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1.
Yuan‐Yuan Xu Xue‐Wei Cao Long‐Yun Fu Tao‐Zhu Zhang Fu‐Jun Wang Jian Zhao 《Journal of peptide science》2019,25(9)
Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell‐penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo‐derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell‐penetrating antimicrobial peptides, including the well‐known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC‐7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC‐5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia‐derived type‐I ribosome‐inactivating protein MAP 30, and the cytotoxicity of the MAP 30‐CAT fusion protein in the tumor cell line SMMC‐7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30‐CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC‐5 was approximately twofold higher than the value for SMMC‐7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38‐fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor‐homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics. 相似文献
2.
The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine,a cell‐penetrating peptide
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Zoltán Bánóczi András Keglevich Ildikó Szabó Ivan Ranđelović Zita Hegedüs Fruzsina L. Regenbach Péter Keglevich Zsófia Lengyel Viktor Háda Áron Szigetvári László Hazai József Tóvári Ferenc Hudecz 《Journal of peptide science》2018,24(10)
Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l )‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l )‐Trp‐Arg8 and Br‐vindoline‐(d )‐Trp‐Arg8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l )‐Trp was more active than conjugate with the (d )‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l )‐Trp‐Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity. 相似文献
3.
LALF32‐51‐E7, a HPV‐16 therapeutic vaccine candidate,forms protein body‐like structures when expressed in Nicotiana benthamiana leaves
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Romana J. R. Yanez Renate Lamprecht Milaid Granadillo Isis Torrens Elsa Arcalís Eva Stöger Edward P. Rybicki Inga I. Hitzeroth 《Plant biotechnology journal》2018,16(2):628-637
High‐risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV‐16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF32‐51), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF32‐51‐E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF32‐51‐E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF32‐51‐E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast‐targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB‐like structures were also seen for the cytoplasmic LG. PB‐like structure formation was confirmed for both LG and LALF32‐51‐E7 by TEM. LALF32‐51‐E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB‐like structures. This study could open a new avenue for the use of LALF32‐51 as a PB‐inducing peptide. 相似文献
4.
Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi
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Melissa Malan June C. Serem Megan J. Bester Albert W. H. Neitz Anabella R. M. Gaspar 《Journal of peptide science》2016,22(1):43-51
Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides 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, 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, 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 glutathione 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. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
5.
Yong Hai Nan Il‐Seon Park Kyung‐Soo Hahm Song Yub Shin 《Journal of peptide science》2011,17(12):812-817
pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
6.
7.
Gaetana Paolella Marilena Lepretti Stefania Martucciello Merlin Nanayakkara Salvatore Auricchio Carla Esposito Maria Vittoria Barone Ivana Caputo 《Cell biology international》2018,42(1):112-120
Alpha‐gliadin peptide 31–43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31–43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31–43 internalization by Caco‐2 cells. In this study, we used a chemical cross‐linker to block peptide 31–43 on cell surface proteins, and pulled‐down peptide‐proteins complexes using antibodies raised against peptide 31–43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31–43 in Coomassie‐stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31–43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31–43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31–43 is able to interact with a membrane‐like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31–43 internalization by cells. 相似文献
8.
Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies
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Jenifer H. Anklesaria Bhalchandra J. Kulkarni Bhakti R. Pathak Smita D. Mahale 《Journal of peptide science》2016,22(6):383-390
Cysteine‐rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94–CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti‐CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti‐CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
9.
Susmita Bandyopadhyay Boon Yee Ng Charmaine Chong Ming Zhen Lim Sonia Kiran Gill Ke Hui Lee J Sivaraman Chiradip Chatterjee 《Journal of peptide science》2014,20(10):811-821
Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
10.
Juliana Mozer Sciani Hugo Vigerelli André Santos Costa Diana Aparecida Dias Câmara Paulo Luiz‐de‐Sá Junior Daniel Carvalho Pimenta 《Journal of peptide science》2017,23(1):68-76
Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell‐penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography‐MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP – interestingly, a classical bradykinin‐potentiating peptide – in the peptide‐rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence‐labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell‐cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label‐free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
11.
Translocation of several fluorescently labeled arginine‐rich peptides into intact plant cells was quantitatively examined in order to investigate the structural factors required for efficient cellular internalization, and thereby, to evaluate the potential of arginine‐rich peptides as intracellular delivery vectors in plants. Cell‐penetrating peptides (CPPs) such as arginine‐rich peptides permit the direct introduction of biologically active macromolecules into plant cytoplasm to manipulate various intracellular processes. While a significant level of adsorption of applied arginine‐rich peptides was observed in the cell walls rich in negative charges, removal of adsorbed peptides by trypsin treatment allowed determination of the amount of internalized peptides in a quantitative manner using spectrofluorometric analysis. The internalization of arginine‐rich peptides depended on the number of arginine residues, and the peptide containing eight arginine residues showed most effective internalization. Besides, the position of small cargoes attached to the arginine‐rich peptides markedly affected the internalization efficiency. The results obtained in this study provide useful information for the development of efficient intracellular delivery tools in plant science. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
12.
