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[Display omitted] Lipopeptides synthesized from the Nucleolar Targeting Peptide (NrTP6) with one, two or four dodecanoic fatty acid (FA) chains, display large head to tail volumes, which together with the number of lipid chains per molecule, impacts their self-assembly behavior. In phosphate buffer (PB), peptide to peptide interactions are triggered by the presence of phosphate ions that act as ionic crosslinkers, affecting the organization of the lipid assemblies. The NrTP6 lipopeptides were synthesized by the solid phase peptide synthesis technique. The critical micellar concentration (CMC) of the lipopeptides was determined in water and PB by pyrene fluorescence. The size and morphology of lipopeptide assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Circular dichroism (CD) was used to study the secondary structures of the lipopeptide assemblies. For NrTP6 lipopeptides with two and four lipid chains, CMCs in water are larger than in PB. TEM images of the lipopeptide assemblies show different morphologies including fibers, rods, and spheres depending on the number of lipid chains, concentration and whether they are assembled in water or PB. CD spectroscopy shows that the peptide conformation, either random or beta, correlates with the morphology of the assemblies. [ABSTRACT FROM AUTHOR]

Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys26Arg34GLP-1 (11–37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity. • N-terminal protein tag in X-Y-D4K-G can promote inclusion body formation and expression level of rLys26Arg34GLP-1 (11-37). • On-column refolding and on-column enzyme cleavage can significantly enhance efficiency and yield of rLys26Arg34GLP-1. • A high yield of 3.0 g/L rLys26Arg34GLP-1 (11–37) was obtained through bio-fermentation. • The full-length semaglutide was successfully obtained through fragmented condensation coupling. [ABSTRACT FROM AUTHOR]

Through the l - to d - type amino acid substitution and attachment of solubilizing tags, the novel proteolysis-resistant and water-soluble fluorescent probes were developed, which could achieve the sensitive "turn on" sensing of heparin. [Display omitted] • The first systematic structural optimizations were conducted on the promising fluorescent probe TPE-1 for heparin sensing. • The novel d -type peptide based fluorescent probes were developed for the highly sensitive "turn on" sensing of heparin. • The representative heparin probe XH-6 was demonstrated to possess extremely high proteolytic stability and good solubility. Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d -type peptide fluorescent probe XH-6 indicated that d -type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d -type peptides based fluorescent probes. [ABSTRACT FROM AUTHOR]

Dengue virus infection depends on its fusion with the host membrane, where the binding occurs through interaction between proteins on the virus cell surface and specific viral receptors on target membranes. This process is mediated by the fusion peptide located between residues 98 and 112 (DRGWGNGCGLFGKGG) that forms a loop in domain II of dengue E glycoprotein. In this study, we evaluated the role of fusion peptide surrounding regions (88–97 and 113–123) of the Dengue 2 subtype on its interaction with the membrane and fusion activity. These sequences are important to stabilize the fusion peptide loop and increase fusion activity. Three peptides, besides the fusion peptide, were synthesized by SPPS using the Fmoc chemical approach. The first contains the fusion peptide and the C-terminal region of the loop (sequence 98–123); another contains the N-terminal region (88–112) and the larger peptide contains both regions (88–123). The peptides were able to interact with a model membrane. Differences in morphology of the monolayer promoted by the peptides were assessed by Brewster Angle Microscopy (BAM). Our data indicated that the C-terminal region of fusion peptide loop is more efficient in promoting fusion and interacting with the membrane than the N-terminal sequence, which is responsible for the electrostatic initial interaction. We propose a 2-step mechanism for the interaction of the dengue virus fusion peptide with the host membrane, where the N-terminal sequence docks electrostatically on the headgroups and then the C-terminal interacts via hydrophobic forces in the acyl chains. [Display omitted] • 2-step mechanism for dengue virus fusion peptide. • N-terminal region can be related to the initial interaction in the membrane. • C-terminal is important for dengue virus fusion peptide. [ABSTRACT FROM AUTHOR]

The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization. • Morpholino-peptide based oligomers show a preferred secondary structure. • Morpholino-glycine based oligomers form complexes with DNA in a 2: 1 molar ratio. • The complex with DNA is stabilized by hydrophobic contacts among bases and H-bonds between the peptide backbone. [ABSTRACT FROM AUTHOR]

[Display omitted] • The synthetic strategies and side reaction mechanisms involved in the synthesis of N -methylated peptides. • The impact of backbone N -methylation on peptide pharmacokinetic properties, such as membrane permeability, metabolic stability and binding affinity and selectivity. • Case studies illustrating the successful application of N -methylated peptides in drug discovery and development. • Current challenges and future directions in the field of backbone N -methylated peptides. Peptide-based drugs have garnered considerable attention in recent years owing to their increasingly crucial role in the treatment of diverse diseases. However, the limited pharmacokinetic properties of peptides have hindered their full potential. One prominent strategy for enhancing the druggability of peptides is N -methylation, which involves the addition of a methyl group to the nitrogen atom of the peptide backbone. This modification significantly improves the stability, bioavailability, receptor binding affinity and selectivity of peptide drug candidates. In this review, we provide a comprehensive overview of the advancements in synthetic methods for N -methylated peptide synthesis, as well as the associated limitations. Moreover, we explore the versatile effects of N -methylation on various aspects of peptide properties. Furthermore, we emphasize the efforts dedicated to N -methylated peptide pharmaceuticals that have successfully obtained marketing approval. [ABSTRACT FROM AUTHOR]

