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Beatriz Cristina Pecoraro Sanches,1 Camila Aguiar Rocha,1 Jose Gregorio Martin Bedoya,1 Vinicius Luiz da Silva,2 Patrícia Bento da Silva,3 Ana Marisa Fusco-Almeida,4 Marlus Chorilli,3 Jonas Contiero,2 Edson Crusca,1 Reinaldo Marchetto1 1São Paulo State University (UNESP), Institute of Chemistry, Department of Biochemistry and Organic Chemistry, Araraquara, SP, Brazil; 2São Paulo State University (UNESP), Institute of Biosciences, Department of General and Applied Biology, Rio Claro, SP, Brazil; 3São Paulo State University (UNESP), School of Pharmaceutical Sciences, Department of Drugs and Medicines, Araraquara, SP, Brazil; 4São Paulo State University (UNESP), School of Pharmaceutical Sciences, Department of Clinical Analysis, Araraquara, SP, BrazilCorrespondence: Reinaldo MarchettoUNESP Institute of Chemistry, Rua Prof. Francisco Degni, 55, Araraquara, 14.800-060, SP, BrazilTel +55 16 3301 9670Fax +55 16 3322 2308Email reinaldo.marchetto@unesp.brBackground: Antimicrobial resistance poses substantial risks to human health. Thus, there is an urgent need for novel antimicrobial agents, including alternative compounds, such as peptides derived from bacterial toxin-antitoxin (TA) systems. ParELC3 is a synthetic peptide derived from the ParE toxin reported to be a good inhibitor of bacterial topoisomerases and is therefore a potential antibacterial agent. However, ParELC3 is inactive against bacteria due to its inability to cross the bacterial membranes. To circumvent this limitation we prepared and used rhamnolipid-based liposomes to carry and facilitate the passage of ParELC3 through the bacterial membrane to reach its intracellular target - the topoisomerases.Methods and Results: Small unilamellar liposome vesicles were prepared by sonication from three formulations that included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. ParELC3 was loaded with high efficiency into the liposomes. Characterization by DLS and TEM revealed the appropriate size, zeta potential, polydispersity index, and morphology. In vitro microbiological experiments showed that ParELC3 loaded-liposomes are more efficient (29 to 11 μmol·L− 1) compared to the free peptide (> 100 μmol·L− 1) at inhibiting the growth of standard E. coli and S. aureus strains. RL liposomes showed high hemolytic activity but when prepared with POPC and Chol this activity had a significant reduction. Independently of the formulation, the vesicles had no detectable cytotoxicity to HepG2 cells, even at the highest concentrations tested (1.3 mmol·L− 1 and 50 μmol·L− 1 for rhamnolipid and ParELC3, respectively).Conclusion: The present findings suggest the potential use of rhamnolipid-based liposomes as nanocarrier systems to enhance the bioactivity of peptides.Keywords: rhamnolipid liposomes, biosurfactants, bioactive peptides, antimicrobial activity

Introduction: This in situ study investigated the protective effect of a solution containing statherin-derived peptide (StatpSpS) against enamel intrinsic erosion (ERO)., Methods: Fifteen volunteers wore appliances containing 2 bovine specimens. The samples were subjected to ERO with HCl, mimicking dental ERO by intrinsic acid. The volunteers participated in 3 phases (double-blind and crossover): (1) deionized water (negative control); (2) commercial solution containing SnCl2/NaF/AmF (800 ppm Sn+2, 500 ppm F-, pH 4.5) (positive control); (3) solution containing 1.88 × 10-5m StatpSpS. Four times a day, the volunteers administered one drop of the solutions (50 μL, 1 min) on each specimen. After the treatment, erosive challenges were performed extraorally with 0.01 m HCl (pH 2.0, 4 times/day, 1 min, 150 mL). Enamel wear was assessed by profilometry. Data were analyzed by one-way RM-ANOVA/Bonferroni's tests (p < 0.05)., Results: In terms of the treatments, both the commercial solution (SnCl2/NaF/AmF) and StatpSpS significantly reduced the wear when compared to the negative control (p < 0.01), without significant differences between them (p > 0.05)., Conclusion: The solution containing StatpSpS demonstrated protection against enamel intrinsic erosive wear. This study marks a significant advancement in the prevention of intrinsic erosive wear, utilizing StatpSpS in acquired pellicle engineering procedures., (© 2024 S. Karger AG, Basel.)

