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Research Square Sep 2023Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane...
Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane selectivity is critical to understanding their function and enabling their therapeutic use. Past attempts to decipher these rules have failed because they cannot interrogate adequate peptide sequence variation. To overcome this problem, we develop deep mutational surface localized antimicrobial display (dmSLAY), which reveals comprehensive positional residue importance and flexibility across an antimicrobial peptide sequence. We apply dmSLAY to Protegrin-1, a potent yet toxic antimicrobial peptide, and identify thousands of sequence variants that positively or negatively influence its antibacterial activity. Further analysis reveals that avoiding large aromatic residues and eliminating disulfide bound cysteine pairs while maintaining membrane bound secondary structure greatly improves Protegrin-1 bacterial specificity. Moreover, dmSLAY datasets enable machine learning to expand our analysis to include over 5.7 million sequence variants and reveal full Protegrin-1 mutational profiles driving either bacterial or mammalian membrane specificity. Our results describe an innovative, high-throughput approach for elucidating antimicrobial peptide sequence-structure-function relationships which can inform synthetic peptide-based drug design.
PubMed: 37790501
DOI: 10.21203/rs.3.rs-3280212/v1 -
BioRxiv : the Preprint Server For... Sep 2023Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane...
Antimicrobial peptides commonly act by disrupting bacterial membranes, but also frequently damage mammalian membranes. Deciphering the rules governing membrane selectivity is critical to understanding their function and enabling their therapeutic use. Past attempts to decipher these rules have failed because they cannot interrogate adequate peptide sequence variation. To overcome this problem, we develop deep mutational surface localized antimicrobial display (dmSLAY), which reveals comprehensive positional residue importance and flexibility across an antimicrobial peptide sequence. We apply dmSLAY to Protegrin-1, a potent yet toxic antimicrobial peptide, and identify thousands of sequence variants that positively or negatively influence its antibacterial activity. Further analysis reveals that avoiding large aromatic residues and eliminating disulfide bound cysteine pairs while maintaining membrane bound secondary structure greatly improves Protegrin-1 bacterial specificity. Moreover, dmSLAY datasets enable machine learning to expand our analysis to include over 5.7 million sequence variants and reveal full Protegrin-1 mutational profiles driving either bacterial or mammalian membrane specificity. Our results describe an innovative, high-throughput approach for elucidating antimicrobial peptide sequence-structure-function relationships which can inform synthetic peptide-based drug design.
PubMed: 37547010
DOI: 10.1101/2023.07.28.551017 -
Frontiers in Microbiology 2018Protegrin (PG) belongs to the antimicrobial peptide cathelicidin family. To date, five protegrin sequences have been identified in pigs, PG-1 to PG-5. Of these, PG-1...
Protegrin (PG) belongs to the antimicrobial peptide cathelicidin family. To date, five protegrin sequences have been identified in pigs, PG-1 to PG-5. Of these, PG-1 exhibits potent antimicrobial activity against a broad range of antibiotic-resistant microorganisms as well as viruses. However, the other potential role(s) of PG beyond antimicrobial has largely been unexplored. The aim of this study was to use nonpathogenic yeast to express antimicrobially active recombinant protegrin (rPG-1). Additionally, the effect of PG-1 on cell migration and proliferation was also examined using pig intestinal epithelial cells as a model. Highest level of rPG-1 (104 ± 11 μg/mL) was detected at 24 h in fermentation culture medium. Similar to rPG-1, 0.8 ± 0.10 g/L of proform PG-1 (rProPG-1) and 0.2 ± 0.02 g/L of the PG-1 cathelin domain (rCath) was detected in fermentation culture medium. Resulting recombinant PG-1 and cleaved rProPG-1 exerted antimicrobial activity against DH5α at the same level as chemically synthesized PG-1. Enhanced cell migration was observed ( < 0.05) in groups treated with rProPG-1, rCath, and rPG-1 compared to the control. Furthermore, rPG-1 was stable at temperatures ranging from 25°C to 80°C. In summary, biologically active recombinant protegrin in its pro-, cathelin-, and mature- forms were successfully expressed in suggesting potential feasibility for future therapeutic applications.
PubMed: 30319593
DOI: 10.3389/fmicb.2018.02300 -
Biochimica Et Biophysica Acta Jul 2012Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell...