[Display omitted] • Comparison of guanidinylation reagents in on-resin transformations. • 1 H -triazole- and 4-nitro-1H-pyrazole-1-[N,N'-bis(tert -butoxycarbonyl)]carboxamidine. • Unprecedented incorporation of 4,4-difluoroproline in an antimicrobial peptide. • Oncocin analogue displaying three 4,4,-difluoroproline residues was best in series. • Low micromolar minimal inhibitory concentrations in E. coli and K. pneumoniae. Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc- RPRPPR) of the AMP oncocin (i.e., VDKPPYLP RPRPPR RIYNR-NH 2) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1 H -triazole-1-[ N,N′ -bis(tert -butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N -terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Arg → Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans -4-hydroxy- l -proline (Hyp) or 4,4-difluoro- l -proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l -Arg → d -Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Pro → Dfp modifications proved to possess the best activity profile: MICs of 1–2 µg/mL against E. coli and Klebsiella pneumoniae , less than 1% hemolysis at 800 µg/mL, and an IC 50 above 1280 µg/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure–activity studies aimed at optimization of Pro-rich AMPs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Peptide nucleic acids (PNAs) have shown tremendous potential in enormous chemical and biological applications. • New designs of PNA analogs have been developed to overcome some limitations and to improve the properties of original PNAs. • Progress in the backbone and chemical modifications developed over the past few years to improve PNA function is summarized. • Recent advances in the PNA therapeutic and diagnostic applications have also been discussed in the manuscript. Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs) are synthesized by attaching modified peptide backbones generally derived from repetitive N -2-aminoethyl glycine units in place of the regular phosphodiester backbone and represent synthetic impersonator of nucleic acids that offers an exciting research field due to their fascinating spectrum of biotechnological, diagnostic and potential therapeutic applications. The semi-rigid peptide nucleic acid backbone serves as a nearly-perfect template for attaching complimentary base pairs on DNA or RNA in a sequence-dependent manner as described by Watson-Crick models. PNAs and their analogues are endowed with exceptionally high affinity and specificity for receptor sites, essentially due to their polyamide backbone's uncharged and flexible nature. The present review compiled various strategies to modify the polypeptide backbone for improving the target selectivity and stability of the PNAs in the body. The investigated biological activities carried out on PNAs have also been summarized in the present review. [ABSTRACT FROM AUTHOR]

• Post-synthetic hooking of photocatalyst Rose Bengal and organocatalyst L-proline in an amine functionalized POF. • The modified framework works in a highly synergistic manner as dual photo-organo catalyst. • The bifunctional catalyst works under environmentally green visible light and can be recycled up to 10 cycles. • The high yield of the products in C-H functionalization is maintained for a wide range of tertiary amine substrates. • Based on experimental findings, a plausible reaction mechanism for the oxidative Mannich reaction is proposed. Photoredox and organo-catalysis denote powerful construction tools for new classes of carbon–carbon bonds, where incisive amalgamation of both the approaches over a single, recyclable platform can bring about synergic and eco-friendly reactions under mild conditions. Aiming at enamine-based photoredox catalysis for atom-economic and metal-free sp3 C H activation, an amide-based two-dimensional (2D) porous organic framework (POF) is devised. The pendent NH 2 groups are judiciously anchored with two catalytic stations viz. Rose Bengal and L-proline, through stepwise variation of solid phase peptide synthesis. The dual-catalyst engineered POF represents a fully organic material that synergistically performs visible-light triggered oxidative Mannich reaction to produce biorelevant heterocycle β-amino ketone in excellent yield at room temperature, using oxygen as clean and selective oxidant. Importantly, activity of this bi-functionalized catalyst compares favorably well to individual homogeneous counterparts. The covalently modified framework demonstrates economic viability via gram-scale synthesis besides admirable reusability, and proves to be effective for nineteen varieties of substrates. The photocatalytic path is detailed from efficient energy transfer from host polymer to substrate in light of experimental and theoretical studies, which provides proof-of-concept to the photo-organo combined mechanism. The material benefits heterogenising two homogeneous catalysts, besides excluding additional steps of conventional Mannich reactions, and offers a step-forward to smart and green cross-dehydrogenative coupling reactions. [ABSTRACT FROM AUTHOR]

Antheraea mylitta , a tropical non-mulberry silkworm, is cultivated for tasar silk production in India. Several defense molecules including few antimicrobial peptides (AMPs) and proteins have been identified from this insect. Here, we have isolated and purified an antimicrobial tri-peptide by sequential chromatographic separation procedures. The amino acid sequence of the peptide was determined as NH 2 -Gln-Ala-Lys-COOH (QAK) using MALDI MS/MS fragmentation analysis. Further, the peptide was synthesized in vitro following solid phase chemistry of peptide synthesis and acetylated by acetic anhydride reaction. Antimicrobial activities of non-acetylated and acetylated QAK were tested against both Escherichia coli and Staphylococcus aureus bacteria. Acetylated peptide inhibited bacterial growth more effectively and its minimum inhibitory concentration (MICs) was found lower than non-acetylated peptide. SEM studies revealed more membrane damage and release of intracellular materials like β-galactosidase enzyme from acetylated peptide treated bacteria in comparison to non-acetylated QAK. At MIC, acetylated peptide did not show any significant hemolytic activity against rabbit erythrocytes. The results suggest that acetylated-QAK is a promising new antimicrobial peptide and can be used for therapeutic purpose. Image 1 • Purification of an antimicrobial peptide from hemolymph of A. mylitta. • Sequence determination as Q-A-K and chemical synthesis of this peptide. • QAK was acetylated and acetylated form showed higher antibacterial activity. • No significant hemolytic activity was observed for the peptide. • Peptide disrupts bacterial membrane and releases intracellular materials. [ABSTRACT FROM AUTHOR]