Bacterial resistance has become a serious public health problem in recent years, thus encouraging the search for new antimicrobial agents. Here, we report an antimicrobial peptide (AMP), called PEPAD, which was designed based on an encrypted peptide from a Kunitz-type plant peptidase inhibitor. PEPAD was capable of rapidly inhibiting and eliminating numerous bacterial species at micromolar concentrations (from 4μM to 10 μM), with direct membrane activity. It was also observed that the peptide can act synergistically with ciprofloxacin and showed no toxicity in the G. mellonella in vivo assay. Circular dichroism assays revealed that the peptide's secondary structure adopts different scaffolds depending on the environment in which it is inserted. In lipids mimicking bacterial cell membranes, PEPAD adopts a more stable α-helical structure, which is consistent with its membrane-associated mechanism of action. When in contact with lipids mimicking mammalian cells, PEPAD adopts a disordered structure, losing its function and suggesting cellular selectivity. Therefore, these findings make PEPAD a promising candidate for future antimicrobial therapies with low toxicity to the host., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Antimicrobial peptides (AMP) represent an alternative in the treatment of fungal infections associated with countless deaths. Here, we report a new AMP, named KWI-19, which was designed based on a peptide encrypted in the sequence of an Inga laurina Kunitz-type inhibitor (ILTI). KWI-19 inhibited the growth of Candida species and acted as a fungicidal agent from 2.5 to 20 μmol L -1 , also showing synergistic activity with amphotericin B. Kinetic assays showed that KWI-19 killed Candida tropicalis cells within 60 min. We also report the membrane-associated mechanisms of action of KWI-19 and its interaction with ergosterol. KWI-19 was also characterized as a potent antibiofilm peptide, with activity against C. tropicalis. Finally, non-toxicity was reported against Galleria mellonella larvae, thus strengthening the interest in all the bioactivities mentioned above. This study extends our knowledge on how AMPs can be engineered from peptides encrypted in larger proteins and their potential as candicidal agents., Competing Interests: Declaration of competing interest The authors declare no competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated., Methods: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics., Results: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05)., Conclusions: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques., (© 2024 S. Karger AG, Basel.)

