Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Evolutionary algorithms and de novo peptide design

One of the goals of computational chemistry is the automated de novo design of bioactive molecules. Despite significant progress in computational approaches to ligand design and efficient evaluation of binding energy, novel procedures for ligand design are required. Evolutionary computation provides a new approach to this design issue. This paper presents an automated methodology for computer-aided peptide design based on evolutionary algorithms. It provides an automatic tool for peptide de novo design, based on protein surface patches defined by user. Regarding the restrictive constrains of this problem a special emphasis has been made on the design of the evolutionary algorithms implemented.     

In situ polycondensation of amino acid modified liposomes and their properties

Three amphiphilic amino acids based on glutamic acid, i.e. S-[1-carboxy-2-([N-bistetradecyl- -glutamate]carbon-yl)ethyl]cysteine (1), S-[1-carboxy-2-([N-bishexadecyl- -glutamate]carbonyl)ethyl]cysteine (2), S-[1-carboxy-2-([N-bis-octadecyl- -glutamate]carbonyl)ethyl]cysteine (3), were synthesized. The aggregation behavior of them in water or buffer solution was studied. It was found that upon hydration and sonication in water, they could form stable liposomes. This kind of amino acid modified liposome was then polycondensed locally on the liposome surface to form a polypeptide-surfaced liposome and the peptide formation was detected by Fr-IR, GPC, etc. The effect of polycondensation of amino acid on the properties of liposomes were studied by detecting the phase transition temperatures with DSC or measuring the leakage of the encapsulated fluorescent probe from the liposomes. It was observed that the phase transition temperatures of the peptide liposomes moved down and the polycondensation of amino acid moieties obviously increased the leakage of the encapsulated molecules.     

Potentiometric urea biosensor based on BSA embedded surface modified polypyrrole film

A potentiometric biosensor based on bovine serum albumin (BSA) embedded surface modified polypyrrole has been developed for the quantitative estimation of urea in aqueous solution. The enzyme, urease (Urs), was covalently linked to free amino groups present over the BSA embedded modified surface of the conducting polypyrrole film electrochemically deposited onto an indium–tin-oxide (ITO) coated glass plate. The biosensor has been characterized by UV–visible, infrared spectroscopy and SEM. Potentiometric and spectrophotometric response of the enzyme electrode (Urs/BSA-PPy/ITO) were measured as a function of urea concentration in Tris–HCl buffer (pH 7.0). It has been found that the electrode responds to low urea concentration with wider range of detection. The electrode showed a linear response range of 6.6 × 10⁻⁶ to 7.5 × 10⁻⁴ M urea. The response time is about 70–90 s reaching to a 95% steady-state potential value and 75% of the enzyme activity is retained for about 2 months. These results indicate an efficient covalent linkage of enzyme to free amino groups of the BSA molecules over the surface of polypyrrole film, which leads to high enzyme loading, an increased lifetime stability of the electrode and an improved wide range of detection of low urea concentration in aqueous solution.     

Novel peptides derived from egg white protein inhibiting alpha-glucosidase

The aim of this study was to search and identify potential anti-diabetic peptides with α-glucosidase and α-amylase inhibitory activities. After the Alcalase hydrolysis, egg white protein hydrolysates were purified and identified by LC–MS–MS. Eight identified peptides were further synthesized by the Fmoc solid-phase synthesis. The anti-diabetic activities of these synthetic peptides were assessed using enzymatic inhibitory assays against the α-glucosidase and α-amylase. Among the eight peptides, peptide RVPSLM was discovered as a potential α-glucosidase inhibitor with an IC₅₀ value at 23.07 μmol L⁻¹. However, it did not exhibit a visible or detectable inhibitory efficiency on the α-amylase. These studies indicate the potential of using egg white protein hydrolysates as a functional food product with the anti-diabetic activity.     

ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical,Non-Canonical,and Alternative Routes of Inflammasome Activation in Human THP1 Cells