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P 1 ′ and/or P 2 ′ of their substrates (mimicked by the Ala-Ala/P1′-P2′ residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP- Asp -Ala-Tyr-PABA, CPP- Arg -Ala-Tyr-PABA, CPP-Ala- Asp -Tyr-PABA, CPP-Ala- Arg -Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP. • Electrostatic differences mapped at the NEL and TOP binding sites were used to design new selective inhibitors. • Inhibitors containing Asp residues at P 1 ′ and P 2 ′ positions presented higher selectivity to NEL. • Inhibitors containing Arg residues at P 1 ′ and P 2 ′ positions presented higher selectivity to TOP. • NEL R470E and TOPE469R mutants confirmed the major contribution of this site for inhibitor binding. • Molecular docking supported the results and provided directions for future developments. [ABSTRACT FROM AUTHOR]

[Display omitted] • β-glucans are carbohydrate polymers that have been used as immunostimulants. • β-glucans stimulate several cellular and humoral mechanisms. • β-glucans increase protection against a wide range of pathogens. • β-glucans improve the production rate of economically important species. • β-glucans could be related to priming innate immunity. β-glucans (βGs) are carbohydrate polymers linked by β-1,3, 1,4 or 1,6 bonds, they have been used to protect against potential pathogens and prevent lethal diseases. The immune system possesses several receptors that identify a wide range of structures and trigger cellular and humoral mechanisms. However, the mechanisms by which βGs activate the immune system of invertebrate organisms have not been fully clarified. This review is focused on evaluating the effect of βGs on innate immune system in invertebrates. βGs stimulate different cellular and humoral mechanisms, such as phagocytosis, oxygen species production, extracellular trap formation, proPO system, and antimicrobial peptide synthesis, moreover, βGs increase survival rate and decrease pathogen load in several species. [ABSTRACT FROM AUTHOR]

Abstract The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S21 protein of phage ϕ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S2168, and negative-dominant mutant form, S21IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ] 222 /[θ] 208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics. Highlights • First in vitro synthesis of S21 pinholins using solid phase peptide synthesis. • Successful preparation of a functional pinholin system in vitro. • First biophysical study of a full length functional pinholin system. • Spectroscopic results offer the opportunity for more in depth spectroscopic studies. [ABSTRACT FROM AUTHOR]

The oncogenic transcriptional corepressor Nac1 contains a conserved POZ protein–protein interaction module that mediates homodimerization or heterodimerization with itself or other POZ proteins. The dimerization has been recognized as an attractive target for cancer therapy. Here, we attempted to (i) discover those potential binding partners of Nac1 in the human genome, (ii) derive key peptide segments from the complex interface of Nac1 with its putative partners, and (iii) improve the peptide binding affinity to Nac1 POZ domain. In the procedure, Nac1 POZ dimerization with 136 human POZ domains was modeled, simulated and analyzed at atomic level to elucidate structural basis, energetic property and dynamics behavior. Two hotspot regions, namely α1-helix and α2/α3-hairpin, at the dimerization interface were identified that are responsible for stabilizing the formed POZ–POZ dimer complexes. The α1-helix and α2/α3-hairpin were stripped from the interface to derive their respective isolated SIP peptides, which, however, exhibited a large flexibility and intrinsic disorder in free state, and thus would incur a considerable penalty upon rebinding to Nac1 POZ domain. By carefully examining the natively folded structures of α1-helix and α2/α3-hairpin in protein context and their interaction modes with the domain, we rationally designed a hydrocarbon bridge and a disulfide bond separately for the two peptides in order to constrain their conformational flexibility in free state, thus largely minimizing the flexibility penalty. Consequently, three α1-helix peptides derived from Nac1, Miz1 and Slx4 were stapled by all-hydrocarbon bridge, while four α2/α3-hairpin peptides derived from Nac1, Bacd1, Klh28 and Mynn were cyclized by disulfide bond. Binding affinity analysis revealed that, as designed, these peptides were converted from non- or weak binders to moderate or good binders of Nac1 POZ domain upon the stapling and cyclization. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the innate immune responses of invertebrates have been less studied. In this study, Sp TNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. Sp TNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of Sp TNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of Sp TNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that Sp TNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (Sp LITAF) can regulate the expression of Sp TNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of Sp LITAF were found to be regulated by WSSV infection. Knocking down of Sp LITAF increased the WSSV copy number and expression of VP28 gene. Taken together, these results proved the protective function of Sp TNF, which is regulated by Sp LITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis. • Sp TNF is initially down-regulated but subsequently up-regulated in hemocytes of mud crabs during WSSV infection. • Sp TNF enhances mud crab immunity against WSSV infection by activating the apoptosis, NF-κB pathway, and AMP synthesis. • Sp LITAF can regulate the expression of Sp TNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. • Expression and nuclear translocation of Sp LITAF are regulated by WSSV infection. • Sp LITAF can regulate the replication of WSSV in mud crabs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Synthesis and cleavage characterization of GSH and cathepsin B responsive fluorinated polyarginine (PFC-PR) • Compared with non-fluorinated analogue, the fluorinated peptide exhibited improved siRNA binding ability, enhanced cellular uptake and better serum resistance capacity. • PFC-PR could effectively deliver VEGF -siRNA into HeLa cells, inducing anti-cancer therapy. Small interfering RNA (siRNA) can be exploited to silence specific genes associated with cancer development, and successful siRNA therapy is highly dependent on the efficiency of the siRNA delivery vector. Herein, a well-designed novel redox- and enzyme-responsive fluorinated polyarginine (PFC-PR) was developed to be used as an anti-cancer siRNA carrier. The multiple guanidine groups could provide positive charges and bind with siRNA efficiently, and further fluorination modification enhanced the interaction with siRNA, resulting in a more stable PFC-PR/siRNA nanocomplex, improving serum tolerance, and promoting cellular uptake and endosome escape. Meanwhile, the PFC-PR was responsive to overexpressed cathepsin B and high levels of glutathione in cancer cells, conferring its ability to enhance siRNA release within cancer cells and making it cancer-targeting. Consequently, PFC-PR showed good biocompatibility and high gene silencing efficiency, which could inhibit cancer cell growth when delivered the siRNA targeting vascular endothelial growth factor, suggesting that it can be potentially used for anti-cancer gene therapy applications. [ABSTRACT FROM AUTHOR]