Objectives: This study evaluated the protective impact of acquired enamel pellicle (AEP) engineering with statherin-derived peptide (StatpSpS), considering different AEP formation times., Design: A total of 120 native human enamel specimens were divided into 2 main groups: 1) No AEP engineering and 2) AEP engineering with StatpSpS (pretreatment for 1 min; 37 °C, under agitation). Each group was further divided into 4 subgroups: No pellicle, or 1, 60-and-120 min AEP formation times (human saliva; 37 °C). The specimens were then subjected to an erosive challenge (1% citric acid; pH 3.6; 1 min; 25 °C). This procedure was repeated for 5 cycles. Relative surface reflection intensity (%SRI) was measured and scanning electron microscopy (SEM) of the enamel surface was done., Results: All AEP engineering groups protected against initial dental erosion in comparison with No pellicle (p < 0.001), likewise all groups with AEP, independent of engineering or formation times (p 0.001). Furthermore, engineering with StatpSpS even without the presence of AEP protected the enamel when compared to the No engineering/No pellicle group (p < 0.0001). No difference was observed regarding the protection from the different AEP formation times (p > 0.05). Regarding the SEM analysis, in the "No AEP engineering & No AEP" group, a more severe effect of citric acid was observed, with more enamel prism heads and scratches on the surface when compared with the other groups., Conclusions: AEP provides almost instant protection at formation times even as short as 1 min, protecting the native enamel against erosion. Treatment with StatpSpS by itself provides similar protection as the AEP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The effect of gels containing a statherin-derived peptide (Stn) on the protection against enamel and dentin erosive tooth wear (ETW) in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 15 and 18/group for enamel and dentin, respectively) that were treated with Chitosan or Carboxymethylcellulose (CMC) gels containing Stn15pSpS at 1.88 × 10-5 M or 3.76 × 10-5 M. Chitosan or CMC gels without active ingredients served as negative controls, while chitosan gel containing 1.23% F (as NaF) and acidulated phosphate fluoride gel (1.23% F) served as positive controls. The gels were applied on the specimens for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The gels were applied again during pH cycling, 2 times/day for 4 min after the first and last erosive challenges. Enamel and dentin loss (μm) were assessed by contact profilometry. Scanning electron microscopy (SEM) was analyzed using a cold field emission. Data were analyzed by two-way ANOVA (for chitosan and CMC gels, separately) and Tukey's multiple comparison test. SEM images showed changes to enamel topography after application oft the gels containing Stn or F. Regarding CMC-based gels, for enamel, none of the treatments significantly reduced ETW in comparison with placebo; for dentin, however, gels containing Stn, regardless the concentration, significantly reduced the ETW. Moreover, Chitosan-based gels, regardless the Stn concentration, were able to protect enamel and dentin against ETW. Gels containing Stn might be a new approach to protect against ETW., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The effect of solutions containing a statherin-derived peptide (Stn15pSpS) on the protection against enamel erosion in vitro was evaluated. Bovine enamel specimens were divided into 4 groups (n = 15/group): (1) deionized water (negative control), (2) Elmex Erosion Protection™ (positive control), (3) 1.88 × 10-5 M Stn15pSpS, and (4) 3.76 × 10-5 M Stn15pSpS. The solutions were applied on the specimens for 1 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH-cycling protocol 4 times/day, for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The solutions were applied again during pH-cycling, 2 times/day for 1 min after the first and last erosive challenges. Enamel loss (μm) was assessed by contact profilometry. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05). The best protection against erosion was conferred by Elmex Erosion Protection that significantly differed from all the other treatments, followed by the solutions containing Stn15pSpS, regardless of the concentration. However, 3.76 × 10-5 M Stn15pSpS did not differ from the negative control. The solution containing the lower concentration of Stn15pSpS protected against erosion in vitro, which should be confirmed using protocols that more closely resemble the clinical condition., (© 2023 S. Karger AG, Basel.)

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: This study investigated the mechanism of action of different proteins/peptides (separately or in combination), focusing on how they act directly on the native enamel surface and on modifying the salivary pellicle., Methods: A total of 170 native human enamel specimens were prepared and submitted to different treatments (2 h; 37 °C): with deionized water, CaneCPI-5, Hemoglobin, Statherin, or a combination of all three proteins/peptides. The groups were subdivided into treatment acting on the enamel surface (NoP - absence of salivary pellicle), and treatment modifying the salivary pellicle (P). Treatment was made (2 h; 37 °C) in all specimens, and later, for P, the specimens were incubated in human saliva (2 h; 37 °C). In both cases, the specimens were immersed in 1% citric acid (pH 3.6; 2 min; 25 °C). Calcium released from enamel (CaR) and its relative surface reflection intensity (%SRI) was measured after 5 cycles. Between-group differences were verified with two-way ANOVA, with "presence of pellicle" and "treatment" as factors (α = 0.05)., Results: The presence of pellicle provided better protection regarding %SRI (p < 0.01), but not regarding CaR (p = 0.201). In relation to treatment, when compared to the control group, all proteins/peptides provided significantly better protection (p < 0.01 for %SRI and Car). The combination of all three proteins/peptides demonstrated the best protective effect (p < 0.01 for %SRI)., Conclusion: Depending on the protein or peptide, its erosion-inhibiting effect derives from their interaction with the enamel surface or from modifying the pellicle, so a combination of proteins and peptides provides the best protection., Clinical Significance: The present study opens a new direction for a possible treatment with a combination of proteins for native human enamel, which can act directly on the enamel surface as well on the modification of the salivary pellicle, for the prevention of dental erosion., (Copyright © 2021. Published by Elsevier Ltd.)

Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS., (© 2021 S. Karger AG, Basel.)

