Histatin 5-Inspired Short-Chain Peptides Selectively Combating Pathogenic Fungi with Multifaceted Mechanisms.

Short-chain antifungal peptides (AFPs) inspired by histatin 5 have been designed to address the problem of antifungal drug resistance. These AFPs demonstrate remarkable antifungal activity, with a minimal inhibitory concentration as low as 2 µg mL-1. Notably, these AFPs display a strong preference for targeting fungi rather than bacteria and mammalian cells. This is achieved by binding the histidine-rich domains of the AFPs to the Ssa1/2 proteins in the fungal cell wall, as well as the reduced membrane-disrupting activity due to their low amphiphilicity. These peptides disrupt the nucleus and mitochondria once inside the cells, leading to reactive oxygen species production and cell damage. In a mouse model of vulvovaginal candidiasis, the AFPs demonstrate not only antifungal activity, but also promote the growth of beneficial Lactobacillus spp. This research provides valuable insights for the development of fungus-specific AFPs and offers a promising strategy for the treatment of fungal infectious diseases.

Full structural ensembles of intrinsically disordered proteins from unbiased molecular dynamics simulations.

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD (HREMD) led to unbiased and accurate ensembles, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of varying sequence properties using two recently optimized force fields, indicating the general applicability of HREMD for IDPs. We further demonstrate that, unlike HREMD, standard MD can reproduce experimental NMR chemical shifts, but not small-angle scattering data, suggesting chemical shifts are insufficient for testing the validity of IDP ensembles. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues). The results suggest that the major hurdle of generating an accurate unbiased ensemble for IDPs has now been largely overcome.

Human salivary histatin-1 (Hst1) promotes bone morphogenetic protein 2 (BMP2)-induced osteogenesis and angiogenesis.

Large-volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin-1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2-induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 µg Hst1 per sample and 0 or 2 µg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2-induced new bone formation in a dose-dependent manner. Co-administration of 500 µg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2-induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large-volume bone defects.

Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control-scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide.

The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

Reweighting ensemble probabilities with experimental histogram data constraints using a maximum entropy principle.

Entropy maximization methods that update a probability distribution P 0(x) to a new distribution P(x) with the use of externally known, averaged constraints find use in diverse areas. Jaynes developed a Maximum Entropy Procedure (MEP) that is an objective approach to incorporate external data to update P 0(x) to P(x). In this work, we consider the MEP in the context of external data known from a probability distribution versus that from a mean and a few higher moments. An immediate problem is that the conventional iterative Lagrange multiplier method, which relies on inverting a certain covariance matrix, is not applicable here because the covariance matrix is not invertible. We introduce an indicator function method that does not suffer from this problem. It leads to an analytic solution to this version of a MEP. As an example, a previously generated ensemble of peptide conformations used to characterize an intrinsically disordered protein is analyzed. The external constraint is on the radius of gyration probability distribution, p(RG), of this peptide. Ensemble observables such as geometric, shape characteristics, the residue end-to-end distance distribution, the all atom-pair distribution function related to the scattering intensity, the polyproline II content, and NMR 3JHNHα three bond couplings are evaluated with the initial and updated ensembles. Some observables are found to be insensitive and others sensitive to the external information. An example of a 24-residue peptide, histatin 5, where an experimentally derived p(RG) is available, is also analyzed.

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds.

Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form ß-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αß folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αßß fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.

In vitro assay to estimate tea astringency via observing flotation of artificial oil bodies sheltered by caleosin fused with histatin 3.

Astringency, a sensory characteristic of food and beverages rich in polyphenols, mainly results from the formation of complexes between polyphenols and salivary proteins, causing a reduction of the lubricating properties of saliva. To develop an in vitro assay to estimate the astringency of oolong tea infusion, artificial oil bodies were constituted with sesame oil sheltered by a modified caleosin fused with histatin 3, one of the human salivary small peptides. Aggregation of artificial oil bodies was induced when they were mixed with oolong tea infusion or its major polyphenolic compound, (-)-epigallocatechin gallate (EGCG) of 100µM as observed in light microscopy. The aggregated artificial oil bodies gradually floated on top of the solution and formed a visible milky layer whose thickness was in proportion to the concentrations of tea infusion. This assay system was applied to test four different oolong tea infusions with sensory astringency corresponding to their EGCG contents. The result showed that relative astringency of the four tea infusions was correlated to the thickness of floated artificial oil bodies, and could be estimated according to the standard curve generated by simultaneously observing a serial dilution of the tea infusion with the highest astringency.

Targeting and synergistic action of an antifungal peptide in an antibiotic drug-delivery system.