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue. [ABSTRACT FROM AUTHOR]

The concept that life on Earth began as an RNA world has been built upon extensive experimentation demonstrating that many of the building blocks required for living cells could be synthesized in the laboratory under conditions approximating our primordial world. Many of the building blocks for life have also been found in meteorites indicating that meteors may have been a source for these molecules, or more likely, that they represent the chemical library present in most/all bodies in the universe after the big bang. Perhaps the most important support for the concept comes from the fact that some RNA species possess catalytic activity, ribozymes, and that RNA could be reverse transcribe to DNA. The thrust of numerous papers on this topic has been to explore how the available molecules on Earth, at its birth, gave rise to life as we know it today. This paper focuses more on a reverse view of the topic. The “how” molecular building blocks were synthesized is not addressed nor how the “first” RNA molecules were synthesized. We can clearly speculate on the variable environmental conditions and chemistry available on Earth billions of years ago. However, we can never truly replicate the changing conditions or know the chemical composition of Earth at the beginning of time. We can, however, confirm that over millions, perhaps billions of years the basic building blocks for life accumulated sufficiently to initiate evolution to an RNA world followed by our RNA/DNA world. Here we are attempting to take the information from our current knowledge of biology and by inference and extrapolation work backward to hypothesize biological events in the march forward from RNA to DNA. It is proposed that the primordial replicating RNA cell, the ribocyte, evolved from liposomes encompassing required reactants and products for “life” and that ribonucleopeptide complexes formed membrane pores to support bidirectional ion and molecular transport to maintain biological functions and osmolarity. Circular RNA, circRNA, is proposed as a critical stable RNA molecule that served as the genetic precursor for the switch to DNA and the replication of circRNA by a rolling circle mechanism gave rise to the RNA complexity required for the genetic functions of the cell. The replicating ribocyte would have required protein synthesis as well as RNA replication and a model for non-coded and primordial coded protein synthesis is proposed. Finally, the switch from the RNA to the DNA world would have involved the synthesis of an RNA:DNA hybrid prior to the formation of dsDNA. If the hybrid was a circular molecule that ultimately yielded a circular dsDNA molecule, it could predict that the primordial DNA cell would evolve into a bacterial cell with a single circular chromosome. One would hope that continued speculation of the origin of life will spur new directions of research that may never fully answer the questions of the past but add to our ability to regulate potentially harmful biological events in the present and in the future. [ABSTRACT FROM AUTHOR]

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola , proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites. [ABSTRACT FROM AUTHOR]

This study was done to isolate novel antioxidant peptides from Chinese chestnut protein (CCP) hydrolysed with alcalase. A series of purification steps including ultrafiltration, gel filtration chromatography, anion exchange chromatography, and reverse-phase high-performance liquid chromatography were used to purify the CCP. ABTS radical scavenging capacity was assessed to evaluate antioxidant activity. Five antioxidant peptides, VYTE, TKGQ, MMLQK, TPAIS, and VSAFLA, with molecular weights of 590.20, 592.17, 745.29, 647.20, and 606.34 Da, respectively, were obtained from the most active fractions and characterized using nano-liquid chromatography electrospray ionisation-tandem mass spectrometry. VYTE and VSAFLA showed the highest ABTS radical scavenging capacity with IC 50 values of 0.13 ± 0.01 and 0.28 ± 0.04 mg/mL, respectively ( P  < .05). Moreover, the five novel antioxidant peptides had good antioxidant activity after synthesis and simulated digestion. These novel peptides may have potential applications as functional foods or nutraceutical compounds. [ABSTRACT FROM AUTHOR]

In this work we evaluated the mode of action of six new synthesized peptides ( Met-Asp-Asn ; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe–Leu ), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3–8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates. [ABSTRACT FROM AUTHOR]

Soy, milk and egg white proteins are commonly added to meat products. Proteins of non-meat origin, are applied to improve the water-binding capacity of meat and the texture of the product, as well as for economic reasons as meat substitutes. Detection of low amounts of these proteins is relevant to food safety and human health due to their high allergenicity. In this study, we demonstrate that a nano-LC-Q-TOF-MS/MS method can be used to reliably detect non-meat proteins derived from functional protein additives in various commercial poultry meat products (sausages, frankfurters, pâtés). A large number of heat-stable peptide markers, unique to soy (43 peptides), milk (9 peptides) and egg white (12 peptides), were detected. The source of the peptides were main allergenic proteins, namely soy glycinin and β-conglycinin, milk α-S1-casein and the whey protein β-lactoglobulin, as well as egg white ovotransferrin and lysozyme C. These proteins appeared to be relatively resistant to technological operations used in meat processing including roasting and sterilisation. The mass spectrometry method presented, enables reliable monitoring of the presence of allergenic proteins in severely processed meat products, providing a valuable alternative to PCR and ELISA tests. [ABSTRACT FROM AUTHOR]