Objectives: To evaluate, in vivo: 1) proteomic alterations in the acquired enamel pellicle (AEP) after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), statherin-derived peptide (StN15) or their combination before the formation of the AEP and subsequent erosive challenge; 2) the protection of these treatments against erosive demnineralization., Materials and Methods: In 5 crossover phases, after prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with: deionized water-1, 0.1 mg/mL CaneCPI-5-2, 1.0 mg/mL HB-3, 1.88 × 10 -5 M StN15-4 or their combination-5. AEP was formed (2 h) and enamel biopsy (10 μL, 1%citric acid, pH 2.5, 10 s) was performed on one incisor for calcium analysis. The same acid was applied on the vestibular surfaces of the remaining teeth. The acid-resistant proteins within the remaining AEP were collected. Samples were quantitatively analyzed by label-free proteomics., Results: Treatment with the proteins/peptide, isolated or combined, increased several acid-resistant proteins in the AEP, compared with control. The highest increases were seen for PRPs (32-fold, StN15), profilin (15-fold, combination), alpha-amylase (9-fold; StN15), keratins (8-fold, CaneCPI-5 and HB), Histatin-1 (7-fold, StN15), immunoglobulins (6.5-fold, StN15), lactotransferrin (4-fold, CaneCPI-5), cystatins, lysozyme, protein S-100-A9 and actins (3.5-fold, StN15), serum albumin (3.5-fold, CaneCPI-5 and HB) and hemoglobin (3-fold, StN15). Annexin, calmodulin, keratin, tubulin and cystatins were identified exclusively upon treatment with the proteins/peptide, alone or combined. Groups 2, 3 and 4 had significantly lower Ca released from enamel compared to group 1 (Kruskal-Wallis/Dunn's, p < 0.05)., Conclusions: Treatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion., Clinical Significance: Our results show, for the first time, that treatment with proteins/peptide remarkably increases acid-resistant proteins in the AEP, protecting against erosive demineralization. These findings open an avenue for a new preventive approach for erosive demineralization, employing acquired pellicle engineering procedures that may in the future be incorporated into dental products., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: In the present study, we used an in vitro initial intrinsic erosion model to evaluate: (experiment 1) the influence of the degree of serine (Ser) phosphorylation of peptides containing the 15 N-terminal residues of statherin and (experiment 2) the effect of different concentrations of the peptide with the best performance in experiment 1 on initial enamel erosion., Design: Bovine enamel specimens were divided into 6 groups (n = 15/group) for each experiment. In experiment 1, the peptides evaluated (at 1.88 × 10 -5 M) were: not phosphorylated (StatSS), phosphorylated in Ser2 (StatpSS), phosphorylated in Ser3 (StatSpS) phosphorylated in Ser2 and Ser3 (StatpSpS). Phosphate buffer and human recombinant statherin were used as negative and positive controls, respectively. In experiment 2, StatpSpS was evaluated at different concentrations: 0.94, 1.88, 3.76 and 7.52 × 10 -5 M. Phosphate buffer and 0.1 mg/mL CaneCPI-5 were employed as negative and positive controls, respectively. In each experiment, the specimens were incubated with the solutions for 2 h, then the AEP was allowed to form (under human pooled saliva) for 2 h. The specimens were then challenged with 0.01 M HCl for 10 s. Demineralization was evaluated by percentage of surface hardness change (%SHC). Data were analyzed by ANOVA and Tukey's test (p < 0.05)., Results: In experiment 1, only StatpSpS significantly reduced the % SHC in comparison with control. In experiment 2, 1.88 × 10 -5 M StatpSpS significantly reduced the %SHC in comparison with control., Conclusions: This is the first study showing that statherin-derived peptide might protect against intrinsic erosion., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMP) are molecules with a broad spectrum of activities that have been identified in most living organisms. In addition, synthetic AMPs designed from natural polypeptides have been largely investigated. Here, we designed a novel AMP using the amino acid sequence of a plant trypsin inhibitor from Adenanthera pavonina seeds (ApTI) as a template. The 176 amino acid residues ApTI sequence was cleaved in silico using the Collection of Antimicrobial Peptides (CAMP R3 ), through the sliding-window method. Further improvements in AMP structure were carried out, resulting in adepamycin, an AMP designed from ApTI. Adepamycin showed antimicrobial activity from 0.9 to 3.6 μM against Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Moreover, this peptide also displayed activity against Candida albicans and Candida tropicalis. No toxic effects were observed on healthy human cells. Studies on the mechanism of action of adepamycin were carried out using an E. coli and C. tropicalis. Adepamycin triggers membrane disturbances, leading to intracellular nucleic acids release in E. coli. For C. tropicalis, an initial interference with the plasma membrane integrity is followed by the formation of intracellular reactive oxygen species (ROS), leading to apoptosis. Structurally, adepamycin was submitted to circular dichroism spectroscopy, molecular modeling and molecular dynamics simulations, revealing an environment-dependent α-helical structure in the presence of 2,2,2- trifluoroethanol (TFE) and in contact with mimetic vesicles/membranes. Therefore, adepamycin represents a novel lytic AMP with dual antibacterial and antifungal properties., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1 H and 15 N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH 2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B 1 and B 2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1 H and 15 N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For H α and H N correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

