pinecone/core-2020-05-10-deduplication huggingface dataset:
Low-generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher-generation (G4-G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site-specifically attached. Therefore, we propose that lower-generation asymmetric dendrimers, which are likely devoid of toxicity and to which site-specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide-based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd
Improving membrane binding as a design strategy for amphipathic peptide hormones: 2-helix variants of PYY3-36

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic a-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed a-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide–micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide–membrane interactions
A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori

Defensins are a class of cysteine-rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well-conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its gamma-motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram-positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram-negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP-1 cells. Finally, SolyC exerts an anti-inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF-alpha and IFN-gamma
The mapping of linear B-cell epitope regions in desmoglein \ud
1 and 3 proteins : Recognition of immobilized peptides by \ud
pemphigus patients’ serum autoantibodies

Desmosomal transmembrane glycoproteins desmoglein 1and desmoglein 3 are targets of life-threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus \ud
foliaceus(PF). In these diseases pemphigus autoantibodies are produced against Dsg1 and \ud
Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of \ud
desmosomal cell-cell adhesion, and clinically, to the presence of blisters and erosions. \ud
Identification, characterization and detailed analysis of the binding sites of autoantibodies \ud
have an outstanding importance in understanding theimmunopathology of the disease and \ud
also in the design of novel diagnostics. \ud
Here we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins \ud
extracellular parts recognized by IgG-type serum autoantibodies of patients with  PV andPF. \ud
In our study overlapping pentadecapeptides were synthesized on hydroxypropylmethacrylate \ud
pins based on the results of in silicopredictions. To detect the interaction between theserum \ud
autoantibodies and the immobilized synthetic peptides, modified ELISA (Enzyme Linked \ud
Immunosorbent Assay) was performed with pin-attached peptides testing the serum samples \ud
of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110, \ud
aa196-220, aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and four \ud
possible epitope regions (aa64-78, aa330-344, aa375-399, aa446-460) within the Dsg3 protein \ud
sequence using these methods. Our data showed that  serum autoantibodies of patients, \ud
previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear \ud
epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in \ud
modified ELISAs
Antimicrobial peptides from plants: stabilization of the core of a tomato defensin by intramolecular disulfide bond

Cysteine-containing antimicrobial peptides of diverse phylogeny share a common structural signature, the γ core, characterized by a strong polarization of charges in two antiparallel β sheets. In this work, we analyzed peptides derived from the tomato defensin SolyC07g007760 corresponding to the protein γ core and demonstrated that cyclization of the peptides, which results in segregation of positive charges to the turn region, produces peptides very active against Gram negative bacteria, such as Salmonella enterica and Helicobacter pylori. Interestingly, these peptides show very low hemolytic activity and thus represent a scaffold for the design of new antimicrobial peptides
Analysis of the haptoglobin binding region on the apolipoprotein A-I-derived P2a peptide

Apolipoprotein A-I (ApoA-I) is the main protein component of the high density lipoproteins and it plays an important role in the reverse cholesterol transport. In particular, it stimulates cholesterol efflux from peripheral cells toward liver and activates the enzyme lecithin-cholesterol acyltransferase (LCAT). Haptoglobin (Hpt), a plasma α2-glycoprotein belonging to the family of acute-phase proteins, binds to ApoA-I inhibiting the stimulation of the enzyme LCAT. Previously, we reported that a synthetic peptide, P2a, binds to and displaces Hpt from ApoA-I restoring the LCAT cholesterol esterification activity in the presence of Hpt. Here, we investigate the molecular determinants underlining the interaction between Hpt and P2a peptide. Analysis of truncated P2a analogs showed that P2a sequence can only be slight reduced in length at the N-terminal to preserve the ability of binding to Hpt. Binding assays showed that charged residues are not involved in Hpt recognition; actually, E146A and D157A substitutions increase the binding affinity to Hpt. Biological characterization of the corresponding P2a peptide analogs, Apo146 and Apo157, showed that the two peptides interfere with Hpt binding to HDL and are more effective than P2a peptide in rescue LCAT activity from Hpt inhibition. This result suggests novel hints to design peptides with anti-atherogenic activity
Structural and functional characterization of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to
evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant,
and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the
carboxy-terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted
(Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15mg/ml) to both Gram-positive
and Gram-negative bacteria, whereas the parent peptide only exhibited Gram-positive antibacterial activity. The Os peptide
was found to be two-fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus
aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer,
suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing
of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data
indicated that in a membrane-mimicking environment, the cysteine-containing peptide has a higher a-helical content.
Both peptides were found to be non-toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity
and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications
and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted.Medical Research Council of South Africa,the National Research Foundation of South Africa, and the University of Pretoriahttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-1387hb201
Role of water in protein folding, oligomerization, amyloidosis and miniprotein