BACKGROUND
Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. In this study, we compare the cell death-inducing activities of four β-hairpin AMPs (gomesin, protegrin, tachyplesin and polyphemusin II) along with their linear analogs in the human erythroleukemia K562 cell line to investigate the relationship between their structure and activity.
METHODS
K562 cells were exposed to AMPs. Morphological and biochemistry alterations were evaluated using light microscopy, confocal microscopy and flow cytometry.
RESULTS
Gomesin and protegrin displayed cytotoxic properties that their linear counterparts did not. Tachyplesin and polyphemusin II and also their linear analogs induced cell death. We were able to distinguish two ways in which these AMPs induced cell death. Lower concentrations of AMPs induced controlled cell death mechanisms. Gomesin, tachyplesin and linear-tachyplesin promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation, but was also inhibited by necrostatin-1. Gomesin and protegrin induced cell death was dependent on intracellular Ca2+ mechanisms and the participation of free radicals was observed in protegrin induced cell death. Polyphemusin II and its linear analog mainly induced necrosis. Conversely, treatment with higher concentrations of AMPs primarily resulted in cell membrane disruption, but with clearly different patterns of action for each AMP tested.
CONCLUSION
Different actions by β-hairpin AMPs were observed at low concentrations and at higher concentrations despite the structure similarity.
GENERAL SIGNIFICANCE
Controlled intracellular mechanism and direct membrane disruption were clearly distinguished helping to understand the real action of AMPs in mammalian cells.
Topics: Antimicrobial Cationic Peptides; Apoptosis; Calcium; Caspases; Cell Membrane; Humans; K562 Cells; Necrosis; Reactive Oxygen Species
PubMed: 22425533
DOI: 10.1016/j.bbagen.2012.02.015 -
The Journal of Physical Chemistry. B Oct 2017Protegrin-1 is an 18-residue β-hairpin antimicrobial peptide (AMP) that has been suggested to form transmembrane β-barrels in biological membranes. However,...
Protegrin-1 is an 18-residue β-hairpin antimicrobial peptide (AMP) that has been suggested to form transmembrane β-barrels in biological membranes. However, alternative structures have also been proposed. Here, we performed multimicrosecond, all-atom molecular dynamics simulations of various protegrin-1 oligomers on the membrane surface and in transmembrane topologies. The membrane surface simulations indicated that protegrin dimers are stable, whereas trimers and tetramers break down. Tetrameric arcs remained stably inserted in lipid membranes, but the pore water was displaced by lipid molecules. Unsheared protegrin β-barrels opened into β-sheets that surrounded stable aqueous pores, whereas tilted barrels with sheared hydrogen bonding patterns were stable in most topologies. A third type of observed pore consisted of multiple small oligomers surrounding a small, partially lipidic pore. We also considered the β-hairpin AMP tachyplesin, which showed less tendency to oligomerize than protegrin: the octameric bundle resulted in small pores surrounded by six peptides as monomers and dimers, with some peptides returning to the membrane surface. The results imply that multiple configurations of protegrin oligomers may produce aqueous pores and illustrate the relationship between topology and putative steps in protegrin-1's pore formation. However, the long-term stability of these structures needs to be assessed further.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Membrane; Cell-Penetrating Peptides; Models, Biological; Peptides
PubMed: 28879767
DOI: 10.1021/acs.jpcb.7b06591 -
Microbiology and Immunology Aug 2012Early defence mechanisms of innate immunity respond rapidly to infection against HIV-1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive... (Review)
Review
Early defence mechanisms of innate immunity respond rapidly to infection against HIV-1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive immune responses against persistent HIV infection. Recent research has highlighted the intrinsic roles of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G, tripartite motif-containing protein 5, tetherin, sterile α-motif and histidine/aspartic acid domain-containing protein 1 in restricting HIV-1 replication. Likewise, certain endogenously secreted antimicrobial peptides, namely α/β/θ-defensins, lactoferrins, secretory leukocyte protease inhibitor, trappin-2/elafin and macrophage inflammatory protein-3α are reportedly protective. Whilst certain factors directly inhibit HIV, others can be permissive. Interferon-λ3 exerts an anti-HIV function by activating Janus kinase-signal transducer and activator of transcription-mediated innate responses. Morphine has been found to impair intracellular innate immunity, contributing to HIV establishment in macrophages. Interestingly, protegrin-1 could be used therapeutically to inhibit early HIV-1 establishment. Moreover, chloroquine inhibits plasmacytoid dendritic cell activation and improves effective T-cell responses. This minireview summarizes the recently identified targets for innate immunity-mediated therapies and outlines the challenges that lie ahead in improving treatment of HIV infection.