Candida yeast infections are the fourth leading cause of death worldwide. Peptides with antimicrobial activity are a promising alternative treatment for such infections. Here, the antifungal activity of a new antimicrobial peptide-PEP-IA18-was evaluated against Candida species. PEP-IA18 was designed from the primary sequence of profilin, a protein from Spodoptera frugiperda, and displayed potent activity against Candida albicans and Candida tropicalis , showing a minimum inhibitory concentration (MIC) of 2.5   µM. Furthermore, the mechanism of action of PEP-IA18 involved interaction with the cell membrane (ergosterol complexation). Treatment at MIC and/or 10   ×   MIC significantly reduced biofilm formation and viability. PEP-IA18 showed low toxicity toward human fibroblasts and only revealed hemolytic activity at high concentrations. Thus, PEP-IA18 exhibited antifungal and anti-biofilm properties with potential applicability in the treatment of infections caused by Candida species.

Currently 75-88% of fungal infections are caused by Candida species, and Candida albicans is the main microorganism that causes these infections, especially oral candidiasis. An option for treatment involves the use of the antifungal peptide Histatin 5 (Hst 5), which is naturally found in human saliva but undergoes rapid degradation when present in the oral cavity, its site of action. For this reason, it is important to develop a way of applying this peptide to the oral lesions, which promotes the gradual release of the peptide. In the present study, we have evaluated the development of liposomes of different lipid compositions, loaded with the peptide as a way to promote its release slowly and gradually, preserving its antifungal potential. For this, the peptide 0WHistatin 5, an analog of the peptide Hst 5, was synthesized, which contains the amino acid tryptophan in its sequence. The solid phase synthesis method was used, followed by cleavage and purification. The liposomes were produced by thin film hydration technique in three different lipid compositions, F1, F2, and F3 and were submitted to an extrusion and sonication process to standardize the size and study the best technique for their production. The liposomes were characterized by dynamic light scattering, and tests were performed to determine the encapsulation efficiency, release kinetics, stability, and evaluation of antifungal activity. The extruded liposomes presented average size in the range of 100 nm, while sonicated liposomes presented a smaller size in the range of 80 nm. The encapsulation efficiency was higher for the sonicated liposomes, being 34.5% for F1. The sonicated F3 presented better stability when stored for 60 days at 4°C. The liposomes showed the ability to release the peptide for the total time of 96 h, with the first peak after 5 h, and a further increase of the released after 30 h. Time-kill assay showed that the liposomes were able to control yeast growth for 72 h. The data suggest that the liposomes loaded with 0WHistatin 5 maintained the action of the peptide and were able to limit the growth of C. albicans , being a suitable system for use in the treatment of oral candidiasis.

In the article mentioned above an author's name was misspelled.

Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI 1-31 ) or StII (StII 1-30 ) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1-11 of StI (StI 12-31 ) or 1-10 of StII (StII 11-30 ). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII 1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI 1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII 1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1-10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU - 145 (prostate adenocarcinoma) and healthy fibroblasts HGF - 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII 1-30 and StII 16-35 . They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII 1-30 partly inserts into the micelle, while StII 16-35 lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged., (© 2017 Wiley Periodicals, Inc.)

Human dihydroorotate dehydrogenase (HsDHODH) enzyme has been studied as selective target for inhibitors to block the enzyme activity, intending to prevent proliferative diseases. The N-terminal microdomain seems to play an important role in the enzyme function. However, the molecular mechanism of action and dynamics of this region are not totally understood yet. This study analyzes the interaction and conformation in model membranes of HsDHODH microdomain using peptide analogues containing the paramagnetic amino acid TOAC at strategic positions. In buffer solution, the analogues presented a disordered conformation, but acquired a high content of α-helical structure in membrane mimetics, which was found to be lipid dependent. The microdomain peptide structure in micelles showed a very different peptide conformation when compared to the reported crystal structure, displaying a conformational flexibility of its helices, promoted by the connecting loop, which might be functionally relevant. Electron spin resonance in membrane compositions containing POPC, POPE, and cardiolipin showed that interaction of the analogues was enhanced by the presence of cardiolipin, indicating that the microdomain preferentially interacts with cardiolipin-containing membranes. Therefore, the great flexibility of the microdomain and the cardiolipin affinity should be considered in further studies aimed at finding new inhibitory compounds to fight proliferative diseases.

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13 C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH 2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B 1 and B 2 is dependent on the type of atom and amino acid studied. For H N , N and C α the first order pressure coefficient B 1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13 C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

Antimicrobial peptides are recognized candidates with pharmaceutical potential against epidemic emerging multi-drug resistant bacteria. In this study, we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to determine the unknown structure and evaluate the interaction with dodecylphosphatidylcholine (DPC) and sodium dodecylsulphate (SDS) micelles with three W 6 -Hylin-a1 analogs antimicrobial peptides (HyAc, HyK, and HyD). The HyAc, HyK, and HyD bound to DPC micelles are all formed by a unique α-helix structure. Moreover, all peptides reach the DPC micelles' core, which thus suggests that the N-terminal modifications do not influence the interaction with zwiterionic surfaces. On the other hand, only HyAc and HyK peptides are able to penetrate the SDS micelle core while HyD remains always at its surface. The stability of the α-helical structure, after peptide-membrane interaction, can also be important to the second step of peptide insertion into the membrane hydrophobic core during permeabilization. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms (1)H(α), (13)C(α) and (13)C' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The polynomial pressure coefficients B 1 and B 2 are dependent on the type of amino acid studied. The coefficients of a given nucleus show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure are also weakly correlated.

A cyclic peptide, jatrophidin I, was isolated from the latex of Jatropha curcas L. Its structure was elucidated by extensive 2D NMR spectroscopic analysis, with additional conformational studies performed using Molecular Dynamics/Simulated Annealing (MD/SA). Jatrophidin I had moderate protease inhibition activity when compared with pepstatin A; however, the peptide was inactive in antimalarial, cytotoxic and antioxidant assays., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The pressure dependence of the one-bond indirect spin-spin coupling constants (1)J(N-H) was studied in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (with Xxx being one of the 20 proteinogenic amino acids). The response of the (1)J(N-H) coupling constants is amino acid type specific, with an average increase of its magnitude by 0.6 Hz at 200 MPa. The variance of the pressure response is rather large, the largest pressure effect is observed for asparagine where the coupling constant becomes more negative by -2.9 Hz at 200 MPa. The size of the J-coupling constant at high pressure is positively correlated with its low pressure value and the β-propensity, and negatively correlated with the amide proton shift and the first order nitrogen pressure coefficient and the electrostatic solvation free energy.