Dysregulated inflammasome activation and interleukin (IL)-1β production are associated with several inflammatory disorders. Three different routes can lead to inflammasome activation: a canonical two-step, a non-canonical Caspase-4/5- and Gasdermin D-dependent, and an alternative Caspase-8-mediated pathway. Natriuretic Peptides (NPs), Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP), binding to Natriuretic Peptide Receptor-1 (NPR-1), signal by increasing cGMP (cyclic guanosine monophosphate) levels that, in turn, stimulate cGMP-dependent protein kinase-I (PKG-I). We previously demonstrated that, by counteracting inflammasome activation, NPs inhibit IL-1β secretion. Here we aimed to decipher the molecular mechanism underlying NPs effects on THP-1 cells stimulated with lipopolysaccharide (LPS) + ATP. Involvement of cGMP and PKG-I were assessed pre-treating THP-1 cells with the membrane-permeable analogue, 8-Br-cGMP, and the specific inhibitor KT-5823, respectively. We found that NPs, by activating NPR-1/cGMP/PKG-I axis, lead to phosphorylation of NLRP3 at Ser295 and to inflammasome platform disassembly. Moreover, by increasing intracellular cGMP levels and activating phosphodiesterases, NPs interfere with both Gasdermin D and Caspase-8 cleavage, indicating that they disturb non-canonical and alternative routes of inflammasome activation. These results showed that ANP and BNP anti-inflammatory and immunomodulatory actions may involve the inhibition of all the known routes of inflammasome activation. Thus, NPs might be proposed for the treatment of the plethora of diseases caused by a dysregulated inflammasome activation.     

Cathode modification with peptide nanotubes (PNTs) incorporating redox mediators for azo dyes decolorization enhancement in microbial fuel cells

To enhance azo dye reduction in cathode of microbial fuel cells (MFCs) and power generation, a novel cathode modification method was developed on carbon paper (CP) through immobilization of redox mediators (RMs) with self-assembled peptide nanotubes (PNTs) as the carrier. Results showed that the optimum peptide concentration for PNT self-assembly on electrode and Orange II decolorization in MFCs was 2 mg mL^?1. The PNT/RMs/CP electrodes exhibited higher electrocatalytic activities than PNT or RM solely modified electrodes and raw carbon paper electrode. MFCs loaded with the riboflavin (RF)/PNT modified cathode (PNT/RF/CP) or anthraquinone-2, 6-disulfonate (AQDS)/PNT modified cathode (PNT/AQDS/CP) showed an enhanced decolorization rate to Orange II compared to that with the control electrode, with the reduction kinetic constants increased by 1.3 and 1.2 folds, respectively. Furthermore, the MFCs with the PNT/AQDS/CP cathode and PNT/RF/CP cathode generated a higher maximum power density of 55.5 mW m^?2 and 72.6 mW m^?2, respectively, compared to the control (15.5 mW m^?2). The PNT/RMs modification could reduce cathode total internal resistance and accelerate electron transfer from electrodes to dyes, which may result in the enhanced performance of MFCs.     

Angiotensin-I converting enzyme inhibitory and antioxidant activity of bioactive peptides produced by enzymatic hydrolysis of skin from grass carp (Ctenopharyngodon idella)

Grass carp skin pieces were homogenized in water and hydrolyzed by Alcalase®, collagenase, proteinase K, and/or trypsin at their optimum conditions. Samples were taken at various degrees of hydrolysis and were evaluated for antioxidant, antimicrobial, and angiotensin-converting enzyme inhibitory activities. Alcalase and collagenase completely hydrolyzed the skin with different rates, and released peptides with antioxidant and angiotensin-converting enzyme-inhibitory activity. These activities increased linearly with increasing degrees of hydrolysis. Subsequent incubation of the collagenase hydrolysates with trypsin slightly increased the antioxidant activity. Proteinase K, although only partially hydrolyzing the skin, also catalyzed the release of peptides with antioxidant and angiotensin-converting enzyme-inhibitory activities. These results show that skin by-products from grass carp can be a source of bioactive peptides produced by a one-step reaction. Such hydrolysates may be applied in food products to prolong shelf life and provide beneficial effects on blood pressure.     

Application of mixed self-assembled monolayers (Mixed SAMs) for nucleic acid detection

Mixed self-assembled monolayers (Mixed SAMs) consisting of 8-Ferrocenyl-1-octanethiol and 6-Mercapto-1-hexanol (FcOT:MCH) with probe PNA on gold electrodes were fabricated by using two-step after the optimization of immobilization temperature of FcOT:MCH SAMs. Using AC voltammetry, a novel nucleic acid detection platform, with mixed SAMs, was proposed. A negative formal potential shift was observed after complementary ssDNA hybridization while there was no significant difference after non-complementary ssDNA hybridization. Compatible results were obtained with the measurement of formal potential differences between mixed SAMs and target DNA (complementary and non-complementary DNA) in different target DNA concentrations. The formal potential difference between mixed SAMs immobilization and complementary ssDNA hybridization was measured in different ionic strength concentrations.