The essential involvement of water in most fundamental extra-cellular and intracellular processes of proteins is critically reviewed and evaluated in this article. The role of water in protein behavior displays structural ambivalence; it can protect the disordered peptide-chain by hydration or helps the globular chain-folding, but promotes also the protein aggregation, as well (see: diseases). A variety of amyloid diseases begins as benign protein monomers but develops then into toxic amyloid aggregates of fibrils. Our incomplete knowledge of this process emphasizes the essential need to reveal the principles of governing this oligomerization. To understand the biophysical basis of the simpler in vitro amyloid formation may help to decipher also the in vivo way. Nevertheless, to ignore the central role of the water's effect among these events means to receive an uncompleted picture of the true phenomenon. Therefore this review represents a stopgap role, because the most published studies—with a few exceptions—have been neglected the crucial importance of water in the protein research. The following questions are discussed from the water's viewpoint: (i) interactions between water and proteins, (ii) protein hydration/dehydration, (iii) folding of proteins and miniproteins, (iv) peptide/protein oligomerization, and (v) amyloidosis
Synthesis of a polymyxin derivative for photolabeling studies in the gram-negative bacterium Escherichia coli

The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics
Structural Characterization of Triple Transmembrane Domain Containing Fragments of a Yeast G Protein-Coupled Receptor in an Organic: Aqueous Environment by Solution-State NMR Spectroscopy

This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second and third transmembrane domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3 was investigated by solution NMR in trifluorethanol/water: TM1-TM3 contains helical transmembrane domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from E. coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast GPCR can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids
Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern.

Article first published online: 27 APR 2015La1 is a 73-residue cysteine-rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N-acylurea approach with Fmoc-SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom
Characterization of Desmoglein-3 Epitope Region Peptides as Synthetic Antigens: Analysis of their in vitro T-cell Stimulating Efficacy, Cytotoxicity, Stability and their Conformational Features

Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189–205, Dsg3/206–222, Dsg3/342–358, and Dsg3/761–777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure–activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342–358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192–205, Dsg3/763–777, and Dsg3/764–777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were themost capable to distinguish between healthy and PV donors
Facile and selective covalent grafting of an RGD-peptide to electrospun scaffolds improves HUVEC adhesion

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly-ε-caprolactone or poly(L-lactic acid-co-ɛ-caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly-Arg-Gly-Asp-Ser-Pro motifs per chain and a p-azido-Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly-ε-caprolactone embeddedwith adhesive peptideswere produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24h with a dosedependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results
Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides (AMPs) are small, cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several AMPs are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin (OsDef2), were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 μM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione (GSH), a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 μM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 μM and 25 μM for melittin and polymyxin B, respectively. In addition, Os, Os-C and GSH protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 μM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties.Medical Research Council (MRC), the National Research Foundation (NRF) of South Africa and the University of Pretoria.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-13872017-01-31hb201
Synthesis, characterisation and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells

Targeted tumour therapy is in the focus of recent cancer research. GnRH analogues are able to deliver anticancer agents selectively into tumour cells which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared and the investigated types of cancers are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II, and -III analogues which successfully used for drug targeting. In this manuscript we investigated these analogues solubility, stability, passive membrane permeability and compared their cellular uptake by various cancer cells. \ud
We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (MDCK). Each of the three conjugates were taken up by GnRH-I receptor expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells.\ud
In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH based drug delivery systems on various cells and offer novel information about these peptides