Amphotericin B (AmB) has been widely used against fungal infections throughout almost the entire body, including the skin, nails, oral cavity, respiratory tract, and urinary tract. However, the development of AmB-loaded nanoparticles demands a novel technique that reduces its toxicity and other associated problems. Here, we developed a pH-responsive and redox-sensitive polymer-based AmB-delivery carrier system. In particular, this system was functionalized by conjugation with the antifungal peptide histatin 5, which acts both as a targeting ligand and a synergistic antifungal molecule against Candida albicans, a major systemic fungal pathogen of humans. Our results in vitro and in vivo suggest that this drug-delivery system may serve as a novel tool to facilitate the use of antimicrobial peptides as targeting ligands to pathogenic microbes, which would open new avenues of investigation in the field of drug delivery.

Interactions of histatin-3 and histatin-5 with actin.

BACKGROUND: Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and -5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. RESULTS: Histatin-3 and -5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and -5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and -5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. CONCLUSIONS: Both histatin-3 and -5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8 amino acid sequence at the C-terminus of histatin-3. Extracellular actin might regulate histatin activity in the oral cavity, which should be the subject of further investigation.

Peptídeos peptidomiméticos da película adquirida do esmalte: efeitos no crescimento de cristal de hidroxiapatita / Peptidomimetics of acquired enamel pellicle peptides: effects on hydroxyapatite crystal growth

Os peptídeos da estaterina (DR9) e da histatina 3 (RR14), que ocorrem naturalmente na película in vivo, amplificam o efeito inibitório do crescimento de cristais de hidroxiapatita, função relacionada à remineralizarão do esmalte e formação de cálculos dentários. A hipótese da duplicação/hibridação de domínios funcionais dos peptídeos DR9 da estaterina e RR14 da histatina 3 foi testada. Para isto, os peptídeos peptidomiméticos (DR9-DR9, DR9-RR14), além deles individualmente e suas proteínas intactas (DR9, RR14, estaterina e histatina 3) foram estudados em sete concentrações diferentes para avaliar o efeito da inibição do crescimento de cristais de hidroxiapatita. Foi utilizado um ensaio colorimétrico de microplaca para quantificar o crescimento de cristais de hidroxiapatita. As experiências foram feitas em triplicata e a concentração inibitória (IC50) foi estabelecida para cada grupo. A IC50 foi calculada para todos os peptídeos e proteínas testados. A histatina 3 e o RR14 não atingiram o valor de IC50. O DR9- RR14 atingiu o valor de IC50 a 3,80 M. Como esperado, DR9 e DR9-DR9 demonstraram um efeito inibitório significativo na atividade de crescimento de cristais, atingindo o valor de IC50 a 2,82 M e 1,07 M, respectivamente. A estaterina atingiu o valor de IC50 a 2,50 M. Na análise estatística, foram aplicados os testes ANOVA e Student-Newman-Keuls para comparações por pares, para comparar os valores entre os grupos. O DR9-DR9 amplificou o efeito inibitório do crescimento de cristais de hidroxiapatita quando comparado com DR9 único (p <0,05), demonstrando que a multiplicação do domínio funcional é uma forte tendência evolutiva da proteína. De forma interessante, o peptídeo híbrido DR9-RR14 demonstrou um efeito inibitório intermediário quando comparado com outros dois grupos: DR9 único e DR9-DR9. Este estudo utilizou a abordagem peptidomimética para investigar uma via potencial de evolução da proteína relacionada com a duplicação/hibridação dos constituintes peptídicos naturais da película adquirida de esmalte. O conhecimento obtido por meio dos resultados deste trabalho pode fornecer uma base para o desenvolvimento de peptídeos sintéticos para uso terapêutico, tanto contra cárie dentária, como para a doença periodontal.(AU)

The statherin and histatin 3 peptides (DR9 and RR14 respectively), which occur naturally in the film in vivo, amplify the inhibitory effect for the growth of hydroxyapatite crystals, a function related to remineralization of the enamel and formation of dental calculi. The hypothesis of duplication/hybridization of functional domains of the DR9 peptides of the statherin and RR14 of histatin 3 was tested. For this, the peptidomimetic peptides (DR9-DR9, DR9-RR14), in addition to them individually and their intact proteins (DR9, RR14, statherin and histatin 3) were studied at seven different concentrations to evaluate the effect of growth inhibition of hydroxyapatite crystals. A colorimetric assay of microplate was used to quantify the growth of hydroxyapatite crystals. The experiments were done in triplicate and the inhibitory concentration (IC50) was established for each group. The IC50 was calculated for all peptides and proteins tested. Histatin 3 and RR14 did not reach the IC50 value. DR9-RR14 reached the IC50 value at 3.80 M. As expected, DR9 and DR9-DR9 demonstrated a significant inhibitory effect on crystal growth activity, reaching the IC50 value at 2.82 M and 1.07 M, respectively. Statherin reached the IC50 value at 2.50 M. ANOVA and Student-Newman-Keuls tests for paired comparisons were applied to compare the values between the groups. DR9-DR9 amplified the inhibitory effect of hydroxyapatite crystal growth when compared to single DR9 (p <0.05), demonstrating that the multiplication of the functional domain is a strong protein evolution pathway. Interestingly, the hybrid peptide DR9-RR14 demonstrated an intermediate inhibitory effect when compared to other two groups: single DR9 and DR9-DR9. This study utilized the peptidomimetic approach to investigate a potential pathway of protein evolution related to duplication/hybridization of the natural peptidic constituents of the acquired enamel film. The knowledge obtained through the results of this work can provide a basis for the development of synthetic peptides for therapeutic use, both against dental caries and for periodontal disease.(AU)