Poor bioavailability of food-derived bioactive peptides impedes their application in functional foods. This study focuses on understanding the impact of net charge of whey peptides, separated by electrodialysis with ultrafiltration membrane (EDUF), on encapsulation efficiency (EE) and surface properties of the peptide-loaded liposomes. EDUF resulted in two fractions; anionic whey peptides (AWP) with a relative abundance of 30.4% anionic amino acid residues, and cationic whey peptides (CWP) with 20.39% cationic amino acid residues. Although free amino group of CWP was higher, average peptide length and molecular weight were similar in both fractions. AWP liposomes had slightly lower encapsulation efficiency than encapsulated CWP. Encapsulated CWP had larger mean particle diameter and higher polydispersity index. However, there was no apparent effect of peptide net charge on liposome surface charge (ζ-potential). FTIR spectra of the CWP- and AWP-loaded liposomes were similar and showed peptide interaction with the liposome phosphate groups. The peptide net charge did not appear to influence their distribution in different regions of the nanocapsules. The EE and physicochemical properties of the liposomes may be influenced by a combination of peptide net charge, homogeneity, and surface hydrophobicity. These findings are relevant to developing efficient liposomal systems for bioactive peptide delivery. [ABSTRACT FROM AUTHOR]

Hypothesis The incorporation of a succinic acid-derived moiety in amino acid derivatives would favor an intramolecular catalysis of a deamidation reaction. Such reaction would permit controlled disassembly of molecular hydrogelators and the use of the hydrogels for controlled release of actives. Experimental Low molecular weight hydrogelators containing a succinic acid-derived moiety were prepared by conventional organic synthesis procedures. Hydrogels were examined by electron microscopy and 1 H NMR studies were carried out to evaluate the solubility in water of the hydrogelators and the deamidation reaction. Liberation of Rose Bengal entrapped in the hydrogels was monitored by UV–Vis spectroscopy. Findings Molecular hydrogels formed by pseudopeptidic derivatives of l -valine suffer a thermal deamidation reaction, leading to partial disassembly. The succinic acid-derived moiety present in the gelators is responsible of intramolecular catalysis of a deamidation reaction. Such neighboring group effect is reminiscent of biochemical processes such as protein deamidation and self-excision of inteins. It has been found that the thermodynamic equilibrium of the deamidation reaction is regulated by the efficiency of hydrogelation. As a proof of concept, the thermally promoted deamidation is applied to controlled release of Rose Bengal. [ABSTRACT FROM AUTHOR]

The cytotoxic peptide LVTX-8, recently isolated from spider Lycosa vittata , is considered as a potential precursor for further anticancer drug development. In this study, a series of novel LVTX-8-based analogues and cytotoxic conjugates were rationally designed, synthesized, and systematically evaluated. Compared with the parent LVTX-8, peptides 825 and 827 not only possessed higher proteolytic stability, but also exhibited significantly improved anticancer durability and greatly reduced hemolysis. [Display omitted] • Ten spider toxin peptide LVTX-8-based analogues were designed and efficiently synthesized. • Their anticancer efficacy, serum stability, hemolytic activity, time-kill kinetics, anticancer mode of action, and subcellular localizations were evaluated systematically. • In particular, peptides 825 and 827 not only possessed higher proteolytic stability, but also exhibited significantly improved anticancer durability. • In addition, peptides 825 and 827 exhibited greatly reduced hemolysis, as well as higher selectivity to cancer cells than to human erythrocytes. Cytotoxic peptides derived from spider venoms have been considered as promising candidates for anticancer treatment. The novel cell penetrating peptide LVTX-8, which is a 25-residue amphipathic α-helical peptide isolated from spider Lycosa vittata , exhibited potent cytotoxicity and is a potential precursor for further anticancer drug development. Nevertheless, LVTX-8 may be easily degraded by multiple proteases, inducing the proteolytic stability problem and short half-life. In this study, ten LVTX-8-based analogs were rationally designed and the efficient manual synthetic method was established by the DIC/Oxyma based condensation system. The cytotoxicity of synthetic peptides was systematically evaluated against seven cancer cell lines. Seven of the derived peptides exhibited high cytotoxicity towards tested cancer in vitro , which was better than or comparable to that of natural LVTX-8. In particular, both N -acetyl and C-hydrazide modified LVTX-8 (8 2 5) and the conjugate methotrexate (MTX)-GFLG-LVTX-8 (8 2 7) possessed more durable anticancer efficiency, higher proteolytic stability, as well as lower hemolysis. Finally, we confirmed that LVTX-8 could disrupt the integrity of cell membrane, target the mitochondria and reduce the mitochondrial membrane potential to induce the cell death. Taken together, the structural modifications were conducted on LVTX-8 for the first time and the stability significantly improved derivatives 825 and 827 may provide useful references for the modifications of cytotoxic peptides. [ABSTRACT FROM AUTHOR]

[Display omitted] • N-terminal modified peptides preferentially bind with anionic lipid bilayer membrane, which could be used for bio-analysis. • The anti-cancer activities of both peptides were evaluated against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero. • The peptide is largely unordered to ordered structure and non-toxic towards normal cell. • In-silico studies suggested that the peptides have good binding interactions with the proteins (PDB ID: 2W3L, 5FC4, and 5GWK). The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2 , did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers. [ABSTRACT FROM AUTHOR]

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein “murine double minute 2” (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities. [ABSTRACT FROM AUTHOR]

This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300 mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine. [ABSTRACT FROM AUTHOR]

Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes. [ABSTRACT FROM AUTHOR]

A toolbox containing diverse Arg N -glycosylated peptides (glucose, galactose, 2-acetamino-2-deoxy-glucose, xylose, ribose, mannose, rhamnose, lactose and maltose), as well as the corresponding specific antibodies was develop. [Display omitted] Among posttranslational modifications, atypical arginine N- glycosylation has drawn increasing interest due to its fundamental role in various cellular procedures and signaling pathways. The efficient synthesis of arginine N- glycosylated substrates, as well as the generation of specific antibodies, remains challenging. This work describes the efficient synthesis of diverse arginine N -glycosylated peptides, in a process termed silver-promoted solid-phase glycosylation (SGG). There are two key features of the SSG strategy: (i) robust synthesis of gram-scale S-alkyl-isothiourea glycosyl donors facilitates the subsequent SSG procedure and (ii) the simultaneous introduction of both the side-chain sugar motif and arginine residue. Notably, our findings, combined with our previous results, provide a toolbox containing diverse S -alkyl-isothiourea glycosyl donors (glucose, galactose, mannose, ribose, xylose, lactose and maltose) as well as the corresponding Arg N -glycosylated peptides. In addition, our toolbox is shown to help investigate specific antibodies and identify multiple potent and precise biochemical tools for exploring arginine N -glycosylation. [ABSTRACT FROM AUTHOR]