Topics: HIV Infections; HIV-1; Humans; Immunity, Innate; Immunologic Factors; Immunotherapy
PubMed: 22900503
DOI: 10.1111/j.1348-0421.2012.00485.x -
PloS One 2014Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious...
Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Chlorocebus aethiops; Dengue; Dengue Virus; Dose-Response Relationship, Drug; Escherichia coli; Female; Gene Expression; Inclusion Bodies; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Peptides; Protein Refolding; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 2; Serine Endopeptidases; Survival Analysis; Vero Cells; Viral Nonstructural Proteins
PubMed: 24722532
DOI: 10.1371/journal.pone.0094561 -
Biomedicines Nov 2023Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer...
Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer effects of nerve growth factor (NGF), cathelicidin (LL-37), and protegrin-1 (PG-1) with chemotherapy (temozolomide, doxorubicin, carboplatin, cisplatin, and etoposide) in the glioblastoma U251 cell line to overcome the limitations of conventional chemotherapy and to guarantee specific treatments to succeed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cell viability and to determine the cytotoxic effects of NGF, LL-37, and PG-1 and their combination with chemotherapy in U251 cells. Synergism or antagonism was determined using the combination index (CI) method. Caspase-3 activity was evaluated spectrophotometrically using a caspase-3 activity assay kit. Apoptosis was analyzed with flow cytometry using propidium iodide (PI) and YO-PRO-1. NGF and the peptides showed a strong cytotoxic effect on U251 glioma cells in the MTT test (IC 0.0214, 3.1, and 26.1 μM, respectively) compared to chemotherapy. The combination of PG-1 + etoposide had a synergistic effect on apoptosis of U251 glioma cells. It should be noted that the cells were in the early and late stages of apoptosis, respectively, compared with the control cells. The caspase-3 activation analysis revealed that the caspase-3 level was not significantly ( > 0.05) increased in U251 cells following PG-1 with etoposide treatment compared with that in the untreated cells, suggesting that the combination of PG-1 and etoposide may induce caspase-independent apoptosis in U251 cells. NGF, LL-37, and PG-1 represent promising drug candidates as the treatment regimen for GBM. Furthermore, the synergistic efficacy of the combined protocol using PG-1 and etoposide may overcome some of the typical limitations of the conventional therapeutic protocols, thus representing a promising approach for GBM therapy.
PubMed: 38002009
DOI: 10.3390/biomedicines11113009 -
Communications Biology Nov 2022Antimicrobial peptides (AMPs) are cationic antibiotics that can kill multidrug-resistant bacteria via membrane insertion. However, their weak activity limits their...
Antimicrobial peptides (AMPs) are cationic antibiotics that can kill multidrug-resistant bacteria via membrane insertion. However, their weak activity limits their clinical use. Ironically, the cationic charge of AMPs is essential for membrane binding, but it obstructs membrane insertion. In this study, we postulate that this problem can be overcome by locating cationic amino acids at the energetically preferred membrane surface. All amino acids have an energetically preferred or less preferred membrane position profile, and this profile is strongly related to membrane insertion. However, most AMPs do not follow this profile. One exception is protegrin-1, a powerful but neglected AMP. In the present study, we found that a potent AMP, WCopW5, strongly resembles protegrin-1 and that the match between its sequence and the preferred position profile closely correlates with its antimicrobial activity. One of its derivatives, WCopW43, has antimicrobial activity comparable to that of the most effective AMPs in clinical use.
Topics: Amino Acids; Anti-Bacterial Agents; Antimicrobial Peptides; Cations
PubMed: 36347951
DOI: 10.1038/s42003-022-04164-4 -
Peptides Sep 2017Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in...
Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre-existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL-37, the porcine cathelicidin Protegrin-1, and the ovine cathelicidin SMAP-29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL-37 does not induce apoptosis or necrosis in HRV-infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection.
Topics: Animals; Antimicrobial Cationic Peptides; Antiviral Agents; Blood Proteins; Cathelicidins; Epithelial Cells; Humans; Respiratory Tract Infections; Rhinovirus; Sheep; Swine
PubMed: 28764966
DOI: 10.1016/j.peptides.2017.07.013