Sticholysin II (St II) is the most potent cytolysin produced by the sea anemone Stichodactyla helianthus, exerting hemolytic activity via pore formation in membranes. The toxin's N-terminus contains an amphipathic α-helix that is very likely involved in pore formation. We have previously demonstrated that the synthetic peptide StII(1-30) encompassing the 1-30 segment of St II forms pores of similar radius to that of the protein (around 1 nm), being a good model of toxin functionality. Here we have studied the functional and conformational properties of fluorescent analogs of StII(1-30) in lipid membranes. The analogs were obtained by replacing Leu residues at positions 2, 12, 17, and 24 with the intrinsically fluorescent amino acid Trp (StII(1-30L2W), StII(1-30L12W), StII(1-30L17W), or StII(1-30L24W), respectively). The exchange by Trp did not significantly modify the activity and conformation of the parent peptide. The blue-shift and intensity enhancement of fluorescence in the presence of membrane indicated that Trp at position 2 is more deeply buried in the hydrophobic region of the bilayer. These experiments, as well as assays with water-soluble or spin-labeled lipid-soluble fluorescence quenchers suggest an orientation of StII(1-30) with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins., (Copyright © 2013 Wiley Periodicals, Inc.)

Recently the peptide Hy-a1 (IFGAILPLALGALKNLIK), with antimicrobial activity, was isolated from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to evaluate four analogues with introduction of acetyl group, Asp or Lys at the N-terminus of antimicrobial peptide Hy-al to supply information about the relationship of structure-biological activity. The antimicrobial activities were assayed by measuring growth inhibition of four species of bacteria and four species of fungus. The hemolytic activity was also tested. The peptide containing Trp instead of Leu in position 6 (for fluorescence studies) presented MIC values comparable to wild type sequence: 32 μmol L(-1) , 32 μmol L(-1) , 8 μmol L(-1) , and 2 μmol L(-1) for E. coli, P. aeruginosa, S. aureus, and B. subtilis, respectively. Two peptides with this modification and containing one acetyl group or Asp residue at the N-terminal region showed activities only against Gram-positive bacteria. Different results were observed when the residue added was Lys. In this case, the activity against the microorganisms was sustained or increased. Conformational properties were investigated by CD techniques in water, TFE, and in zwitterionic micelles (LPC). The CD experiments demonstrated that, in water, the peptides had a random structure, but in TFE and LPC solutions they acquired an ordered structure, composed mainly by α-helix. However, these data have no relationship with activity against Gram-positive bacteria. These results showed that the N-terminal region of the peptide Hy-a1 has key roles in its antibacterial action toward different types of bacteria., (Copyright © 2010 Wiley Periodicals, Inc.)

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog") led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH(2)). The alpha-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC(50)=18 microM). Similarity analysis using PSI-BLAST revealed 50-44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).

Sticholysins I and II (St I and St II) are cytolysins produced by the sea anemone Stichodactyla helianthus. In spite of their 93% sequence homology, St II is more hemolytic against human erythrocytes than St I. In order to establish the possible causes of this difference, we studied the hemolytic activity of synthetic peptides containing sequences from the N-termini of both proteins. The results demonstrated that the differences in hemolytic activity of the toxins could be ascribed at least partly to differences in their N-termini.

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.

The SPPS methodology has continuously been investigated as a valuable model to monitor the solvation properties of polymeric materials. In this connection, the present work applied HRMAS-NMR spectroscopy to examine the dynamics of an aggregating peptide sequence attached to a resin core with varying peptide loading (up to 80%) and solvent system. Low and high substituted BHAR were used for assembling the VQAAIDYING sequence and some of its minor fragments. The HRMAS-NMR results were in agreement with the swelling of each resin, i.e. there was an improved resolution of resonance peaks in the better solvated conditions. Moreover, the peptide loading and the attached peptide sequence also affected the spectra. Strong peptide chain aggregation was observed mainly in highly peptide loaded resins when solvated in CDCl3. Conversely, due to the better swelling of these highly loaded resins in DMSO, improved NMR spectra were acquired in this polar aprotic solvent, thus enabling the detection of relevant sequence-dependent conformational alterations. The more prominent aggregation was displayed by the VQAAIDYING segment and not by any of its intermediary fragments and these findings were also corroborated by EPR studies of these peptide-resins labelled properly with an amino acid-type spin probe., (Copyright 2005 European Peptide Society and John Wiley & Sons, Ltd.)