[Display omitted] • A small peptide library of a de novo -designed peptide was synthesized via the solid phase peptide synthesis for screening. • Eight peptides bearing different aspartic acid-rich macrolactam rings exhibited improved antibacterial activities. • Mechanistic studies revealed that they could destroy the integrity of bacterial envelope, leading to cytoplasm leakage and rapid dissipation of membrane potential. • Peptides zp90 with a macrolactam ring of (KaDGD) demonstrated preferential interaction with calcium ions. In this work, by capping a macrolactam ring at the C -terminus of a de novo -designed peptide, namely zp80 , we have constructed a small peptide library via the solid phase peptide synthesis for screening. Eight peptides bearing different aspartic acid-rich macrolactam rings but the same linear (IIRR) 4 unit exhibited improved antibacterial activities, hemolytic activity, and selectivity index. Mechanistic studies revealed that they could destroy the integrity of bacterial envelope, leading to cytoplasm leakage and rapid dissipation of membrane potential. One of these peptides, zp90 with a macrolactam ring of (KaDGD), demonstrated preferential interaction with calcium ions at a stoichiometric ratio of 1:1, promoting the affinity of designed peptides to bacterial membrane. Overall, this work provides a feasible strategy for medicinal chemists to further develop potent, selective, and multifunctional de novo -designed antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24–40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1–37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli . Furthermore, when tested on HKL in combination with the immunostimulant β-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture. [ABSTRACT FROM AUTHOR]

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB − phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE. [ABSTRACT FROM AUTHOR]

[Display omitted] • The search for domain-specific ACE inhibitors has been the focus of this report. • Ac-Ang (2–7)-NH 2 is a potential analogue to develop effective cACE inhibitors. • First dynamics simulation report of an Ang (1–7) derivative with both ACE domains. • Molecular dynamics simulations show Val3 and Tyr4 interactions with ACE subsites. • Our results can facilitate the development of domain-selective ACE inhibitors. The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1–7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1–7), sequentially removing the N -terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2–7)-NH 2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S 3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2–7)-NH 2 to develop effective cACE inhibitors. [ABSTRACT FROM AUTHOR]

Alzheimer disease (AD) is the most prevalent form of dementia. Loss of hippocampal synapses is the first neurodegenerative event in AD. Synaptic loss has been associated with the accumulation in the brain parenchyma of soluble oligomeric forms of amyloid β peptide (Aβ1-42 wt ). Clinical observations have shown that a mutation in the APP protein (A673V) causes an early onset AD-type dementia in homozygous carriers while heterozygous carriers are unaffected. This mutation leads to the formation of mutated Aβ peptides (Aβ1-42 A2V ) in homozygous patients, while in heterozygous subjects both Aβ1-42 wt and Aβ1-42 A2V are present. To better understand the impact of the A673V mutation in AD, we analyzed the synaptotoxic effect of oligomers formed by aggregation of different Aβ peptides (Aβ1-42 wt or Aβ1-42 A2V ) and the combination of the two Aβ1-42 MIX (Aβ1-42 wt and Aβ1-42 A2V ) in an in vitro model of synaptic injury. We showed that Aβ1-42 A2V oligomers are more toxic than Aβ1-42 wt oligomers in hippocampal neurons, confirming the results previously obtained in cell lines. Furthermore, we reported that oligomers obtained by the combination of both wild type and mutated peptides (Aβ1-42 MIX ) did not exert synaptic toxicity. We concluded that the combination of Aβ1-42 wt and Aβ1-42 A2V peptides hinders the toxicity of Aβ1-42 A2V and counteracts the manifestation of synaptopathy in vitro. Finally we took advantage of this finding to generate a cell-permeable peptide for clinical application, by fusing the first six residues of the Aβ1-42 A2V to the TAT cargo sequence (Aβ1-6 A2V TAT(D)). Noteworthy, the treatment with Aβ1-6 A2V TAT(D) confers neuroprotection against both in vitro and in vivo synaptopathy models. Therefore Aβ1-6 A2V TAT(D) may represent an innovative therapeutic tool to prevent synaptic degeneration in AD. [ABSTRACT FROM AUTHOR]

Microsphere-based controlled release technologies have been utilized for the long-term delivery of proteins, peptides and antibiotics, although their synthesis poses substantial challenges owing to formulation complexities, lack of scalability, and cost. To address these shortcomings, we used the electrospray process as a reproducible, synthesis technique to manufacture highly porous (>94%) microspheres while maintaining control over particle structure and size. Here we report a successful formulation recipe used to generate spherical poly(lactic-co-glycolic) acid (PLGA) microspheres using the electrospray (ES) coupled with a novel thermally induced phase separation (TIPS) process with a tailored Liquid Nitrogen (LN 2 ) collection scheme. We show how size, shape and porosity of resulting microspheres can be controlled by judiciously varying electrospray processing parameters and we demonstrate examples in which the particle size (and porosity) affect release kinetics. The effect of electrospray treatment on the particles and their physicochemical properties are characterized by scanning electron microscopy, confocal Raman microscopy, thermogravimetric analysis and mercury intrusion porosimetry. The microspheres manufactured here have successfully demonstrated long-term delivery (i.e. 1 week) of an active agent, enabling sustained release of a dye with minimal physical degradation and have verified the potential of scalable electrospray technologies for an innovative TIPS-based microsphere production protocol. [ABSTRACT FROM AUTHOR]