The Influence of Chronic Wound Extracts on Inflammatory Cytokine and Histatin Stability.

Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.

Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp.

The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1-2, 2-4 and 2-4 µg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation.

A study of procyanidin binding to Histatin 5 using Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and molecular simulations.

Tannins act as antioxidants, anticarcinogens, cardio-protectants, anti-inflammatory and anti-microbial agents and bind to salivary peptides by hydrophilic and hydrophobic mechanisms. Electrospray Ionization Mass Spectrometry (ESI-MS) has been used to assess both hydrophilic and hydrophobic components of noncovalent binding in protein complexes. In the present study, direct infusion Electrospray-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ES-FTICR MS) is used to assess relative binding affinities of procyanidin tannin stereoisomers for salivary peptides arising from aqueous solutions. The condensed tannins procyanidin B1, B2, B3, and B4 demonstrate significantly different binding affinities for the salivary peptide Histatin 5. Rigid docking combined with molecular dynamics optimization is used to investigate procyanidin-Histatin 5 binding mechanisms and as a basis to rationalize trends found in the corresponding ES-FTICR MS experiments. The relative binding affinities of the four procyanidin rotamers are different in the gas and liquid phases. The simulation results indicate that many of the same contact points are made in both phases, but there is a increase in strong electrostatic interactions and an decrease in π-π contacts upon transfer from the liquid to the gas phase. The simulations reveal that the tannin interactions can make close contacts with a variety of amino acid residues on the peptide.

Histatins: salivary peptides with copper(II)- and zinc(II)-binding motifs: perspectives for biomedical applications.

Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

Sortase A as a tool for high-yield histatin cyclization.

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 µM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.

Titanium immobilized with an antimicrobial peptide derived from histatin accelerates the differentiation of osteoblastic cell line, MC3T3-E1.

The objective of this study was to evaluate the effect of titanium immobilized with a cationic antimicrobial peptide (JH8194) derived from histatin on the biofilm formation of Porphyromonas gingivalis and differentiation of osteoblastic cells (MC3T3-E1). The titanium specimens (Ti) were immobilized with JH8194, according to the method previously described. The colonization of P. gingivalis on JH8194-Ti was significantly lower than that on control- and blocking-Ti. JH8194-Ti enhanced the mRNA expressions of Runx2 and OPN, and ALPase activity in the MC3T3-E1, as compared with those of control- and blocking-Ti. These results, taken together, suggested the possibility that JH8194-Ti may be a potential aid to shorten the period of acquiring osseointegration.

Candida albicans biofilm formation on peptide functionalized polydimethylsiloxane.

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.

In vitro reactive oxygen species production by histatins and copper(I,II).

The ability of the histidine-rich peptides, histatin-5 (Hst-5) and histatin-8 (Hst-8), to support the generation of reactive oxygen species during the Cu-catalyzed oxidation of ascorbate and cysteine has been evaluated. High levels of hydrogen peroxide (70-580 mol/mol Cu/h) are produced by aqueous solutions containing Cu(II), Hst-8 or Hst-5, and a reductant, either ascorbate or cysteine, as determined by the postreaction Amplex Red assay. When the reactions are conducted in the presence of superoxide dismutase, the total hydrogen peroxide produced is decreased, more so in the presence of the peptides (up to 50%), suggesting the intermediacy of superoxide in these reactions. On the other hand, the presence of sodium azide or sodium formate, traps for hydroxyl radicals, has no appreciable effect on the total hydrogen peroxide production for the Cu-Hst systems. EPR spin-trapping studies using 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in the cysteine-Cu(II) reactions reveal the formation of the CYPMPO-hydroperoxyl and CYPMPO-hydroxyl radical adducts in the presence of Hst-8, whereas only the latter was observed with Cu alone.