BmK86-P1 (i.e., Lys19 and Ile21) were the key residues that interacted with the hKv1.2 channel. In addition, channel chimeras and mutagenesis experiments revealed that three amino acids (i.e., Gln357, Val381 and Thr383) of the hKv1.2 channel were responsible for BmK86-P1 selectivity. This research uncovered a new bioactive peptide from traditional Chinese scorpion medicinal material that has desirable thermostability and Kv1.2 channel-specific activity, which strongly suggests that thermally processed scorpions are novel peptide resources for new drug discovery for the Kv1.2 channel-related ataxia and epilepsy diseases.

The rapid development of the first instar telson with venom secretion highlights the remarkable survival ability of scorpions.

The scorpion venom system plays a critical role in capturing prey and defending against predators. In this study, the rapid developmental process of the first instar telson was first presented. The small amount of venom in the first instar could be stored well by the distorted and blocked venom ducts, which disappeared in the older scorpions. This special developmental process of the first instar telson revealed the notable survival ability of scorpions.

Different pharmacological properties between scorpion toxin BmKcug2 and its degraded analogs highlight the diversity of K+ channel blockers from thermally processed scorpions.

Novel degraded potassium channel-modulatory peptides were recently found in thermally processed scorpions, but their pharmacological properties remain unclear. Here, we identified a full-length scorpion toxin (i.e., BmKcug2) and its four truncated analogs (i.e., BmKcug2-P1, BmKcug2-P2, BmKcug2-P3 and BmKcug2-P4) with three conserved disulfide bonds in processed scorpion medicinal material by mass spectrometry. The pharmacological experiments revealed that the recombinant BmKcug2 and BmKcug2-P1 could selectively inhibit the human Kv1.2 and human Kv1.3 potassium channels, while the other three analogs showed a much weaker inhibitory effect on potassium channels. BmKcug2 inhibited hKv1.2 and hKv1.3 channels, with IC50 values of 45.6 ± 5.8 nM and 215.2 ± 39.7 nM, respectively, and BmKcug2-P1 inhibited hKv1.2 and hKv1.3, with IC50 values of 89.9 ± 9.6 nM and 1142.4 ± 64.5 nM, respectively. The chromatographic analysis and pharmacological properties of BmKcug2 and BmKcug2-P1 boiled in water for different times further strongly supported their good thermal stability. Structural and functional dissection indicated that one amino acid, i.e., Tyr36, determined the differential affinities of BmKcug2 and four BmKcug2 analogs. Altogether, this research investigated the different pharmacological properties of BmKcug2 and its truncated analogs, and the findings highlighted the diversity of K+ channel blockers from various scorpion species through thermal processing.

ImKTx96, a peptide blocker of the Kv1.2 ion channel from the venom of the scorpion Isometrus maculates.

Scorpion venom contains diverse bioactive peptides that can recognize and interact with membrane proteins such as ion channels. These natural toxins are believed to be useful tools for exploring the structure and function of ion channels. In this study, we characterized a K+-channel toxin gene, ImKTx96, from the venom gland cDNA library of the scorpion Isometrus maculates. The peptide deduced from the ImKTx96 precursor nucleotide sequence contains a signal peptide of 27 amino acid residues and a mature peptide of 29 residues with three disulfide bridges. Multiple sequence alignment indicated that ImKTx96 is similar with the scorpion toxins that typically target K+-channels. The recombined ImKTx96 peptide (rImKTx96) was expressed in the Escherichia coli system, and purified by GST-affinity chromatography and RP-HPLC. Results from whole-cell patch-clamp experiments revealed that rImKTx96 can inhibit the current of the Kv1.2 ion channel expressed in HEK293 cells. The 3D structure of ImKTx96 was constructed by molecular modeling, and the complex formed by ImKTx96 interacting with the Kv1.2 ion channel was obtained by molecular docking. Based on its structural features and pharmacological functions, ImKTx96 was identified as one member of K+-channel scorpion toxin α-KTx10 group and may be useful as a molecular probe for investigating the structure and function of the Kv1.2 ion channel.

Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels.

Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain α-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7±6.6nM for rNav1.4, 454.2±50.1nM for hNav1.5 and 30.9±10.3µM for hNav1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNav1.4 and hNav1.5 channels toward a more negative direction and present the typical features of a ß-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNav1.4 and hNav1.5 channels. The V1/2 values of the steady-state inactivation were altered to be more positive for rNav1.4 and more negative for hNav1.5. Moreover, the recovery of the hNav1.5 channel from inactivation was more significantly delayed than that of the rNav1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both α- and ß-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.