Bacteria and fungi use non-ribosomal peptide synthetases (NRPSs) to produce peptides of broad structural diversity and biological activity, many of which have proven to be of great importance for human health. The impressive diversity of non-ribosomal peptides originates in part from the action of tailoring enzymes that modify the structures of single amino acids and/or the mature peptide. Studying the interplay between tailoring enzymes and the peptidyl carrier proteins (PCPs) that anchor the substrates is challenging owing to the transient and complex nature of the protein–protein interactions. Using sedimentation velocity (SV) methods, we studied the collaboration between the PCPs and cytochrome P450 enzyme that results in the installation of β-hydroxylated amino acid precursors in the biosynthesis of the depsipeptide skyllamycin. We show that SV methods developed for the analytical ultracentrifuge are ideally suited for a quantitative exploration of PCP–enzyme equilibrium interactions. Our results suggest that the PCP itself and the presence of substrate covalently tethered to the PCP together facilitate productive PCP–P450 interactions, thereby revealing one of nature's intricate strategies for installing interesting functionalities using natural product synthetases. [ABSTRACT FROM AUTHOR]

Ustiloxins were found recently to be the first example of cyclic peptidyl secondary metabolites that are ribosomally synthesized in filamentous fungi. In this work, two function-unknown genes ( ustYa / ustYb ) in the gene cluster for ustiloxins from Aspergillus flavus were found experimentally to be involved in cyclization of the peptide. Their homologous genes are observed mainly in filamentous fungi and mushrooms. They have two “HXXHC” motifs that might form active sites. Computational genome analyses showed that these genes are frequently located near candidate genes for ribosomal peptide precursors, which have signal peptides at the N-termini and repeated sequences with core peptides for the cyclic portions, in the genomes of filamentous fungi, particularly Aspergilli , as observed in the ustiloxin gene cluster. Based on the combination of the ustYa / ustYb homologous genes and the nearby ribosomal peptide precursor candidate genes, 94 ribosomal peptide precursor candidates that were identified computationally from Aspergilli genome sequences were classified into more than 40 types including a wide variety of core peptide sequences. A set of the predicted ribosomal peptide biosynthetic genes was experimentally verified to synthesize a new cyclic peptide compound, designated as asperipin-2a, which comprises the amino acid sequence in the corresponding precursor gene, distinct from the ustiloxin precursors. [ABSTRACT FROM AUTHOR]

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for g rass i nduced g ene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum. [ABSTRACT FROM AUTHOR]

The successful synthesis and solid state NMR characterization of silica-based organic–inorganic hybrid materials is presented. For this, collagen-like peptides are immobilized on carboxylate functionalized mesoporous silica (COOH/SiO x ) materials. A pre-activation of the silica material with TSTU ( O -( N -Succinimidyl)- N , N , N ′, N ′-tetramethyluronium tetrafluoroborate) is performed to enable a covalent binding of the peptides to the linker. The success of the covalent immobilization is indicated by the decrease of the 13 C CP-MAS NMR signal of the TSTU moiety. A qualitative distinction between covalently bound and adsorbed peptide is feasible by 15 N CP-MAS Dynamic Nuclear Polarization (DNP). The low-field shift of the 15 N signal of the peptide's N-terminus clearly identifies it as the binding site. The DNP enhancement allows the probing of natural abundance 15 N nuclei, rendering expensive labeling of peptides unnecessary. [ABSTRACT FROM AUTHOR]

We performed shock experiments simulating natural comet impacts in an attempt to examine the role that comet impacts play in peptide synthesis. In the present study, we selected a mixture of alanine ( dl -alanine), water ice, and silicate (forsterite) to make a starting material for the experiments. The shock experiments were conducted under cryogenic conditions (77 K), and the shock pressure range achieved in the experiments was 4.8–25.8 GPa. The results show that alanine is oligomerized into peptides up to tripeptides due to the impact shock. The synthesized peptides were racemic, indicating that there was no enantioselective synthesis of peptides from racemic amino acids due to the impact shock. We also found that the yield of linear peptides was a magnitude higher than those of cyclic diketopiperazine. Furthermore, we estimated the amount of cometary-derived peptides to the early Earth based on two models (the Lunar Crating model and the Nice model) during the Late Heavy Bombardment (LHB) using our experimental data. The estimation based on the Lunar Crating model gave 3 × 10 9 mol of dialanine, 4 × 10 7 mol of trialanine, and 3 × 10 8 mol of alanine-diketopiperazine. Those based on the Nice model, in which the main impactor of LHB is comets, gave 6 × 10 10 mol of dialanine, 1 × 10 9 mol of trialanine, and 8 × 10 9 mol of alanine-diketopiperazine. The estimated amounts were comparable to those originating from terrestrial sources (Cleaves, H.J., Aubrey, A.D., Bada, J.L. [2009]. Orig. Life Evol. Biosph. 39, 109–126). Our results indicate that comet impacts played an important role in chemical evolution as a supplier of linear peptides, which are important for further chemical evolution on the early Earth. Our study also highlights the importance of icy satellites, which were formed by comet accumulation, as prime targets for missions searching for extraterrestrial life. [ABSTRACT FROM AUTHOR]

Here we report a unique method of ribosomally synthesizing fused tricyclic peptides. Flexizyme-assisted in vitro translation of a linear peptide with the N-terminal chloroacetyl group and four downstream cysteines followed by the addition of 1,3,5-tris(bromomethyl)benzene results in selective production of the fused tricyclic peptide. This technology can be used for the ribosomal synthesis of fused tricyclic peptide libraries for the in vitro selection of bioactive peptides with tricyclic topology. [ABSTRACT FROM AUTHOR]