Interactions between chensinin‐1, a natural antimicrobial peptide derived from Rana chensinensis,and lipopolysaccharide
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Lipopolysaccharide (LPS) plays a critical role in the pathogenesis of sepsis caused by gram‐negative bacterial infections. Therefore, LPS‐neutralizing molecules would have important clinical applications. Chensinin‐1, a novel antimicrobial peptide with atypical structural features, was found in the skin secretions of the Chinese brown frog Rana chensinensis. To understand the role of LPS in the bacterial susceptibility to chensinin‐1 and to investigate its anti‐endotoxin effects, the interactions of chensinin‐1 with LPS were investigated in this study using circular dichroism, in situ IR, isothermal titration calorimetry, and zeta potential. This study is the first to use in situ IR spectroscopy to evaluate the secondary structural changes of this peptide. The capacity of chensinin‐1 to block the LPS‐dependent cytokine secretion of macrophages was also investigated. Our results show that chensinin‐1 can form α‐helical structures in LPS suspensions. LPS can affect the antimicrobial activity of chensinin‐1, and chensinin‐1 was able to mitigate the effects of LPS. These data may facilitate the development of antimicrobial peptides with potent antimicrobial and anti‐endotoxin activities. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 719–726, 2015. 相似文献
13.
The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study
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Begum Alaybeyoglu Bilge Gedik Uluocak Berna Sariyar Akbulut Elif Ozkirimli 《Journal of peptide science》2017,23(5):374-383
Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
14.
Lezaan Prinsloo Alex Naidoo June Serem Helena Taute Yasien Sayed Megan Bester Albert Neitz Anabella Gaspar 《Journal of peptide science》2013,19(5):325-332
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 0.94–15 µg/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 α‐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. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
15.
Cyclization of a cell‐penetrating peptide via click‐chemistry increases proteolytic resistance and improves drug delivery
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Ines Neundorf 《Journal of peptide science》2016,22(6):421-426
In this work we report synthesis and biological evaluation of a cell‐penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne‐azide click reaction. Cell viability studies in several cell‐lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF‐7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
16.
Platelet‐activating factor (PAF) is an important mediator of anaphylaxis and is therefore an anti‐anaphylactic drug target. We recently reported that synthetic N‐terminally biotinylated peptides (BP4‐BP29) inhibit PAF by directly interacting with PAF and its metabolite/precursor lyso‐PAF. In this study, we investigated whether the biotinylated peptides can inhibit anaphylactic reactions in vivo. In mouse models of anaphylaxis, one of the peptides, BP21, markedly and dose‐dependently inhibited hypothermia with a maximum dose–response within 30 min after administration, even at doses 20 times lesser than doses of the known PAF antagonist CV‐3988. In contrast, the anti‐hypothermic effect of BGP21, in which the Tyr‐Lys‐Asp‐Gly sequence in BP21 was modified to a Gly‐Gly‐Gly‐Gly sequence, was less than that of BP21. The alanine scanning and shuffling the amino acid residues of BP4 (Tyr‐Lys‐Asp‐Gly) demonstrated that the Tyr‐Lys‐Asp‐Gly consensus sequence is important for the inhibitory effect of the peptide on hypothermia. BP21 also suppressed vascular permeability during anaphylaxis with a maximum dose–response within 30 min of administration. In a rat model of hind paw oedema, BP21 significantly inhibited the oedema induced by PAF but not that induced by the other pro‐inflammatory mediators, such as histamine, serotonin, and bradykinin. Tryptophan fluorescence measurements showed that BP21 interacted with PAF, but not with histamine, serotonin, or bradykinin. In contrast, BGP21 did not interact with PAF. These results suggest that biotinylated peptides, especially BP21, can specifically and markedly inhibit anaphylactic reactions in vivo and that this involves direct interaction of its Tyr‐Lys‐Asp‐Gly region with PAF. Therefore, a biotinylated peptide, BP21, can be used as novel potential anti‐anaphylactic drugs targeting PAF. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
17.
Philippe Laporte Béatrice Satiat‐Jeunemaître Isabel Velasco Tibor Csorba Willem Van de Velde Anna Campalans Joszef Burgyan Miguel Arevalo‐Rodriguez Martin Crespi 《The Plant journal : for cell and molecular biology》2010,62(1):24-38
Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen‐fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so‐called nodulin genes are induced, including large families that encode small peptides. Using a three‐hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA‐binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single‐stranded RNA‐binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA‐binding peptides. 相似文献
18.
The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 310‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH2 was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and 13C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/310‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]222/[θ]207, as a reporter of α‐/310‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc. 相似文献
19.
Molecular dissection of the interactions of an antitumor interleukin‐2‐derived mutein on a phage display‐based platform
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Gertrudis Rojas Tania Carmenate Kalet Leon 《Journal of molecular recognition : JMR》2015,28(4):261-268
A mutein with stronger antitumor activity and lower toxicity than wild‐type human interleukin‐2 (IL‐2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL‐2 receptor alpha chain (thought to be critical for both IL‐2‐driven expansion of T regulatory cells and IL‐2‐mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL‐2‐derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage‐displayed IL‐2 variants to induce proliferation of the IL‐2‐dependent CTLL‐2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display‐based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL‐2 and related cytokines, accelerating the development of cytokine‐derived therapeutics. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
20.
Eunjung Lee Jin‐Kyoung Kim Soyoung Shin Ki‐Woong Jeong Juneyoung Lee Dong Gun Lee Jae‐Sam Hwang Yangmee Kim 《Journal of peptide science》2011,17(10):675-682
Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9‐mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR‐NH2), 9Pbw3 (RLWLAIWRR‐NH2), and 9Pbw4 (RLWLAWKRR‐NH2). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559–568). In this study, we investigated the anti‐inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS‐stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp7 as well as optimum level of hydrophobicity may play key roles in the anti‐inflammatory activity of 9Pbw3. In order to design potent anti‐inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3‐D , the all‐D ‐amino acid analog of 9Pbw3. 9Pbw3‐D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation‐induced cytokine production in LPS‐stimulated RAW264.7 cells than 9Pbw3. 9Pbw3‐D inhibited the gene expression of inflammatory‐induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3‐D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti‐inflammatory activities. 9Pbw3‐D with higher anti‐inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. 相似文献