Characterization of pathogenesis genes of Metarhizium anisopliae , will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24 h after infection, when the infected larvae reached the moribund stage (36 h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2 h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12 h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1 , CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1 , CHI2 , and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect. [ABSTRACT FROM AUTHOR]

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, which has been implicated in the development of diseases. In the present study, we synthesized a peptide that entered cells and translocated to the ER. This peptide possessed fluorescein isothiocyanate (FITC), HIV-TAT, mini-αA-crystallin, and KDEL sequences. We demonstrated that this peptide entered cells and translocated to the ER. Time course experiments revealed that this peptide existed in the ER of cos-7 cells for 16 h. Furthermore, we detected the full-length peptide in cells by fluorescent immunostaining followed by SDS-PAGE. The peptide also entered glial and neuronal cells. These results suggest that this peptide has the ability to enter cells and exert chaperone activity at the ER, and provide an insight into the development of new drugs. [ABSTRACT FROM AUTHOR]

Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii ( Mr ). Two short peptides ( Mr MBL-N20: 20 AWNTYDYMKREHSLVKPYQG 39 and Mr MBL-C16: 307 GGLFYVKHKEQQRKRF 322 ) were synthesized from the Mr MBL polypeptide. The purity of the Mr MBL-N20 (89%) and Mr MBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. Mr MBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the Mr MBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, Mr MBL-C16 peptide produced the highest activity compared to the Mr MBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The Mr MBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the Mr MBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which is involved in antimicrobial mechanism. Moreover, Mr MBL derived Mr MBL-N20 and Mr MBL-C16 peptides are important antimicrobial peptides for the recognition and eradication of viral and bacterial pathogens. [ABSTRACT FROM AUTHOR]

The objective of this study was to identify antioxidative peptides from peptic hydrolysates of half-fin anchovy (HAHp). Furthermore, the possible synergistic effects of identified peptides were evaluated. HAHp were separated using CM FF ion exchange and Sephadex G 25 chromatography. HAHp1-2, one peptide fraction with the highest 1, 1-diphenyl-2-pycrylhydrazyl (DPPH) scavenging activity, was subjected to RP-HPLC coupled with ESI under positive ionization mode. Six peptides, AGDDARPA (771.35 Da), ESAGLHE (741.33 Da), NKVKGELD (901.45 Da), TLWSGAGQTVNT (1233.63 Da), EMSAGLHE (872.37 Da) and WRKKDPLND (1171.60 Da), were identified by ESI-MS/MS analysis. Peptides AGDDARPA (HAHp1-2Ⅰ), ESAGLHE (HAHp1-2Ⅱ), NKVKGELD (HAHp1-2Ⅲ), EMSAGLHE (HAHp1-2Ⅳ) and WRKKDPLND (HAHp1-2Ⅴ) were synthesized to determine in vitro antioxidative activity. Results showed that HAHp1-2Ⅳ and HAHp1-2Ⅴ displayed higher DPPH radical scavenging activities than other three peptides and l -carnosine. Peptide HAHp1-2Ⅲ demonstrated similar reducing power ability as l -carnosine. All synthetic peptides had moderate or negligible metal chelating activities. Additionally, the enhanced DPPH radical scavenging activities or reducing power abilities were observed for the mixture of HAHp1-2Ⅲ, HAHp1-2Ⅳ or HAHp1-2Ⅴ, indicating the possible synergistic effects. These results suggested that the antioxidative peptides derived from HAHp could be used as potential antioxidants in functional foods and nutraceuticals. [ABSTRACT FROM AUTHOR]

Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) ( 1 ) and Gd-DOTA-c(RGD-ACH-K) ( 2 ), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo . Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyK peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images. [ABSTRACT FROM AUTHOR]

The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA–EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20 μM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA–EntE as well as for EntA oligomerization. [ABSTRACT FROM AUTHOR]

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver. [ABSTRACT FROM AUTHOR]

[Display omitted] • Insulin-like androgenic gland factor (IAG) form the marbled crayfish Procambarus virginalis and its disulfide isomer were chemically synthesized. • CD spectral analysis demonstrated that the Asn-linked glycan moiety attached at the B chain did not affect the peptide conformation. • Administration of the synthetic IAG suppressed the oocyte maturation, indicating that the synthetic IAG had the biological function in vivo. Insulin-like androgenic gland factor (IAG) from the marbled crayfish Procambarus virginalis is an insulin-like heterodimeric peptide composed of A and B chains and has an Asn-linked glycan at the B chain. IAG is considered to be a male sex hormone inducing the sex differentiation to male in decapod crustacean, although there is no report on the function of IAG peptide in vivo. In order to characterize P. virginalis IAG, we chemically synthesized it and evaluated its biological function in vivo. A and B chains were prepared by the ordinary solid-phase peptide synthesis, and three disulfide bonds were formed regioselectively by dimethyl sulfoxide oxidation, pyridylsulfenyl-directed thiolysis and iodine oxidation reactions. An IAG disulfide isomer was also prepared by the same manner. Circular dichroism spectral analysis revealed that the disulfide bond arrangement affected the peptide conformation, which was similar to the other insulin-family peptides analyzed so far. On the other hand, the glycan moiety attached at the B chain had no effect on the peptide secondary structure. Injection of the synthetic IAG and its disulfide isomer to female crayfish did not induce male characteristics on the external morphology, but both peptides suppressed the oocyte maturation in vivo. These results suggest that IAG has a pivotal role on the suppression of female secondary sex characteristics. [ABSTRACT FROM AUTHOR]